Nutrisi Untuk Jantung

Nutrition and
Cardiometabolic Health
Nutrition and
Edited by
Nathalie Bergeron, Patty W. Siri-Tarino, George A. Bray,
and Ronald M. Krauss
CRC Press
Taylor & Francis Group
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Library of Congress Cataloging-in-Publication Data
Names: Bergeron, Nathalie, editor. | Siri-Tarino, Patty W., editor. | Bray,

George A., editor. | Krauss, Ronald M., editor.
Title: Nutrition and cardiometabolic health / [edited by] Nathalie Bergeron,
Patty W. Siri-Tarino, George A. Bray, and Ronald M. Krauss.
Description: Boca Raton : Taylor & Francis, 2017. | Includes bibliographical
references.
Identifiers: LCCN 2017020806 | ISBN 9781498704267 (hardback : alk. paper)
Subjects: | MESH: Obesity, Metabolically Benign--diet therapy |
Cardiovascular Diseases--prevention & control | Risk Factors | Nutrition
Therapy—methods
Classification: LCC RC628 | NLM WD 210 | DDC 616.3/980654--dc23
LC record available at https://lccn.loc.gov/2017020806
Visit the Taylor & Francis Web site at

http://www.taylorandfrancis.com
and the CRC Press Web site at

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Contents
Preface................................................................................................................................................ix
Editors................................................................................................................................................xi
Contributors......................................................................................................................................xv
Section I Energy Balance, Adiposity, and

Chapter 1 Regulation of Food Intake: The Gut–Brain Axis..........................................................3

Surya Panicker Rajeev, Ian W. Seetho, and John P. H. Wilding
Chapter 2 Overeating Behavior and Cardiometabolic Health: Mechanisms and Treatments......23

Ashley E. Mason and Frederick M. Hecht
Chapter 3 Energy Balance and Regulation of Body Weight: Are All Calories Equal?................51
Kevin D. Hall
Chapter 4 Diets for Weight Loss..................................................................................................61

George A. Bray and Patty W. Siri-Tarino
Chapter 5 Weight Loss by Surgical Intervention: Nutritional Considerations and Influence

on Health.....................................................................................................................77
Karim Kheniser and Sangeeta Kashyap
Chapter 6 Physical Activity and Cardiometabolic Health......................................................... 101

Andrea M. Brennan and Robert Ross
Chapter 7 Diet as a Potential Modulator of Body Fat Distribution........................................... 123

Sofia Laforest, Geneviève B. Marchand, and André Tchernof
Chapter 8 Nutritional Considerations for Cardiometabolic Health in Childhood and

Adolescent Obesity...................................................................................................149
Elizabeth Prout Parks, Jennifer Panganiban, Stephen R. Daniels,
and Julie Brothers
Chapter 9 Aging and Cardiovascular Disease: Lessons from Calorie Restriction.....................173

Jasper Most and Leanne M. Redman
v
vi Contents
Section II Dietary Fats and Cardiometabolic Health

Chapter 10 Omega-3 and Omega-6 Fatty Acids: Roles in Cardiometabolic Disease..................193
William S. Harris
Chapter 11 Evolving Role of Saturated Fatty Acids....................................................................209

Patty W. Siri-Tarino and Ronald M. Krauss
Chapter 12 Effects of Dietary Trans Fatty Acids on Cardiovascular Risk...................................223

Ronald P. Mensink
Section III D
ietary Carbohydrates and
Chapter 13 Epidemiologic and Mechanistic Studies of Sucrose and Fructose in Beverages
and Their Relation to Obesity and Cardiovascular Risk...........................................237
George A. Bray
Chapter 14 Effects and Mechanisms of Fructose-Containing Sugars in the Pathophysiology

of Metabolic Syndrome.............................................................................................251
Kimber L. Stanhope and Peter J. Havel
Chapter 15 Dietary Carbohydrate Restriction in the Management of NAFLD and

Metabolic Syndrome.................................................................................................275
Grace Marie Jones, Kathleen Mulligan, and Jean-Marc Schwarz
Chapter 16 Dietary Starches and Grains: Effects on Cardiometabolic Risk................................297

Nathalie Bergeron and Ronald M. Krauss
Section IV Dietary Protein and Cardiometabolic Health

Chapter 17 Interaction of Dietary Protein and Energy Balance...................................................317
Eveline A. Martens, Richard D. Mattes, and Margriet S. Westerterp-Plantenga
Chapter 18 A Protein-Centric Perspective for Skeletal Muscle Metabolism and

Cardiometabolic Health.............................................................................................333
Donald K. Layman
Chapter 19 Protein Sources, CVD, Type 2 Diabetes, and Total Mortality..................................349

Peter Clifton
Contents vii
Section V D
ietary Food Groups, Patterns,
and Cardiometabolic Health
Chapter 20 Consumption of Foods, Food Groups, and Cardiometabolic Risk............................373

Edward Yu and Frank B. Hu
Chapter 21 Dietary Patterns and Cardiometabolic Disease.........................................................397

Elizabeth M. Cespedes Feliciano and Frank B. Hu
Chapter 22 The Mediterranean Diet to Prevent Type 2 Diabetes and Cardiovascular Disease...421
Michel de Lorgeril
Chapter 23 The DASH Diet.........................................................................................................431

Catherine M. Champagne
Chapter 24 Nut Consumption and Coronary Heart Disease (CHD) Risk and Mortality.............449
Christina Link, Alyssa Tindall, Jordi Salas-Salvadó, Caitlin Lynch, and
Penny Kris-Etherton
Chapter 25 Dairy Product Consumption, Dairy Fat, and Cardiometabolic Health......................481

Benoît Lamarche
Chapter 26 Paleolithic Diets........................................................................................................493

Staffan Lindeberg, Maelán Fontes Villalba, Pedro Carrera-Bastos, and
Lynda Frassetto
Chapter 27 Fasting Intermittently or Altering Meal Frequency: Effects on Plasma Lipids.........517

John F. Trepanowski and Krista A. Varady
Section VI Other Nutritional Influences

of Cardiometabolic Health
Chapter 28 Early-Life Nutrition, Epigenetics, and Later Cardiometabolic Health.....................531

Mark H. Vickers, Clare M. Reynolds, and Clint Gray
Chapter 29 Gene–Diet Interactions..............................................................................................555

Silvia Berciano and Jose M. Ordovas
viii Contents
Chapter 30 Gut Microbiome: Its Relationship to Health and Its Modulation by Diet.................571
Brian J. Bennett and Katie A. Meyer
Chapter 31 Alcohol: Associations with Blood Lipids, Insulin Sensitivity, Diabetes,

Clotting, CVD, and Total Mortality..........................................................................593
Charlotte Holst and Janne Schurmann Tolstrup
Chapter 32 Endocrine Disrupting Chemicals, Obesogens, and the Obesity Epidemic............... 603
Raquel Chamorro-Garcia and Bruce Blumberg
Index��615
Preface
Nutrition is the major environmental influence on metabolic systems that impact cardiovascular
health and disease. Past decades have seen major advances in the identification of specific dietary
effects on these systems. However, as this knowledge has grown, and the tools for studying these
effects have become more diverse and powerful, there has been growing appreciation of the com-
plexities and challenges facing those seeking to gain an in-depth yet comprehensive understanding
of dietary effects on cardiometabolic health. Intensifying this concern is the imperative of address-
ing the global increase in the incidence of cardiovascular disease, coupled with the diet-related
metabolic conditions—dyslipidemia, diabetes, and obesity—that play key roles in its pathogenesis.
In preparing this textbook, we have called on the expertise of scientists across a broad range of
topics and disciplines to assemble information aimed at researchers, clinicians, and other health
professionals who have interests in this important field.
The chapters in the first section of the textbook are clustered around the theme of energy balance
and adiposity as they relate to cardiometabolic health. An overview of the regulatory mechanisms
that determine energy balance is provided in Chapter 1 followed by a chapter on the critical role of
behavior in regulating eating (Chapter 2). The debate of whether “a calorie is a calorie” regardless
of food source is addressed in the next two chapters (Chapters 3 and 4), along with methods for
weight loss. Caloric restriction is addressed in Chapter 4 and bariatric surgery in Chapter 5. The
benefits of physical activity on cardiometabolic health, with or without weight loss, are addressed in
Chapter 6. The effects of diet on body fat distribution and the significance of the more metabolically
active central versus peripheral adiposity are the topics for Chapter 7. This section concludes with
Chapters 8 and 9 on nutritional considerations at different stages of the lifespan—in childhood and
adolescence, and in the elderly.
Sections II through IV of the book are devoted to evaluating macronutrient effects on cardio-
metabolic health. In Section II on dietary fats, the effects of polyunsaturated fatty acids, specifi-
cally omega-3 and omega-6 fatty acids, are discussed in Chapter 10. The role of dietary saturated
fats, along with the need to evaluate them in the context in which they are consumed, is reviewed
in Chapter 11. The effects of trans fatty acids on blood lipids and cardiovascular disease risk, with
consideration for industrially produced vs. ruminant trans fats, are discussed in Chapter 12, the third
and final chapter of this section.
In Section III, Chapters 13 and 14 on dietary carbohydrates and cardiometabolic health pay par-
ticular attention to the role of sugar—consumed in quantities 40 times what was consumed at the
time of the American Revolution in 1776. The quantity and quality of carbohydrates and the role of
carbohydrate restriction in improving metabolic health are reviewed in Chapters 15 and 16.
Section IV’s focus, dietary protein in relation to cardiometabolic health is covered in
Chapters 17 through 19, with a focus on its role in energy balance, skeletal muscle function, and
cardiovascular and diabetes risk.
A renewed interest and focus on whole foods and overall dietary patterns as a means toward
cardiovascular health has led to the development of unique and validated methods of analysis as
presented in Section V, Chapters 20 and 21. The two dietary patterns with the strongest evidence
base for cardioprotective effects, namely, the Mediterranean and DASH diets, are reviewed in
Chapters 22 and 23. Food groups that have been heavily touted and consumed include tree nuts
and dairy foods, and the evidence for effects of these food groups on cardiometabolic health are
reviewed in Chapters 24 and 25. The Section V concludes with Chapters 26 and 27 that present
more debated approaches toward cardiometabolic health, including Paleolithic diets and intermittent
fasting regimens.
ix
x Preface
In the last section of the textbook, Section VI, other nutritional factors impacting cardiometabolic
health are considered. Chapter 28 focuses on the role of nutrition in modulating gene expression
and epigenetics. Chapter 29 reviews gene–diet interactions that may contribute to interindividual
differences in dietary needs and responses. An assessment of the role of the intestinal microbiome
in modulating metabolic traits is provided in Chapter 30. Chapters 31 and 32, the final two chapters
discuss alcohol and endocrine disruptors as diet-related influences on cardiometabolic health.
We are grateful to the individuals who contributed well-researched and incisive chapters to this
textbook. It is our hope that the information assembled here will have value to those who share our
goal of applying nutritional science to reducing the burden of cardiovascular disease.
Editors
Nathalie Bergeron, PhD, is professor of biological sciences at
Touro University California College of Pharmacy and associ-
ate staff scientist in the Atherosclerosis Research Program at
Children’s Hospital Oakland Research Institute. She was trained
in dietetics and nutritional biochemistry and graduated from Laval
University, Canada, with a PhD in nutrition. She pursued her post-
doctoral training at the Cardiovascular Research Institute of the
University of California, San Francisco, where she specialized in
postprandial lipoprotein metabolism. Dr. Bergeron began her aca-
demic career as a research professor at Laval University in 1996.
She was a visiting professor in the Department of Nutritional
Sciences and Toxicology at the University of California, Berkeley, from 2000 to 2002 and joined the
Touro University, California College of Pharmacy, at its inception in 2005. At Touro, Dr. Bergeron
teaches in the areas of pathophysiology of metabolic diseases, as well as nutrition. She also holds a
staff scientist position at the Children’s Hospital Oakland Research Institute. Her research is clini-
cal in nature and focuses on dietary composition, with a special emphasis on carbohydrate quantity
and quality, and its relationship to features of atherogenic dyslipidemia. Her more recent research
activities include looking at variations of the DASH and Mediterranean dietary patterns and their
relationship to cardiometabolic health. Over the course of her academic career, she has received
research grants from the Medical Research Council of Canada, the Heart and Stroke Foundation
of Canada, the American Diabetes Association, and the National Institutes of Health, along with
investigator-initiated funding from the Dairy Farmers of Canada, the Dairy Research Institute, and
the Almond Board of California.
Patty W. Siri-Tarino, PhD, is an associate staff scientist in the

Atherosclerosis Research Program and program director of the
Family Heart & Nutrition Center at the Children’s Hospital Oakland
Research Institute. She earned her undergraduate degree in biol-
ogy at Tufts University, a Master of Science in epidemiology at the
Netherlands Institute of Health Sciences, and a PhD in nutrition and
metabolic biology at Columbia University, where she developed a
transgenic mouse model of insulin resistance, obesity, and dyslip-
idemia. Dr. Siri-Tarino began her postdoctoral work by developing
and conducting studies in humans aimed at understanding variabil-
ity in the postprandial response to high-fat meals and the role of
cholesterol absorption inhibitors in its modulation. She subsequently worked on dietary intervention
studies evaluating macronutrient effects on CVD risk profiles in the context of weight loss and sta-
bility as well as studies evaluating genetic effects on energy metabolism at rest and during exercise.
Dr. Siri-Tarino has spoken nationally and internationally on the role of diet on lipoprotein profiles
as biomarkers of cardiovascular disease and published peer-reviewed journal articles, reviews, book
chapters, and popular media articles on diet, lifestyle, and genetic determinants of heart health. She
is interested in community engagement and education.
xi
xii Editors
George A. Bray, MD, MACP, MACE, is a Boyd professor emeri-

tus at the Pennington Biomedical Research Center of Louisiana
State University in Baton Rouge, Louisiana, and professor of med-
icine emeritus at the Louisiana State University Medical Center
in New Orleans. After graduating from Brown University summa
cum laude in 1953, Dr. Bray entered Harvard Medical School,
graduating magna cum laude in 1957. His postdoctoral training
included an internship at the Johns Hopkins Hospital, Baltimore,
Maryland, a fellowship at the NIH, residence at the University of
Rochester, and fellowships at the National Institute for Medical
Research in London and at the Tufts-New England Medical Center
in Boston. In 1970, he became director of the Clinical Research Center at the Harbor UCLA Medical
Center and the organizer of the First Fogarty International Center Conference on Obesity in 1973.
Dr. Bray chaired the Second International Congress on Obesity in Washington, DC, in 1977. In
1989, he became the first executive director of the Pennington Biomedical Research Center in
Baton Rouge, a post he held until 1999. He is a Master of the American College of Physicians,
Master of the American College of Endocrinology, and Master of the American Board of Obesity
Medicine. Dr. Bray founded the North American Association for the Study of Obesity in 1982 (now
The Obesity Society), and he was the founding editor of its journal, Obesity Research, as well
as cofounder of the International Journal of Obesity and the first editor of Endocrine Practice,
the official journal of the American College of Endocrinologists. He has received many awards
during his medical career, including the Johns Hopkins Society of Scholars Award, Honorary
Fellow of the American Dietetic Association, the Bristol-Myers Squibb Mead-Johnson Award in
Nutrition, the Joseph Goldberger Award from the American Medical Association, the McCollum
Award from the American Society of Clinical Nutrition, the Osborne-Mendel Award from the
American Society of Nutrition, the TOPS Award, the Weight Watchers Award, the Stunkard
Lifetime Achievement Award, and the Presidential Medal from The Obesity Society. During his
50 academic years, he authored or coauthored more than 1900 publications, ranging from peer-
reviewed articles and reviews to books, book chapters, and abstracts reflected in his Hirsch (H)
Index of 89. Dr. Bray has had a long interest in the history of medicine and has written articles
and a book on the history of obesity.
Ronald M. Krauss, MD, is senior scientist and Dorothy Jordan

Endowed Chair at Children’s Hospital Oakland Research Institute,
professor of medicine at UCSF, and adjunct professor of nutri-
tional sciences at UC Berkeley. He earned his undergraduate and
medical degrees from Harvard University with honors and served
his internship and residency in the Harvard Medical Service of
Boston City Hospital. He then joined the staff of the National
Heart, Lung, and Blood Institute in Bethesda, Maryland, first as
clinical associate and then as senior investigator in the Molecular
Disease Branch. Dr. Krauss is board certified in internal medicine,
endocrinology, and metabolism, and is a member of the American
Society for Clinical Investigation, a fellow of the American Society of Nutrition and the American
Heart Association (AHA), and a distinguished fellow of the International Atherosclerosis Society.
He has served on the U.S. National Cholesterol Education Program Expert Panel on Detection,
Evaluation, and Treatment of High Blood Cholesterol in Adults; was the founding chair of the
AHA Council on Nutrition, Physical Activity, and Metabolism; and is a national spokesperson
for the AHA. He has also served on both the Committee on Dietary Recommended Intakes for
Macronutrients and the Committee on Biomarkers of Chronic Disease of the Institute of Medicine of
the National Academy of Sciences. He has received numerous awards, including the AHA Scientific
Editors xiii
Councils Distinguished Achievement Award, the Centrum Center for Nutrition Science Award
of the American Society for Nutrition, the Distinguished Leader in Insulin Resistance from the
International Committee for Insulin Resistance, and the AHA Award of Meritorious Achievement.
In addition, he has been the Robert I. Levy Lecturer of the AHA, the Edwin Bierman Lecturer for
the American Diabetes Association, and the Margaret Albrink Lecturer at West Virginia University
School of Medicine. Dr. Krauss is on the editorial boards of a number of journals and has been
associate editor of Obesity, the Journal of Lipid Research, and the Journal of Clinical Lipidology.
He has published nearly 500 research articles and reviews on genetic, dietary, and drug effects on
plasma lipoproteins and coronary artery disease. Among his accomplishments is the identification
of atherogenic dyslipidemia, a prevalent lipoprotein trait (high triglyceride, low HDL, and increase
in small, dense LDL particles) that is associated with risk of cardiovascular disease and type 2
diabetes. In recent years, his work has focused on interactions of genes with dietary and drug treat-
ments that affect metabolic phenotypes and cardiovascular disease risk.
Contributors
Brian J. Bennett Julie Brothers
Western Human Nutrition Research Center Division of Cardiology
Agricultural Research Service The Children’s Hospital of Philadelphia
United States Department of Agriculture Philadelphia, Pennsylvania
Davis, California
Pedro Carrera-Bastos
Silvia Berciano Center for Primary Health Care Research
Faculty of Pharmacy Lund University
Universidad Autonoma de Madrid, Spain Lund, Sweden
Madrid, Spain
Elizabeth M. Cespedes Feliciano
and
Kaiser Permanente Northern California
Jean Mayer USDA Human Nutrition Research Oakland, California
Center on Aging
Tufts University Raquel Chamorro-Garcia
Boston, Massachusetts Department of Developmental and
Cell Biology
Nathalie Bergeron University of California, Irvine
College of Pharmacy Irvine, California
Touro University California
Vallejo, California Catherine M. Champagne
Pennington Biomedical Research Center
and Baton Rouge, Louisiana
Children’s Hospital Oakland Research Institute
Oakland, California Peter Clifton
Alliance for Research in Exercise, Nutrition
and Activity (ARENA)
Bruce Blumberg
Sansom Institute for Health Science
Department of Developmental and Cell Biology
School of Pharmacy and Medical Sciences
and
University of South Australia
Department of Pharmaceutical Sciences
Adelaide, South Australia, Australia
University of California, Irvine
Irvine, California
Stephen R. Daniels
Department of Pediatrics
George A. Bray University of Colorado School of Medicine
Pennington Biomedical Research Center Aurora, Colorado
Baton Rouge, Louisiana
and Michel de Lorgeril
Laboratoire Cœur et Nutrition
Children’s Hospital Oakland Research Institute Faculté de Médecine
Oakland, California Grenoble, France
Andrea M. Brennan Lynda Frassetto

School of Kinesiology and Health Studies School of Medicine
Queen’s University University of California, San Francisco
Kingston, Ontario, Canada San Francisco, California
xv
xvi Contributors
Clint Gray Karim Kheniser

Liggins Institute and Gravida: National Centre Department of Endocrinology, Diabetes
for Growth and Development and Metabolism
University of Auckland Cleveland Clinic
Auckland, New Zealand Cleveland, Ohio
Kevin D. Hall
National Institute of Diabetes and Digestive and Ronald M. Krauss
Kidney Diseases Children’s Hospital Oakland Research
National Institutes of Health Institute
Bethesda, Maryland Oakland, California
William S. Harris
Penny Kris-Etherton
Department of Internal Medicine
Department of Nutritional Sciences
Sanford School of Medicine
The Pennsylvania State University
University of South Dakota
University Park, Pennsylvania
and
OmegaQuant Analytics, LLC
Sioux Falls, South Dakota Sofia Laforest
School of Nutrition
Peter J. Havel Laval University
Department of Molecular Biosciences and
School of Veterinary Medicine Quebec Heart and Lung Institute
and Québec City, Québec, Canada
Department of Nutrition
University of California, Davis
Benoît Lamarche
Davis, California
Institute of Nutrition and Functional Foods
Frederick M. Hecht School of Nutrition
Department of Medicine Laval University
University of California, San Francisco Québec City, Québec, Canada
San Francisco, California
Charlotte Holst Donald K. Layman
National Institute of Public Health Department of Food Science and Human
University of Southern Denmark Nutrition
Copenhagen, Denmark University of Illinois at Urbana-Champaign
Urbana, Illinois
Frank B. Hu
Staffan Lindeberg
and
Center for Primary Health Care Research
Department of Epidemiology
Lund University
Harvard University School of Public Health
Lund, Sweden
Boston, Massachusetts
Grace Marie Jones Christina Link
College of Medicine Department of Nutritional Sciences
Touro University California The Pennsylvania State University
Vallejo, California University Park, Pennsylvania
Sangeeta Kashyap
Department of Endocrinology, Diabetes Caitlin Lynch
and Metabolism Department of Nutritional Sciences
Cleveland Clinic The Pennsylvania State University
Cleveland, Ohio State College, Pennsylvania
Contributors xvii
Geneviève B. Marchand Jose M. Ordovas

School of Nutrition IMDEA Alimentación
Laval University and
and Centro Nacional de Investigaciones
Quebec Heart and Lung Institute Cardiovasculares
Québec City, Québec, Canada Madrid, Spain
and
Eveline A. Martens
Dutch Kidney Foundation Jean Mayer USDA Human Nutrition Research
Bussum, the Netherlands Center on Aging
Tufts University
Ashley E. Mason Boston, Massachusetts
UCSF Department of Psychiatry
UCSF Osher Center for Integrative Medicine Jennifer Panganiban
San Francisco, California Division of Gastroenterology, Hepatology, and
Nutrition
The Children’s Hospital of Philadelphia
Richard D. Mattes
Philadelphia, Pennsylvania
Department of Nutrition Science
College of Health and Human Sciences Elizabeth Prout Parks
Purdue University The Children’s Hospital of Philadelphia
West Lafayette, Indiana and
Perelman School of Medicine
Ronald P. Mensink The University of Pennsylvania
Department of Human Biology Philadelphia, Pennsylvania
NUTRIM School of Nutrition and Translational
Research in Metabolism Surya Panicker Rajeev
Maastricht University Medical Center Institute of Ageing and Chronic Disease
Maastricht, the Netherlands University of Liverpool
and
Aintree University Hospital NHS Foundation
Katie A. Meyer
Trust
Liverpool, United Kingdom
Nutrition Research Institute
University of North Carolina at Chapel Hill Leanne M. Redman
Chapel Hill, North Carolina Division of Clinical Sciences
Pennington Biomedical Research Center
Jasper Most Baton Rouge, Louisiana
Division of Clinical Sciences
Pennington Biomedical Research Center Clare M. Reynolds
Baton Rouge, Louisiana Liggins Institute and Gravida: National Centre
for Growth and Development
University of Auckland
Kathleen Mulligan
Auckland, New Zealand
College of Medicine
Touro University California Robert Ross
Vallejo, California School of Kinesiology and Health Studies
and
and
Division of Endocrinology and Metabolism
Department of Medicine Department of Medicine
University of California, San Francisco Queen’s University
San Francisco, California Kingston, Ontario, Canada
xviii Contributors
Jordi Salas-Salvadó Janne Schurmann Tolstrup

Hospital Universitari de Sant Joan de Reus National Institute of Public Health
Universitat Rovira i Virgili University of Southern Denmark
Reus, Spain Copenhagen, Denmark
Jean-Marc Schwarz
College of Medicine John F. Trepanowski
Touro University California Department of Kinesiology and Nutrition
Vallejo, California University of Illinois, Chicago
Chicago, Illinois
and
Krista A. Varady
Department of Medicine Department of Kinesiology and Nutrition
University of California, San Francisco University of Illinois, Chicago
San Francisco, California Chicago, Illinois
Ian W. Seetho Mark H. Vickers
Institute of Ageing and Chronic Disease Liggins Institute and Gravida: National Centre
University of Liverpool for Growth and Development
and University of Auckland
Aintree University Hospital NHS Foundation Auckland, New Zealand
Trust
Liverpool, United kingdom Maelán Fontes Villalba
Center for Primary Health Care Research
Patty W. Siri-Tarino Lund University
Children’s Hospital of Oakland Research Lund, Sweden
Institute
Oakland, California Margriet S. Westerterp-Plantenga
Department of Human Biology
Kimber L. Stanhope School of Nutrition and Translational Research
Department of Molecular Biosciences in Metabolism
School of Veterinary Medicine Maastricht University
and Maastricht, the Netherlands
University of California, Davis John P.H. Wilding
Davis, California Institute of Ageing and Chronic Disease
University of Liverpool
André Tchernof and
School of Nutrition Aintree University Hospital NHS Foundation
Laval University Trust
and Liverpool, United Kingdom
Quebec Heart and Lung Institute
Québec City, Québec, Canada Edward Yu
Alyssa Tindall and
Department of Nutritional Sciences Department of Epidemiology
The Pennsylvania State University Harvard University School of Public Health
University Park, Pennsylvania Boston, Massachusetts
Section I
Energy Balance, Adiposity, and
1 The Gut–Brain Axis
Regulation of Food Intake
Surya Panicker Rajeev, Ian W. Seetho, and John P.H. Wilding
CONTENTS
Introduction.........................................................................................................................................4
Central Nervous System Regulation of Food Intake...........................................................................4
Brainstem Regulation.....................................................................................................................4
Hypothalamus................................................................................................................................6
Arcuate Nucleus........................................................................................................................6
Paraventricular Nucleus.............................................................................................................6
Lateral Hypothalamus...............................................................................................................6
Dorsal Medial Nucleus and Ventromedial Nucleus of the Hypothalamus................................6
Neurotransmitters and Homeostatic Control of Food Intake.........................................................7
Neuropeptide Y and Agouti-Related Peptide............................................................................7
Proopiomelanocortin and Cocaine- and Amphetamine-Regulated Transcript..........................7
Gastrointestinal Signals That Regulate Food Intake...........................................................................8
Gut Hormones..............................................................................................................................10
Ghrelin ....................................................................................................................................10
Cholecystokinin.......................................................................................................................11
Pancreatic Polypeptide............................................................................................................11
Glucagon-Like Peptide-1 and Peptide YY..............................................................................12
Glucagon-Like Peptide-1........................................................................................................12
Peptide YY..............................................................................................................................13
Oxyntomodulin........................................................................................................................14
GIP��14
Amylin ....................................................................................................................................15
Mechanical Mechanisms..............................................................................................................15
The Ileal-Brake Reflex............................................................................................................15
The Role of Mechanoreception in Appetite Control...............................................................15
Effect of Bariatric Surgery on Gut Hormones.........................................................................15
Diet and the Gut–Brain Axis: Are All Macronutrients Equal?................................................16
Conclusion........................................................................................................................................17
References.........................................................................................................................................17
ABSTRACT
Regulation of food intake is a fundamental biological homeostatic process and is regulated at multiple
levels. The process begins with sight, smell, and taste of food, the act of ingestion evoking either plea-
surable or unpleasant sensations via these routes, which can themselves enhance, decrease, or even stop
intake. Once food enters the gastrointestinal tract, it evokes a cascade of responses to its mechanical
and chemical properties that have the function of coordinating orderly digestion, ensuring an appropri-
ate metabolic fate for nutrients, and activating the process of satiation that ultimately leads to the end
of the meal. This process of satiation involves signaling to the brain via neural, humoral, and hormonal
signals from the gut. Brain signals are primarily detected in the brainstem and hypothalamus, which
3
4 Nutrition and Cardiometabolic Health
contain networks of neurons that are able to respond both to these messages and to important long-term
signals of energy stores, most importantly the fat-derived hormone, leptin. These biological processes
are also sensitive to and modulated by higher brain centers that are sensitive to hedonic signals, social
context, and mood. Ultimately body weight is regulated by the net balance between intake and energy
expenditure, the latter also being modifiable by many of the same neurotransmitter signals that influence
food intake. The system seems biased toward weight gain, as a negative energy balance evokes a strong
corrective response with a drive to increase intake and reduce expenditure; in contrast, the response
to excess is weak and may be more easily overridden by hedonic and other higher centers. This may
explain the difficulty many people have in maintaining a healthy weight.
INTRODUCTION
The mechanism of energy homeostasis is complex and influenced by various factors including nutri-
ent availability and loss, hormone levels, genetic factors, and environmental stimuli. The system is
also set in such a way to be more responsive to energy loss than gain [1], which poses a challenge
in combating the obesity pandemic. The fundamental control of intake of energy/food starts with
the sight, smell, and taste of food, the process of digestion in the gut, and finishes in the brain with
physiological interactions at multiple levels.
The “gut–brain axis” describes the channels of communication between the gastrointestinal tract
and the appetite control areas of the central nervous system (CNS). The gut responds to nutrients and
mechanical stretch by generation of neural and hormonal signals to the brain with its homeostatic
and hedonic regions. Apart from the gut and brain, adipose tissue and its hormones, notably leptin,
play a major role in the regulation of appetite control and energy homeostasis.
The brain, through its hypothalamic nuclei as well as brainstem connections, plays an important part
in regulating energy homeostatic mechanisms and hence is a potential target for drug therapy, although
“off target” effects may limit this approach. The hypothalamus has orexigenic and anorexigenic neurons
and can sense nutrient changes and alterations in the hormonal milieu including gut hormones. There
are homeostatic and non-homeostatic systems involved in appetite control. While the former operate
through the hypothalamus, brainstem, and gut, the non-homeostatic (environmental and hedonic) mech-
anisms influence food intake via the corticolimbic system.
The gastrointestinal (GI) system secretes various endocrine hormones and apart from exerting effects
on several aspects of GI function, the gut sends neural and humoral signals to various brain regions,
which are initially processed in the hypothalamus and brainstem. The gut–brain axis is the two-way
interaction between the gut (through neural, nutrient, and hormonal factors) and the CNS (mainly in the
hypothalamus and brainstem) and plays an important role in the maintenance of energy homeostasis by
the human body. Short-term signals including gut hormones and neural pathways (vagal and spinal vis-
ceral afferents) moderate meal intake and satiation. Long-term humoral signals involved in the regulation
of energy homeostasis include leptin, the adipose tissue hormone, and insulin, the pancreatic hormone.
CENTRAL NERVOUS SYSTEM REGULATION OF FOOD INTAKE

In this part of the chapter, we review the literature describing the role of the CNS in the control of
food intake and show how this integrates peripheral signals from the gut and elsewhere to regulate
energy homeostasis (Figure 1.1).
Brainstem Regulation
The CNS mediates energy balance in the body and is a key regulator of energy homeostasis. The
brain receives signals from the gastrointestinal tract via both the nervous system and the circulation.
Vagal afferents from the gut converge at the brainstem dorsal vagal complex (DVC), composed of
the dorsal motor nucleus of the vagus, the area postrema, and nucleus of the tractus solitarius (NTS).
Regulation of Food Intake 5
PVN
DMH PFA
VMH LHA
Third ventricle
ARC (NPY/AgRP and

POMC/CART) Brain stem
(NTS, AP, DVC)
Median eminence
Adipose tissue Leptin
Vagus
Insulin
Pancreas Pancreatic
Polypeptide
Stomach Ghrelin
CCK
GLP-1
Intestine PYY
OXM
FIGURE 1.1 Links between the peripheral signals and central regulation of feeding. Numerous neural path-
ways transmit sensory information from the upper gastrointestinal viscera. Inputs are relayed to the NTS, AP,
and DVC; signals from mechanical and chemical stimuli from the stomach and intestine initially via vagal
afferents. Signals are relayed to the hypothalamus and appetite-regulating areas of the brain. ARC, arcuate
nucleus; AgRP, agouti-related peptide; AP, area postrema; CART, cocaine- and amphetamine-regulated tran-
script; CCK, cholecystokinin; DMH, dorsomedial nucleus of hypothalamus; DVC, dorsal vagal complex;
GLP-1, glucagon-like peptide 1; LHA, lateral hypothalamic area; NPY, neuropeptide Y; NTS, nucleus of trac-
tus solitarius; OXM, oxyntomodulin; POMC, proopiomelanocortin; PP, pancreatic polypeptide; PVN, paraven-
tricular nucleus; PYY, peptide YY; PFA, perifornical area; VMH, ventromedial hypothalamus.
The NTS is in close proximity to the area postrema with an incomplete blood–brain barrier and also
responds to peripheral signals in the circulation as well as vagal afferents from the gastrointestinal
tract [2]. The DVC subsequently projects to the hypothalamus and higher brain centers [3]. Leptin,
insulin, and glucose-sensing receptors are expressed in the brainstem [3,4].
The NTS in the medulla receives afferent gustatory signals via vagal nerve stimulation (e.g.,
from mechanoreceptors detecting gastric distension and chemoreceptors detecting changes in
nutrient composition and pH). The vagus also facilitates transmission of gut hormone signals
such as cholecystokinin (CCK), ghrelin, pancreatic polypeptide (PP), and glucagon-like peptide-1
(GLP-1) that are regulated by the presence of food in the gastrointestinal tract [3,5]. The NTS and
parabrachial nucleus in the brainstem innervate the hypothalamic paraventricular nuclei, arcuate
nuclei, and dorsomedial nucleus of hypothalamus (DMH), as well as the lateral hypothalamic area
(LHA), central nucleus of the amygdala, and nucleus of the stria terminalis. The visceral sensory
cortex integrates taste sensation and communicates with the thalamus, which has projections from
the NTS to mediate perceptions of fullness and satiety.
Hypothalamus
Arcuate Nucleus
Within the hypothalamus, the arcuate nucleus (ARC) at the base of the median eminence in the
floor of the third ventricle integrates neural and hormonal signals regulating peripheral satiety and
adiposity through neuropeptide orexigenic and anorexigenic transmission to other brain regions.
These include orexigenic neuropeptides such as neuropeptide Y (NPY) and Agouti-related peptide
(AgRP) and anorexigenic neuropeptides such as proopiomelanocortin (POMC) and cocaine- and
amphetamine-regulated transcript (CART). Peripheral signals influence the activity of these neuro-
nal populations to change feeding behavior and energy homeostasis [6].
The ARC is composed of neuronal cell bodies that express receptors for peripheral signals such as
gut hormones and adipokines and is accessible to circulating peripheral factors across the incomplete
blood–brain barrier and by carrier-mediated transport. ARC neuronal populations are linked with
second-order neurons in other hypothalamic nuclei that include the paraventricular nucleus (PVN),
LHA, DMH, and ventromedial hypothalamus (VMH). From these nuclei, the second-order neurons
project onto the caudal brainstem, cortex, and limbic system.
Paraventricular Nucleus
NPY/AgRP and POMC/CART neurons in the ARC send projections to the PVN of the hypothala-
mus. The PVN is adjacent to the superior part of the third ventricle in the anterior hypothalamus
and the neurons express anorexigenic peptides corticotrophin-releasing hormone (CRH) and thy-
rotropin-releasing hormone (TRH). The PVN integrates the thyroid and hypothalamic–pituitary–
adrenal axes with nutritional signals, thus allowing for responsiveness to alterations in metabolic
rate and sympathetic activity [7,8]. NPY/AgRP downregulates CRH and TRH while α-melanocyte-
stimulating hormone (α-MSH) increases CRH and TRH expression.
The PVN also contains synaptic terminals for NPY, α-MSH, serotonin (5-HT), noradrenaline, and
opioid peptides and appetite-regulating signals such as ghrelin, orexin A, CCK, and leptin, which can alter
food intake and body weight [9]. The PVN may have an inhibitory role in food intake as hyperphagia is
produced by central administration of NPY into the PVN and by destruction of the PVN [10]. The PVN
has projections to the midbrain, prelocus coeruleus in dorsal pons, and NTS in the ventral medulla [3].
Lateral Hypothalamus
The LHA comprises populations of nuclei that receive projections from the ARC and express the orexi-
genic neuropeptides melanin-concentrating hormone (MCH) and orexin [3]. NPY neurons synapse with
orexin and MCH nuclei in the LHA. MCH levels rise during fasting and stimulate appetite [11]. Excess
MCH expression in transgenic mice leads to obesity [12], while mice with MCH deficiency are lean [13].
Orexins A and B stimulate appetite and are produced by neurons in the LHA that project to the olfactory
bulb, cerebral cortex, thalamus, hypothalamus, brainstem, locus coeruleus, tuberomammillary nucleus,
and raphe nucleus [2]. Glucose-sensing neurons have been found in the LHA, ARC, and ventromedial
nucleus of hypothalamus that respond to fluctuations in local extracellular glucose concentration [3,4].
Dorsal Medial Nucleus and Ventromedial Nucleus of the Hypothalamus
The DMH is dorsal to the VMH and receives neuronal NPY/AgRP projections from the ARC and
projects α-MSH to the PVN [3]. α-MSH activates catabolic pathways to reduce food intake and
enhance energy expenditure.
LHA and perifornical area

PVN
MC4 receptor
Increase NPY Y5 CART Decrease
food intake receptors receptor food intake
+ – +
Arcuate POMC (α-MSH)

NPY AGRP nucleus CART
+ Neurotensin
+
+ –
– –
Ghrelin PYY3–36 GLP–1 Leptin
GI tract (gut hormones) Adipose tissue
FIGURE 1.2 Schematic of the hypothalamic nuclei and interactions with peripheral signals. LHA, lateral
hypothalamic area; PVN, paraventricular nucleus; NPY, neuropeptide Y; AGRP, agouti-related protein; POMC,
pro-opiomelanocortin; CART, cocaine and amphetamine-related transcript; α-MSH, α-melanocyte-stimulating
hormone; MC4, melanocortin 4.
The VMH has connections with the PVN, DMH, and the LHA. In the VMH, brain-derived neu-
rotrophic factor is expressed and acts to suppress food intake through MC4 receptor activation.
Hyperphagia and obesity have been found in mice with selective loss of brain-derived neurotrophic
factor pathways in the VMH and DMH [3] (Figure 1.2).
Neurotransmitters and Homeostatic Control of Food Intake

Neuropeptide Y and Agouti-Related Peptide
The ARC contains NPY and AgRP neuronal populations that express receptors for circulating satiety
signals. Neuronal activation leads to positive energy balance, lower energy expenditure, and increased
food intake. NPY is a neuropeptide that has orexigenic effects mediated by G-protein-coupled recep-
tors (hypothalamic Y1 and Y5 receptors) and levels are associated with nutritional status, with levels
being increased during fasting and reduced following food intake [14]. Studies have shown that the
administration of NPY in the CNS of rats stimulates food intake and increases body weight [15].
The AgRP is expressed in the ARC and is secreted with NPY. AgRP is an antagonist of melanocortin
receptors in the melanocortin system (MC3 and MC4 receptors) and stimulates appetite. Increased
expression occurs in periods of fasting and acts to increase food intake [16].
Proopiomelanocortin and Cocaine- and Amphetamine-Regulated Transcript

The POMC and CART neuronal populations respond to satiety and their activation promotes negative
energy balance, increased energy expenditure, and decreased food consumption. POMC is a precursor
polypeptide of melanocortins such as the α-MSH that act at MC3 and MC4 receptors to control appetite.
Nutritional status regulates mRNA POMC expression and this is decreased during fasting periods and
conversely increased with feeding. Administration of agonists for MC3 and MC4 receptors reduces
food intake and of antagonists at the receptors produces hyperphagia [10]. α-MSH acts as the agonist at
the hypothalamic MC3 and MC4 receptors to suppress appetite [3]. In murine models of POMC defi-
ciency, obesity and decreased metabolic rate occur and these effects are reversed by administration of
melanocortins that suppress food intake. In humans, perturbations such as congenital POMC deficiency
and MC3 and MC4 receptor mutations have been associated with obesity [17].
CART has anorexigenic properties and regulates energy homeostasis as neuronal expression
responds to nutritional status. It modulates the actions of NPY and leptin. Administration of CART
inhibits NPY-induced feeding while anti-CART antibody infusion promotes food intake [18].
However, the effects of CART may depend on signaling location as polymorphisms and altered
levels of CART have been associated with human obesity [19].
Non-Homeostatic CNS Pathways

The orbitofrontal cortex and corticolimbic pathways are responsible for processing somatosensory
stimuli and reward-associated feeding behavior. The very act of eating, coupled with environmental
cues, cognitive and emotional state may have an impact on food intake [20].
The endocannabinoid and opioid systems have receptors within the CNS and have a role in
reward-associated feeding [21]. Endocannabinoids may be linked with feeding behavior, possibly
mediating cravings and desire for food. Endocannabinoids have orexigenic properties, and in
rodents, levels in the hypothalamus increase with fasting and decrease with feeding [15].
The cannabinoid system consists of two major receptor subtypes (CB1 and CB2). Central can-
nabinoid receptors (CB1) are located in regions involved in appetite regulation including the ARC
and LHA and are integral to the stimulatory effects of cannabinoids and endocannabinoids on food
intake. CB1 receptor–deficient mice have reduced appetite and are resistant to diet-induced obe-
sity. In humans, the RIO-Europe trial was a randomized double-blind trial that compared treat-
ment of obese subjects with rimonabant and a calorie-restricted diet versus placebo over 2 years.
Rimonabant was associated with a significant reduction in weight, waist circumference, and the
presence of metabolic syndrome compared with placebo [22]. Rimonabant was approved for the
treatment of obesity in Europe in 2008 but subsequently withdrawn due to concerns about increased
risk of anxiety, depression, and suicide with its use.
Dopamine has central signaling pathways that are mediated by dopamine D1, D2, D3, and D5
receptors. It may have complex effects on feeding and may be associated with reward-associated
behavior [2,23]. It is noteworthy that the D1/D5 receptor antagonist, ecopipam, was withdrawn from
clinical trials for obesity due to depression [24], although the combination of the μ opioid antago-
nist drug naltrexone with the dopamine/noradrenaline reuptake inhibitor bupropion is approved for
obesity treatment [25].
GASTROINTESTINAL SIGNALS THAT REGULATE FOOD INTAKE

The GI tract is the largest endocrine gland in the human body and secretes gut hormones that have
a significant role in the maintenance of energy equilibrium. The main gut hormones that act on the
central hypothalamic and brainstem pathways are ghrelin, CCK, PP, peptide YY (PYY), GLP-1, and
oxyntomodulin (OXM).
A criterion for the physiological endocrine action of gut hormones on energy intake was pro-
posed by Geary [26]. He suggested that
1. Hormone secretion should be associated with changes in eating—secretory effect.

2. Receptors should be expressed at the site of action of the relevant hormone—receptor-
mediated effect.
3. Effects on appetite should be reproduced by the parenteral administration of a hormone,
similar to its endogenous effects—physiological dose–related effect.
4. Removal of the hormone or receptor should prevent the effect on appetite, and replacement
of the hormone should reinstitute the effect—removal and replacement effect.
5. Selective, potent antagonism of the hormone should prevent the effect of endogenous hor-
mone as well as hormone treatment—antagonistic effect.
However, not all the gut hormones identified fit all these criteria.
Gut hormones were originally thought to have significant roles in regulating endocrine and
paracrine aspects of gut function, such as motility, pancreatic exocrine secretion, and gall bladder
contraction, as well as actions as incretins take on pancreatic endocrine activity. However, more
recent work has demonstrated their fundamental role in the maintenance of energy equilibrium as
peripheral signals of energy status (depletion or surge) through communication to the hypothalamus
and brainstem. Local effects of gut hormones on motility such as delay in gastric emptying may also
contribute to changes in energy intake. Mechanical effects include activation of stretch receptors
through gastric distension contributing to decrease in food intake. Thus, neuroendocrine signaling
through the gut–brain axis has a vital role in controlling food intake.
Enteroendocrine cells (EECs) are distributed throughout the gastrointestinal tract and are respon-
sible for the secretion of these peptide hormones. There are open and closed types of EECs that are
located throughout the GI tract. Open EECs are cone-shaped cells with microvilli at their open end
while the closed end lies abutting the basal lamina. The microvilli at the open end sense the macronu-
trients in food via G-protein-coupled receptors (by stimulating the chemosensors on these receptors).
This leads to release of gut hormones that act through endocrine, paracrine, and neural mechanisms
to regulate food intake and energy homeostasis. Closed EECs do not have direct contact with luminal
contents but utilize the neural mechanisms to exert their role. Sensory information from the gastro-
intestinal tract and abdominal viscera as well as taste information from the oral cavity are initially
integrated by the NTS.
Due to their role in energy homeostasis, gut hormones (Table 1.1) are a major therapeutic target
for the development of antiobesity drugs.
TABLE 1.1
Effects of Gut Peptides on Appetite and Other Physiological Effects
Effect on
Gut Hormone Site of Synthesis Food Intake Other Physiological Effects
Ghrelin Gastric fundus + 1. Stimulates GH release from pituitary
2. Increases gastric motility
3. Positive inotropic effect on heart
4. Reduction of glucose-stimulated insulin secretion
CCK Small intestine − 1. Contraction of gall bladder
2. Inhibition of exocrine pancreatic secretions
3. Delayed gastric emptying
PP Endocrine pancreas − 1. Relaxation of gall bladder
2. Inhibition of exocrine pancreatic secretions
3. Delayed gastric emptying
GLP-1 Ileum and proximal − 1. Glucose-induced insulin secretion/incretin effect
colon 2. Inhibits glucagon production
3. Delays gastric emptying
4. Trophic effects on pancreatic beta cell mass
5. Inhibition of gastric acid secretion
6. Cardiovascular effects—increases heart rate
PYY Distal small and − 1. Inhibits gall bladder contraction
large intestine 2. Inhibits exocrine pancreatic secretion
3. Delays gastric emptying
4. Inhibits gastric acid secretion
Oxyntomodulin Distal small and − 1. Delays gastric emptying
large intestine 2. Inhibits gastric acid secretion
GIP K cells of Not known 1. Stimulates insulin secretion
duodenum and 2. Increase beta cell proliferation
jejunum 3. Inhibits beta cell apoptosis
4. Increase lipogenesis
5. Increase bone formation
Gut Hormones
Ghrelin
Ghrelin is the only identified orexigenic hormone. Ghrelin, originally discovered as an endogenous
ligand for the growth hormone secretagogue receptor (GHS-R) [27], is a 28-amino-acid peptide
hormone secreted principally by the X/A like cells in the oxyntic glands of the gastric fundus [28].
Ghrelin is cleaved from preproghrelin by prohormone convertase. Though ghrelin is synthesized in
many tissues, the stomach is the largest source of circulating ghrelin. The presence of an acyl side
chain attached to the serine amino acid at position three of ghrelin is necessary for its orexigenic
effects [27] through binding to GHS-R as well as enhancing its ability to cross the blood–brain
barrier. Ghrelin O-acyltransferase (GOAT) is the enzyme responsible for the acylation of ghrelin.
Des-acylated ghrelin (which lacks the serine-3 acylation) is not orexigenic and reduces food intake
in rodent models with central or peripheral administration [29]. Though des-acyl ghrelin was origi-
nally thought to be the inactive form of ghrelin, recent data has demonstrated its physiological
roles in the regulation of adiposity as well as glucose homeostasis through agonistic action at the
growth hormone secretagogue receptor [30]. Obestatin was identified in 2005 from the ghrelin pre-
cursor [31]. Though initially thought to have opposing effects to ghrelin, the anorexigenic effects of
obestatin were subsequently not proven from clinical studies.
Peripheral administration of acylated ghrelin in rodent models increases food intake [32], and
chronic intracerebroventricular administration induces weight gain [33]. This was replicated in
humans and intravenous administration of ghrelin increased food intake and appetite with no effect
on gastric emptying, suggesting a central role as the possible explanation for its orexigenic effects [34].
This is through its effects on the hypothalamic ARC [33] via the bloodstream, and NTS via the
vagal route. Vagotomy in rats as well as humans abolishes the effects of ghrelin [35–37]. GHS-R-
knockout mice as well as ghrelin-deficient mice are resistant to diet-induced obesity further confirm-
ing the orexigenic effects of ghrelin. Ghrelin-deficient mice also selectively metabolize fat as a fuel,
which provides evidence that ghrelin has other metabolic effects [38].
Circulating ghrelin levels are increased with fasting and fall in the postprandial period [39]. The
macronutrient composition of the meal is important in ghrelin-induced postprandial suppression,
with carbohydrate-rich meals causing more suppression of ghrelin than protein or fat [40].
In obesity, fasting ghrelin levels are low and the postprandial suppression of ghrelin is attenuated
[41]. Moreover, peripheral administration of ghrelin stimulates appetite in obesity [42] and hence,
unlike leptin resistance associated with obesity, the obese are not ghrelin resistant. Interestingly,
patients with Prader–Willi syndrome (a genetic obesity syndrome associated with hyperphagia,
short stature, mental retardation, and pituitary hormone deficiencies) have higher fasting as well as
postprandial ghrelin levels [43–45]. However, the search for ghrelin antagonists for obesity treat-
ment has so far been unsuccessful.
Fasting ghrelin levels are high in anorexia nervosa [46] and in patients who have lost weight
through diet or gastric bypass surgery [47]. Though ghrelin antagonists may not be a successful
therapeutic option in obesity, agonists might be of use in managing select patient groups with
anorexia. Ghrelin agonists have also been shown to be of potential benefit in treating malnutrition
associated with congestive cardiac failure, chronic obstructive pulmonary disease, renal failure
[48], and cancer [49]. However, a concern regarding the use of ghrelin in patients with malignancy
as an appetite stimulant is its potential to stimulate growth hormone and thus other growth factors.
Apart from its effect on energy homeostasis as a short-term signal, other physiological roles
of ghrelin include stimulation of growth hormone release, increase in gastric motility, possible
role in glucose homeostasis, and its positive inotropic effect on the heart [50]. The satiety effect
of glucagon is also thought to be ghrelin-mediated [51]. Ghrelin along with insulin and leptin
acts as a mechanism to combat energy deficit via its orexigenic signal as well as metabolic switch
from carbohydrate to fat metabolism, thus facilitating the storage of carbohydrate. However, these
metabolic effects of ghrelin are more pronounced during stages of energy deficit (i.e., fasting
and starvation) than at the other end of the spectrum (fed and obese states) [52]. The effects
of ghrelin on glucose homeostasis are more pronounced than on body weight [53]. Exogenous
ghrelin administration resulted in reduction of glucose-stimulated insulin secretion [54]. Hence,
ghrelin antagonism could be another untapped therapeutic approach for the management of
type 2 diabetes.
In conclusion, ghrelin plays an important role in the regulation of energy homeostasis through
its gut–brain interaction, acting on homeostatic as well as non-homeostatic centers. Manipulation of
the ghrelin system could be a potential therapeutic approach for the management of obesity, other
eating disorders, and type 2 diabetes but has not been successful so far. Ghrelin agonism, antago-
nism, anti-ghrelin vaccines, des-acyl ghrelin analogues, and GOAT enzyme inhibition are being
investigated and could be potential pharmacotherapies in the future.
Cholecystokinin
CCK was the first gut hormone demonstrated to influence appetite control [55]. Secreted by the
L cells of the duodenum and jejunum after meals, it is involved in contraction of the gall bladder,
inhibition of exocrine pancreatic secretion, delays in gastric emptying, and increases in motility of
the intestine, apart from its currently discussed role as a satiety signal. CCK exerts its gastrointesti-
nal and other physiological effects through CCK1 and CCK2 receptors (previously named as CCKA
and CCKB receptors), which are located in the gastrointestinal tract and select brain centers [56].
CCK1 receptors are mainly located in the pancreas, pylorus, vagus, NTS, and hypothalamus and
contribute to post meal satiety signaling. This is thought to be vagally mediated due to activation of
vagal CCK1 receptors [57]. CCK might also affect mechanoreception, augmenting the local stretch
signals and conveying them to the hypothalamus. According to Geary’s criteria, CCK was thought
to be the gut hormone with the best-established physiological endocrine action [26].
CCK has a short half-life (1–2 minutes) and its levels increase 10–30 minutes after food intake,
returning to basal levels after 3–5 hours [58]. With respect to the macronutrient composition of the meal,
fat and protein are the major stimulants of CCK release, while carbohydrates are weak stimuli [58].
CCK levels in obesity have been shown to differ between studies with some studies showing
increased levels while others had demonstrated opposite results [59,60]. There are data from animal
studies that CCK might work synergistically with leptin [61] and insulin [62] in the maintenance of
energy homeostasis.
CCK infusion (the C-terminal octapeptide of CCK) decreased food intake in lean individuals [63]
suggesting its possible role as an appetite suppressant. The OLETF (Otsuka Long-Evans Tokushima
Fatty rat) lacking the CCK1 receptor was demonstrated to be obese and diabetic with defective con-
trol of meal sizes, and CCK receptor deficiency was thought to be the reason for obesity in these
rodents (along with NPY overexpression) [64]. This suggested that CCK might have a physiological
role in meal intake. However, chronic administration of CCK (2 weeks of intraperitoneal infusion)
in rodent models was not associated with any changes in body weight or food consumption [65], and
trials of CCK agonists in humans have not proceeded beyond phase 2 due to adverse effects. Thus,
the therapeutic potential of CCK as an antiobesity agent may be limited.
Pancreatic Polypeptide
PP is a 36-amino-acid peptide secreted mainly from the endocrine pancreas (by the PP cells of the
islets of Langerhans). It is a member of the PP family, all of which bind to the Y family of receptors.
NPY and PYY are members of the same family. PP is released postprandially and has been demon-
strated to reduce appetite in rodents [66] as well as humans [67]. This is receptor mediated, through
its effect on the Y4 receptors (to which it has the highest affinity) in the brainstem and hypothalamus
[68] as well as vagally [69]. The Y receptors are G-protein-coupled receptors of which there are five
subtypes, Y1–Y5, that are all coupled to adenylate cyclase. Y4 receptors are expressed in abundance
in area postrema in the DVC, and PP exerts its physiological effects by binding to these receptors.
PP has a biphasic mode of release and its levels remain high up to 6 hours postprandially. PP also
exhibits a diurnal rhythm with lowest levels at 02:00 and peaks at 21:00 hours [70]. The release of
PP is stimulated by CCK, hypoglycemia, and exercise and inhibited by somatostatin.
The effect of PP on satiety is dependent on the route of administration with peripheral PP having
anorexic effects while central administration causes orexigenic effects [71]. Transgenic mice overex-
pressing PP were lean with reduction in fat mass, reduction in food intake, delayed gastric emptying, and
reduction in leptin levels [72]. Intraperitoneal administration of PP reduced adiposity in leptin-deficient
ob/ob mice with a favorable metabolic profile for insulin resistance and hyperlipidemia [73].
PP also delays gastric emptying [74] that is thought to contribute to its anorectic effect. Other
physiological effects of PP include inhibition of exocrine pancreatic secretions and inhibition of gall
bladder contraction [75]. The concentration of PP post meal is markedly reduced in obese individu-
als [76] and is increased in patients with anorexia nervosa [77]. Prader–Willi syndrome patients have
a blunted response to PP [78], and exogenous administration (PP infusion) reduces food intake in
this condition restoring PP levels to normal [79].
PP is an anorexigenic peptide and transmits information related to food intake through vagus
to the DVC as well as hypothalamic nuclei, thus playing a role in modulating energy homeostasis
through the gut–brain axis. PP analogues have been investigated as antiobesity agents, but their
therapeutic potential is still uncertain.
Glucagon-Like Peptide-1 and Peptide YY

The enteroendocrine L cells form an important part of the gut–brain axis. They express the preproglu-
cagon gene, which undergoes tissue-specific posttranslational processing, that is, it generates different
hormonal products when processed in different organs, such as the pancreas or intestine. The prohor-
mone convertase 1 is responsible for the production of GLP-1, glucagon-like peptide-2 (GLP-2), and
OXM from proglucagon in the intestine [80]. L cells in the intestine co-secrete different gut peptides
depending on their location. The upper small intestinal cells secrete gastric inhibitory polypeptide
(GIP). The lower small intestinal cells co-secrete GLP-1 and PYY, and these are important for energy
homeostasis. Both GLP-1 and PYY are secreted in a biphasic manner with a first phase of release on
arrival of food in the proximal small intestine, which is neurally mediated, and a second phase when
food reaches the distal intestine through a nutrient receptor–mediated process [81].
Glucagon-Like Peptide-1
The peptide hormone GLP-1 is derived from the posttranslational processing of preproglucagon,
which is also the source of glucagon, GLP-2, and OXM. GLP-1 is produced in the L cells of the
distal small intestine (jejunum) and proximal colon and is secreted following a meal. GLP-1 secre-
tion is proportional to caloric intake, especially the glucose content of the meal. Fat as well as pro-
tein also promotes GLP-1 responses [81]. GLP-1 is secreted 5–30 minutes following meal intake.
It exists in two biologically active forms—GLP-1 (7-37) and GLP-1 (7-36) amide; the latter is the
most abundant form in human circulation.
GLP-1 exerts its effects through the GLP-1 receptor, which is a G-protein-coupled receptor. GLP-1
receptors are widely distributed in the body, including pancreas, gastrointestinal tract, heart, lung, kid-
ney, brain, as well as vascular endothelium and arteriolar smooth muscles. In the brain, these are mostly
expressed in the supraoptic, paraventricular, and arcuate nuclei of the hypothalamus [82], brainstem,
substantia nigra, and striatum. The demonstration of GLP-1 receptors in the brain indicates that the
physiological effects of the GLP-1 system are at least partly, centrally mediated. GLP-1 is expressed in
neurons in the NTS that project to the POMC and amphetamine-regulated transcript (CART) neurons in
the arcuate nuclei of the hypothalamus, as well as to the paraventricular and dorsomedial nuclei.
The binding of GLP-1 to its receptors in the pancreas leads to a cascade of events.
There is an increase in cyclic AMP levels causing an increase in intracellular calcium levels
leading to exocytosis of insulin-containing vesicles [83]. Similar to CCK, ghrelin, and PYY,
the vagus nerve plays a significant role in the effects of GLP-1 on energy intake. Vagotomy
in rodents has been demonstrated to abolish the effects of GLP-1 on food intake and its
hypothalamic effects [84] demonstrating the significance of this gut–brain axis, that is, an intact
vagal–brainstem–hypothalamic connection.
GLP-1 is an incretin hormone and its effect to stimulate insulin secretion in response to oral
glucose is described as the “incretin effect.” GLP-(7-36) and GIP are the incretin hormones and the
circulating levels of both of these have been shown to rise following intake of carbohydrate or mixed
meals. Incretin deficiency has been suggested as part of the pathophysiological process leading to
the development of type 2 diabetes. Consequently, incretin-based therapies are now widely used in
the management of type 2 diabetes.
Apart from its insulin stimulatory and glucagon inhibitory effect, GLP-1 also causes satiation [85],
delays gastric emptying [86], and promotes weight loss in humans by decreasing energy intake [87];
all of these properties make it suitable as a treatment for obesity. It acts synergistically with PYY in
the ileal brake phenomenon, which aids nutrient digestion. GLP-1 also has trophic effects on beta
cells of the pancreas.
GLP-1 levels are low in obesity and increase after weight loss. GLP-1 secretion is low in anorexia
nervosa [88].
Endogenous GLP-1, the most abundant incretin, has, however, a short half-life of 2–3 minutes and
is susceptible to degradation by endopeptidases, notably dipeptidyl peptidase 4 (DPP-IV). Hence,
pharmaceutical development has focused on the synthesis of GLP-1 analogues resistant to the action
of DPP-IV, thus increasing its half-life. Available GLP-1 analogues include exenatide, lixisenatide
(short-acting), liraglutide (intermediate-acting) and exenatide QR, dulaglutide (in development),
and semaglutide (long-acting GLP-1 analogues, which are administered once weekly).
The therapeutic benefits of GLP-1 analogues include a glucose-lowering effect without increasing
the risk of hypoglycemia (since its insulin secretory effect is only seen above blood glucose levels of
3.5–4 mmol/L), weight reduction, moderate reductions in systolic and diastolic blood pressure [89],
and a favorable effect on the lipid profile (reduction in total cholesterol, low density lipoprotein-
cholesterol, triglycerides, free fatty acid, and increase in high density lipoprotein cholesterol) [90].
Exendin-4 (4-39) is a purified derivative from the saliva of Heloderma suspectum, the Gila monster
lizard, and was discovered in 1992. Exendin-4 shares only 53% homology with human GLP-1 but is a
potent analogue and is resistant to the action of DPP-IV. Subsequently, exenatide, a synthetic form of
exendin-4, became the first approved GLP-1 analogue for the treatment of type 2 diabetes in 2005. It has
a short half-life of 2.4 hours and is administered subcutaneously, twice daily. In addition to improving
glycemic control, exenatide treatment reduces body weight in patients with type 2 diabetes [91].
The GLP-1 system is the only successfully manipulated component of the gut–brain axis as
a therapy for the treatment of obesity. Liraglutide, which shares 97% structural homology with
endogenous GLP-1, is the first GLP-1 analogue to get the approval of regulatory authorities as
a weight loss drug. Liraglutide 3 mg along with increased physical activity and reduced caloric
intake resulted in significant weight loss in overweight and obese individuals without type 2
diabetes [92]. Liraglutide-induced weight loss was associated with improvement of overall car-
diometabolic profile, including waist circumference, blood pressure, lipids, and inflammatory
markers. Liraglutide was given FDA approval in December 2014 and EMEA approval in March
2015 and is currently marketed as a weight loss drug, Saxenda (liraglutide 3 mg). Low-dose
liraglutide (0.6–1.8 mg) is marketed as Victoza and has been used for the treatment of type 2
diabetes since 2009. The cardiovascular safety of liraglutide was demonstrated in the LEADER
(Liraglutide and Cardiovascular Outcomes in Type 2 Diabetes) trial, which randomized 9340
patients and followed up for 3.8 years [93].
Peptide YY
PYY is a 36-amino-acid peptide hormone co-secreted postprandially with GLP-1 from the entero-
endocrine L cells of the GI tract. The name PYY is derived from its tyrosine residues at the C and N
terminals. PYY exists in circulation in two forms, PYY1–36, which has affinity to all the Y receptors,
and PYY3–36, the N-terminally truncated form which is the major circulating and biologically active
form of PYY with the highest affinity to the Y2 receptors. Though PYY-expressing cells are distrib-
uted all along the intestine, they are most abundant in the distal part. Data from radioimmunoassay
have demonstrated that PYY levels are low in the duodenum and jejunum, and high in the ileum and
colon with the highest levels in the rectum [94].
PYY is another anorexigenic hormone with low levels in the fasting state and increasing in con-
centration postprandially. PYY levels rise after 30 minutes of food intake, plateau by 1–2 hours,
and remain high for up to 6 hours. This rise in PYY is greatest with protein-rich meals. The satiety-
enhancing effect of dietary protein is thought to be mediated by PYY. Dietary protein increased PYY
levels in mice, resulting in decreased food intake and weight gain. PYY-deleted mice were resistant
to this satiety effect of protein and developed obesity, which was in turn reversed by exogenous PYY
administration [95]. Peripheral administration of PYY3–36 was shown to reduce food intake as well
as weight gain in rodents [96], and these results have been replicated in humans [97]. PYY levels are
low in obesity and there is a blunted postprandial rise in PYY levels. The circulating levels of PYY
are increased in anorexia nervosa and this has been postulated to contribute to decreased food intake
and increased bone turnover in such patients [98]. In women with anorexia nervosa, an increase in
the PYY level is associated with diminished bone mineral density, especially at spine. Fasting PYY
levels are high in inflammatory bowel disease, steatorrhea, tropical sprue, and cardiac cachexia, and
high PYY levels could be a reason for the loss of appetite in such states.
The anorectic effects of PYY could be through the stimulation of the POMC neurons in the ARC
of the hypothalamus or due to inhibition of the NPY neurons [99], which is likely to be Y2 receptor
mediated. Vagotomy can dampen the anorectic action of PYY, thus demonstrating the role of vagus
nerve in this gut–brain communication [84]. Direct administration of PYY into the third ventricle
however has been shown to be orexigenic [100] likely due to the effects of PYY on the NPY neurons
in the hypothalamic arcuate nuclei.
PYY along with GLP-1 mediates the “ileal brake” explained as follows.
Oxyntomodulin
OXM is a 37-amino-acid peptide that derives its name from its inhibitory effect on the oxyntic
glands of stomach. It is secreted by the intestinal L cells along with GLP-1 and PYY by the post-
translational processing of preproglucagon. Apart from the intestinal L cells, OXM is also expressed
in the pancreas and the CNS. OXM exerts its physiological effects via the GLP-1 receptor though
its affinity for GLP-1 receptors is low compared to GLP-1 [101]. The anorectic effect of OXM was
blocked when it was coadministered in rats with exendin 9-39, a GLP-1 antagonist [102]. OXM
signaling through the gut–brain axis is likely to be mediated through the vagus nerve as vagotomy
abolished the effect of OXM on CCK-induced pancreatic secretion [103]. It is secreted postprandi-
ally with levels peaking in 30 minutes and remains elevated for several hours. Its secretion is stimu-
lated by dietary fat [104].
Central (intracerebroventricular) [102] and peripheral (intraperitoneal) [105] administration of
OXM in rats inhibited feeding. OXM also increases energy expenditure apart from its effects on
energy intake, thus affecting long-term energy homeostasis. The anorexic effects of OXM have been
replicated in human studies with OXM reducing both ad libitum energy intake at a buffet meal as
well as 12-hour cumulative energy intake in healthy volunteers [106]. It was also noted that fasting
ghrelin levels were suppressed by OXM, which might be responsible for its anorectic effects.
Similar to GLP-1, OXM is also inactivated by the DPP-IV enzyme and OXM analogues, which
are resistant to the action of this endopeptidase, are under investigation [107].
GIP
GIP is a 42-amino-acid peptide secreted by the K cells in the duodenum and jejunum. GIP is mainly
secreted after the ingestion of carbohydrates and fat [108] depending on the rate of absorption of
nutrients rather than their presence. Fat is the major stimulator of GIP release in humans. Like GLP-1,
GIP is an incretin hormone and is inactivated by the DPP-IV enzyme. GIP does not have a direct
effect on energy intake. GIP-receptor-knockout mice on a high-fat diet have lower respiratory quo-
tient (due to the use of fat as a metabolic fuel) and consequently are resistant to obesity and insulin
resistance [109]. GIP has anabolic and lipolytic effects on adipose tissue.
Amylin
Amylin is co-secreted with insulin from the beta cells of the pancreas in response to food ingestion. In
humans, amylin binds to AMY-receptor subtypes, which are calcitonin receptor complexes with receptor
activity-modifying proteins [110]. In rats, amylin reduced the meal size and meal duration, thus dem-
onstrating its anorectic effects [111]. Meal-induced amylin release activates area postrema neurons in
the brain [112]. It enhances the anorectic effects of CCK, and this is likely to be via modulation in area
postrema. Apart from reducing food intake, amylin delays gastric emptying, decreases gastric secretion,
and reduces postprandial glucagon secretion. As such, pramlintide, a synthetic amylin analogue, is
approved in the United States as an adjunct therapy in type 1 and type 2 diabetes [113,114].
Mechanical Mechanisms
The Ileal-Brake Reflex
The ileal-brake reflex is an inhibitory intestinal control mechanism whereby unabsorbed dietary con-
stituents in ileum and colon inhibit proximal GI motility, thus increasing the digestion and absorp-
tion of nutrients [115]. Both GLP-1 and PYY may contribute to the ileal brake, which can reduce
energy intake. Though fat was thought to be the most potent stimulator of the ileal brake, subsequent
studies illustrated that all three macronutrients (carbohydrates, proteins, and lipids) influenced the
ileal brake [116].
The Role of Mechanoreception in Appetite Control

Apart from communication through gut peptides, the hypothalamus also receives information from
mechanical stimuli due to stretching of the stomach. The gastrointestinal tract is abundantly inner-
vated with mechanoreceptors that can sense the mechanical stretch stimulus due to food intake and
communicate information to the higher centers in the brain through vagal and splanchnic connec-
tions. Animal studies have demonstrated that gastric volume is an important factor for mechanore-
ception, which in turn regulates food intake. Male rats implanted with gastric catheters and pyloric
cuffs were given specific volumes of saline or milk and decreased food intake was shown to be
secondary to the volume or rate of infusion rather than related to a specific nutrient [117]. Because
such techniques cannot be replicated in human studies, evidence for the contribution of mechanical
factors to appetite control is scanty. Nevertheless, human data have demonstrated that energy den-
sity and portion size of food independently contribute to energy intake [118]. Energy-dense foods
thus contribute to overconsumption, and reducing energy density of the diet is one possible dietary
strategy to deal with the public health problem of obesity. Gastric antral area and antral distension
are important in determining satiation as well as satiety [119] in humans.
Effect of Bariatric Surgery on Gut Hormones

Bariatric surgery is the most effective therapeutic intervention for obesity and results in significant,
sustained weight loss as well as metabolic improvement in the majority of patients. The mechanisms
of post-bariatric surgery weight reduction (reviewed in Chapter 7 of this textbook) are not completely
unraveled but are thought to be mediated by more than caloric restriction and malabsorption. Alterations
in circulating gut hormones may contribute; an increase in GLP-1, PYY, and OXM levels has been
observed, and changes in ghrelin and GIP hormones could also play a role in weight reduction.
Bariatric surgical procedures involving the intestine (Roux-en-Y gastric bypass [RYGB] and
biliopancreatic diversion [BPD]) and those that expedite nutrient delivery (sleeve gastrectomy)
resulted in an increase in GLP-1 and PYY levels, whereas no increase was demonstrated after
gastric banding [120]. Hence, rapid delivery of nutrients to the distal gut might be responsible for
this phenomenon. Sleeve gastrectomy increases gastric emptying that in turn could result in fast
passage of nutrients to the intestine, stimulating L cells to release these gut hormones. RYGB and
BPD that are the surgical procedures associated with maximum weight loss had greater elevations in
GLP-1 and PYY levels. Similar reductions in body weight via caloric restriction did not elicit these
hormonal changes, thus demonstrating that these are solely related to bariatric surgery. Elevation in
OXM levels was noticed after RYGB. However, changes in ghrelin levels were inconsistent from
different studies and may be due to differences in surgical approaches.
Hormones from Adipose Tissue

Adipose tissue is an important source of hormones that regulate hypothalamic energy homeo-
stasis. Though leptin is an adipose tissue hormone, it needs mentioning due to its significant
role in the maintenance of energy homeostasis as well as carbohydrate, lipid, and bone metabo-
lism. Leptin is predominantly expressed by white adipose tissue and acts as a circulating satiety
factor, promoting negative energy balance. Circulating leptin levels correspond to fat mass,
thereby reflecting the energy status of an individual. Leptin exerts its effects through the ARC
by stimulation of POMC and CART neurons and inhibition of the NPY and AgRP neurons. The
leptin-deficient ob/ob mouse model was found to be associated with hyperphagia and obesity,
which was reversed with leptin administration. Recombinant leptin is used as a therapeutic
strategy in congenital leptin deficiency (where it reduces body weight and fat mass) [121] and
in congenital as well as acquired lipodystrophy, which is associated with leptin deficiency
(where it improves insulin resistance and decreases triglyceride levels) [122]. However, obesity
and type 2 diabetes are associated with leptin resistance, thus limiting the therapeutic potential
of leptin in these conditions. It should be noted that although circulating leptin levels are asso-
ciated with adiposity, leptin resistance may occur as obese individuals have high leptin levels
(REF). In these individuals, the expected anorexigenic effects of leptin are diminished and may
be due to leptin receptor overstimulation and activation of negative feedback loops that block
leptin signaling contributing to leptin resistance [6].
Adiponectin is also secreted from white adipose tissue but in contrast to leptin, levels are
decreased in obesity and inversely related to adiposity and are increased in fasting. Adiponectin
receptors (AdipoR1 and R2) are expressed in the hypothalamus and induce AMP kinase phosphory-
lation and activity. Adiponectin promotes energy expenditure and fatty acid oxidation. Conversely,
adiponectin deficiency induces insulin resistance and hyperlipidemia. Central administration of
adiponectin increases expression of uncoupling protein 1 in brown adipose tissue, with increases
in brown adipose tissue thermogenesis, thus promoting energy expenditure. Conversely, adiponec-
tin administered peripherally resulted in increased food intake, reduced energy expenditure, and
weight gain.
Circulating resistin levels are higher in obesity and its receptors are expressed in the hypothala-
mus. Resistin acts centrally to decrease food intake, an effect associated with suppression of the
normal fasting-induced increase in NPY/AgRP expression and the normal fasting-induced decrease
in CART expression [123].
Proinflammatory cytokines found in adipose tissue, such as TNF-α and IL-6, also inhibit feeding
and induce thermogenesis by modulating expression of hypothalamic neurotransmitters; they may
play a role in the anorexia associated with febrile illness, but their role in day-to-day appetite regula-
tion is unproven.
Diet and the Gut–Brain Axis: Are All Macronutrients Equal?

The relative proportion of dietary macronutrients (protein, carbohydrate, and fat) as well as the
glycemic index has a significant role in appetite and energy balance mechanisms. A high-protein,
low-carbohydrate, low–glycemic index diet has been demonstrated from numerous clinical stud-
ies to be superior to a normal-protein, high-GI diet for the induction as well as maintenance of
weight loss [124,125]. A high protein intake also promotes a negative energy balance due to
the greater satiating effect of protein compared to other macronutrients. Dietary thermogenesis,
which is a component of total energy expenditure, is also more for protein compared to carbo-
hydrates and fat. The protein content of a meal produces a dose-dependent increase in GLP-1,
PYY, and glucagon levels, which might contribute to the satiety-stimulating effects of this mac-
ronutrient [126].
CONCLUSION
The gut–brain axis is a vital link between the peripheral and central pathways facilitating the com-
plex neuroendocrine regulation of energy homeostasis. Gut hormones have a crucial role in the
maintenance of energy homeostasis. Modulation of one component of the system (GLP-1) has
finally proven to be fruitful as an antiobesity therapy, and others, perhaps with multiple targets for
action, seem likely to follow. As the obesity epidemic continues to be one of the major public health
problems in all parts of the world, unraveling of this complex neuroendocrine network will help us
in understanding appetite control mechanisms and, hopefully, more successful therapeutic strategies
in the future.
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2 Overeating Behavior and
Mechanisms and Treatments
Ashley E. Mason and Frederick M. Hecht
CONTENTS
Abbreviations................................................................................................................................... 24
Introduction...................................................................................................................................... 25
Types of Overeating......................................................................................................................... 25
Mindless Overeating................................................................................................................... 25
Portion Size............................................................................................................................ 26
Proximity and Visibility......................................................................................................... 26
Ambient Factors..................................................................................................................... 26
Palatability.............................................................................................................................. 27
Categorization Cues and Health Halo Effects........................................................................ 27
Distracted Eating.................................................................................................................... 27
Social Influences.................................................................................................................... 28
Stress-Induced Overeating.......................................................................................................... 28
Neurobiological Mechanisms Linking Stress and Overeating............................................... 29
Studies of Stress-Induced Overeating in Humans.................................................................. 30
Stress-Induced Overeating and Cardiometabolic Health....................................................... 30
Compulsive Overeating............................................................................................................... 30
Neurobiological Mechanisms Underlying Compulsive Overeating....................................... 31
Compulsive Overeating and Cardiometabolic Health............................................................ 32
Behavioral Interventions for Overeating.......................................................................................... 32
Environmental Approaches......................................................................................................... 32
Institutional-Level Environmental Interventions.................................................................... 33
Individual-Level Environmental Interventions....................................................................... 34
Environmental Interventions and Cardiometabolic Outcomes............................................... 35
Cognitive-Behavioral Approaches.............................................................................................. 35
Cognitive Restructuring.......................................................................................................... 35
Self-Monitoring...................................................................................................................... 36
Goal Setting and Goal Striving.............................................................................................. 36
Stimulus Control.................................................................................................................... 37
Problem Solving..................................................................................................................... 37
Cognitive-Behavioral Protocols for Binge Eating.................................................................. 38
Cognitive-Behavioral Interventions and Cardiometabolic Outcomes.................................... 38
Independent and Combination Mindfulness Approaches........................................................... 39
Independent Mindfulness Approaches................................................................................... 39
Combination Mindfulness Approaches.................................................................................. 40
Mindfulness Approaches and Cardiometabolic Outcomes.................................................... 40
Conclusion....................................................................................................................................... 41
References........................................................................................................................................ 41
23
ABSTRACT
Environmental and genetic factors are important influences on diet and its cardiometabolic effects,
but overeating and unhealthy eating are ultimately malleable behaviors. Environmental- and indi-
vidual-level factors are therefore key targets for intervention. In this chapter, we review current
behavioral models of three types of overeating: mindless overeating, stress-induced overeating, and
compulsive overeating. We define mindless overeating as overeating that occurs outside of one’s
awareness and without attention. We define stress-induced overeating as overeating that can occur
with or without awareness in response to stress. We define compulsive overeating as eating that
occurs with awareness and that typically involves (1) overeating of highly palatable foods for their
hedonic properties, and (2) experiencing a loss of control over such eating. These forms of overeat-
ing can increase risk of metabolic syndrome. We review evidence that environmental interventions
at both the institutional and individual levels can decrease overeating and improve cardiometabolic
health. These interventions target food environments in which people make most of their food pur-
chasing and consumption decisions and include workplaces, supermarkets, restaurants, and schools.
Cognitive-behavioral intervention approaches, such as cognitive restructuring and self-monitoring
of weight and food consumption, have been demonstrated effective in weight loss in the context of
lifestyle interventions, such as the Diabetes Prevention Program, which typically employ a combi-
nation of such cognitive-behavioral approaches. Mindfulness-based interventions that target over-
eating by enhancing nonjudgmental awareness of eating behaviors and eating related to distressing
emotions also hold promise, although more data are needed to better assess the effectiveness of these
interventions. We conclude that current data support referral of individuals who are overweight or
obese to high-quality behavioral intervention programs that include evidence-based components to
promote improvements in cardiometabolic risk factors. In settings without high-quality group inter-
ventions, referral to a nutritionist with expertise in behavioral weight loss strategies may be helpful.
ABBREVIATIONS
ACTH Adrenocorticotropic hormone
ADHD Attention-deficit/hyperactivity disorder
ANS Autonomic nervous system
BED Binge eating disorder
BES Binge eating scale
CBT Cognitive behavioral therapy
CRH Corticotropin-releasing hormone
DBT Dialectical behavioral therapy
DSM-IV-TR Diagnostic and Statistical Manual for Mental Disorders, 4th edition, Text-Revision
DSM-5 Diagnostic and Statistical Manual for Mental Disorders, 5th edition
GC Glucocorticoid
HbA1C Hemoglobin A1C
HDL High-density lipoprotein
f-MRI Functional magnetic resonance imaging
L-HPA Limbic hypothalamic-pituitary-adrenocortical
LEARN Lifestyle, Exercise, Attitudes, Relationships, Nutrition
MIDUS Midlife in the United States
NAC Nucleus accumbens
NHLBI National Heart, Lung, and Blood Institute
SAM Sympathetic-adrenal-medullary
TSST Trier Social Stress Test
VTA Ventral tegmental area
YFAS Yale Food Addiction Scale
Overeating Behavior and Cardiometabolic Health 25
INTRODUCTION
As described throughout this volume, the qualities and quantities of food that people eat impact
cardiovascular health via several complex and interactive metabolic processes. This chapter focuses
on behavioral factors that promote overeating and intervention techniques that target these fac-
tors. We first review three types of overeating: mindless overeating, stress-induced overeating, and
compulsive overeating. The modern food environment is replete with cues to eat and easy access to
highly palatable foods, which generally contain high levels of sugar, salt, fat, caffeine, and/or flavor
enhancers, and are typically highly processed (Gearhardt et al. 2011). Cues to eat such foods can
operate outside of our awareness and powerfully impact how much we eat, resulting in mindless
overeating (Wansink 2007). In the past three decades, increases in both psychosocial stress and
weight among U.S. adults (Block et al. 2009) may reflect a situation in which some people overeat
highly palatable foods to reduce psychological stress. Habitual stress-induced overeating may carry
particular risk for abdominal adiposity (Dallman 2010), a key risk factor for poor cardiovascular
outcomes (Ashwell, Gunn, and Gibson 2012; Carmienke et al. 2013). Finally, more than one-third
of U.S. adults with obesity who seek weight-loss treatment self-report compulsive overeating, which
involves overeating for the hedonic properties of food and experiencing a loss of control over one’s
eating (Davis and Carter 2009).
We then review behavioral intervention techniques that directly address the processes underlying
each type of overeating. We first review environmental interventions at both the institutional and
the individual level. Such interventions target food environments in which people make most of
their food purchasing and consumption decisions, including the workplace, supermarkets, restau-
rants, and schools (Wansink and Chandon 2014). Next, we review cognitive-behavioral techniques
commonly combined into intervention packages (“lifestyle interventions”) for obesity. These tech-
niques include cognitive restructuring, self-monitoring, and other practices rooted in the theoreti-
cal foundation of cognitive and cognitive behavioral therapy (CBT) (Beck 2011). We also review
the CBTs specifically developed for pathological overeating as observed in binge eating disorder
(BED). Finally, we review mindfulness-based interventions that target overeating by cultivating an
open, nonjudgmental orientation toward present-moment experiences. This serves to disrupt habit-
ual patterns of thoughts, emotions, and behaviors that underpin overeating by promoting awareness
of bodily experiences related to physical hunger, satiety, taste satisfaction, and emotional triggers of
overeating (Kristeller and Wolever 2010). We close this review with suggestions for future interven-
tion development.
TYPES OF OVEREATING
Eighty-three percent of Americans report having overeaten, defined as eating more than one intends
to eat at a given occasion, within the past 10 days (Wansink 2014). Overeating takes many forms;
here, we outline three major types of overeating. We define mindless overeating as overeating that
occurs outside of one’s awareness and without attention. We define stress-induced overeating as
overeating that can occur with or without awareness in response to stress (Adam and Epel 2007;
Tsenkova, Boylan, and Ryff 2013). We define compulsive overeating as eating that occurs (1) with
awareness and that involves overeating of (typically) highly palatable foods for their hedonic prop-
erties, and (2) experiencing a loss of control over such eating (Davis and Carter 2009). Next, we
review these three types of overeating.
Mindless Overeating
Mindless overeating occurs in a myriad of settings, such as at the movie theater, while watching a
baseball game, or when walking past a candy dish in the office for the tenth time. Indeed, people are
unable to detect when they have eaten 15%–20% more or less than they typically do, and the modern
food environment powerfully biases this margin of error toward overeating (Brownell and Horgen
2004). Consequently, people routinely underreport how much they eat: Pooled results from five vali-
dation studies of dietary self-report instruments indicate that people underreport caloric intake by as
much as 28% and that greater body mass index (BMI) is associated with greater levels of underre-
porting (Freedman et al. 2014). These severe misperceptions of how much one has eaten make sense
in light of the tremendous impacts of portion sizes, food proximity and visibility, ambient environ-
ments, palatability, distracted eating, and social influences, among other factors, on eating behavior.
Portion Size
Perception of portion size plays an important role in estimating how much food one is about to eat
and how much food one has eaten. “Portion distortion,” or the tendency to consider a portion to be
smaller or less calorically dense than it actually is, can contribute to overeating and weight gain
(Schwartz and Byrd-Bredbenner 2006). Although people tend to be more accurate when estimating
small portion sizes, they routinely underestimate larger portion sizes by as much as 38% (Wansink
and Chandon 2006). Doubling the size of a fast food hamburger or a product package makes it
appear only 50%–70% larger (Chandon and Ordabayeva 2009). Unfortunately, simple knowledge
of this effect does not reduce this misperception: Estimates by nutritionists and other diet experts
are also affected by portion size cues. In one study, nutrition experts given a larger bowl, relative
to those given a smaller bowl, served themselves 31% more ice cream (Wansink, van Ittersum, and
Painter 2006b). The effects of packaging size on consumption effects exist beyond the impact of
palatability: One study provided moviegoers with medium or large popcorn containers with fresh or
stale, 14-day-old popcorn. Moviegoers given fresh popcorn in large containers ate 45% more than
those given fresh popcorn in medium containers. Similarly, moviegoers given stale popcorn in large
containers ate 33% more than those given stale popcorn in medium containers (Wansink and Kim
2005). Thus, portion size powerfully impacts how much people eat, regardless of expert knowledge
of these effects and the palatability of the food being eaten.
Proximity and Visibility

Food proximity and visibility highly impacts the amount and frequency with which we eat (Sobal
and Wansink 2007). In one classic study, researchers placed dishes of chocolates either within reach-
ing distance (proximately) or 6 ft away (standing required for access) from where secretaries sat
in their offices. Additionally, researchers placed the chocolates in clear dishes (highly visible) or
opaque dishes (less visible). On days when the dishes were within reaching distance and clear, the
secretaries ate 7.7 chocolates; on days when the dishes were within reaching distance and opaque,
they ate 4.6 chocolates; on days when dishes were further away and clear, they ate 5.6 chocolates;
and on days when dishes were further away and opaque, they ate 3.1 chocolates (Wansink, Painter,
and Lee 2006a). Additionally, when the chocolates were within reaching distance, participants
underestimated the amount of chocolates they had actually eaten. These effects have been replicated
in several experimental studies (Sobal and Wansink 2007; Privitera and Creary 2013; Privitera and
Zuraikat 2014). Thus, proximity and visibility each impact not only how much people eat, but also
how much they believe they have eaten.
Ambient Factors
Environmental factors such as lighting, music, temperature, and odor can each impact the types and
quantities of foods we eat outside of awareness (Stroebele and de Castro 2004; Spence 2012). For
example, people dining in the harsh, bright lighting of fast-food restaurants consume more calories
than when they dine in the soft, low lighting of fine dining restaurants (Wansink and van Ittersum
2012). Music loudness is also associated with consumption patterns: People tend to drink more soda
(McCarron and Tierney 1989) and alcohol (Guéguen et al. 2008) when in conditions of loud back-
ground music. Studies report mixed associations between music and rate of eating that vary by type
of music and/or music preference (Caldwell and Hibbert 2002). For example, the slower the tempo
of country western music played in a bar, the more quickly patrons consume their drinks (Bach and
Schaefer 1979). In contrast, the faster the tempo of piano music, the more time patrons spent drinking
soda (McElrea and Standing 1992). Warmer ambient temperatures are associated with selection of less
calorically dense food and reduced food intake overall, which may follow from reduced metabolic
rates observed in warmer environs (Stroebele and de Castro 2004). For example, participants in a caf-
eteria study chose less calorically dense foods in the late spring and summer months relative to the fall
and winter months (Zifferblatt, Wilbur, and Pinsky 1980). Finally, pleasant ambient odors can increase
consumption, whereas noxious odors can decrease consumption: In one study, participants consumed
more Pepsi Cola while watching a movie if they were exposed to a pleasant scent (air freshener) than
if they were exposed to a noxious scent (ammonia; Wadhwa, Shiv, and Nowlis 2008). In sum, ambient
factors such as lighting, music, temperature, and odor—that is, factors over which consumers have
little control when eating outside of the home—can impact eating behavior outside of awareness.
Palatability
The modern food environment is replete with highly processed, highly palatable foods, and consump-
tion of these foods can promote overeating (Sørensen et al. 2003; Gearhardt et al. 2011). For example,
participants who sampled a sweet Hawaiian punch beverage before being allowed to drink as much
Pepsi Cola ad libitum while watching a movie drank significantly more Pepsi Cola than those who
did not sample the Hawaiian punch beforehand (Wadhwa, Shiv, and Nowlis 2008). These findings
suggest that sampling highly palatable food cues stimulates subsequent consumption of similar palat-
able foods. Importantly, the amount of palatable food or drink that people consume correlates with
their ratings of the food’s palatability, but not with ratings of self-reported hunger (Yeomans 1996;
Yeomans and Symes 1999). One study demonstrated this effect by adding highly palatable condiments
to already highly palatable foods: Perceived palatability of highly palatable foods (French fries and
brownies) decreased over time spent eating them; however, the addition of highly palatable condiments
(mayonnaise and vanilla ice cream, respectively) led to renewed increases in perceived palatability and
intake that were independent of self-reported hunger (Brondel et al. 2009). Thus, the innate satiety
system does not regulate eating of highly palatable foods in ways that adjust consumption after caloric
needs have been met; therefore, such hyperpalatability may increase overeating.
Categorization Cues and Health Halo Effects

Perceptions of foods as “healthy” and “unhealthy” significantly impact how much of them people eat.
People often misinterpret specific health claims about foods (Mariotti et al. 2010). This contributes
to the “health halo effect” whereby people overgeneralize or magnify a given food’s healthful prop-
erties. Such halo effects can highly distort beliefs about caloric impact of a proposed snack or meal.
For example, in one study, participants reported that a small snack of an unhealthy food (Snickers
miniature candy, 47 calories) would promote more weight gain than a large snack of healthful foods
(cottage cheese, carrots, and pears, 569 calories; Oakes 2005). Similarly, in another study, partici-
pants perceived a 1000-calorie meal from Subway to have 213 fewer calories than a 1000-calorie
meal from McDonald’s (Chandon and Wansink 2007). Importantly, these misperceptions correlate
with actual intake: Participants ate 35% more oatmeal cookies when they were labeled as a “healthy
snack” relative to when they were labeled as an “unhealthy snack” (Provencher, Polivy, and Herman
2009). Misperceptions of foods as more or less healthful can strongly impact overeating by focusing
people’s attention on perceived quality rather than measurable quantity.
Distracted Eating
Engaging in activities while eating, such as watching TV or listening to music, impacts eating.
For example, people eat 12%–14% more food when they eat lunch while watching TV, and this
increase in eating is not associated with commensurately larger changes in post-meal hunger or sati-
ety (Bellisle, Dalix, and Slama 2004; Hetherington et al. 2006). Auditory distractions exert a similar
effect: Listening to music (Stroebele and de Castro 2006) or a recorded story, such as a detective
novel (Bellisle, Dalix, and Slama 2004), relative to eating in silence, is associated with longer time
spent eating and greater caloric intake. Distracted eating can also promote greater eating throughout
the day. This may follow from the distraction of attention away from a meal, which compromises
the encoding of the memory of that meal. In one study, participants’ abilities to accurately estimate
the amount of food that they had eaten were significantly worse if they had eaten while watching
TV rather than without watching TV (Moray et al. 2007). Poorer memory of a recent meal due
to distracted eating has been linked with greater subsequent snacking: For example, in one study,
women ate lunch while watching TV on one day and ate lunch without distraction on another day.
On both days, women were offered cookies upon returning to the laboratory later in the day. Women
ate significantly more cookies on the day they had eaten lunch while watching TV relative to the
day they ate lunch without distraction (Higgs and Woodward 2009). In contrast, enhancing memory
of a recent meal before an opportunity for an afternoon snack decreases snack consumption (Higgs
2002). Thus, engaging in activities that distract one’s attention away from his or her food can con-
tribute to overeating both at that time, and at later times, suggesting that distracted eating may have
a broader impact on overeating than is typically considered.
Social Influences
Social factors impact the amount of food that people eat (Herman 2015). Two types of social
influences on eating behavior are referred to as social facilitation and social matching, which are
theorized to impact eating behavior via different mechanisms. Social facilitation of eating refers to
the observation that people tend to eat more and for a longer period of time when with other people
than when alone. This effect is commensurate with the size of the group and the relationship with
other diners: Dining with a larger group of people and/or dining with people with whom one is closer
(e.g., friends and family) is associated with eating more. Such increases in intake, which range from
approximately 30%–50% (Herman and Polivy 2005), may be due to increased arousal or emotional-
ity, disinhibition, distraction from consumption monitoring, or longer meal times (Herman, Roth,
and Polivy 2003; Hetherington et al. 2006; Pliner et al. 2006).
Social matching of eating refers to the tendency for people to eat more when their dining part-
ners eat more, and less when their dining partners eat less. Researchers theorize that the desire for
social acceptance, ingratiation concerns, appropriateness, perceived eating norms, and noncon-
scious mimicry can play important roles in this phenomenon (Herman et al. 2005; Robinson et al.
2011; Robinson, Blissett, and Higgs 2013). For example, eating behavior is one important avenue
of impression management (Vartanian, Herman, and Polivy 2007), which involves regulating one’s
own behavior so as to create a specific image for an external audience (Schlenker 1980). Along these
lines, people may undereat or overeat, or may mimic actual eating behavior, so as to gain favor with
their dining partners (Salvy et al. 2007). One early study of this phenomenon examined eating of
crackers among obese and normal-weight people when in the presence of a confederate who ate many
crackers (20 crackers in 20 minutes), few crackers (1 cracker in 20 minutes), or no crackers. All par-
ticipants ate significantly more crackers when with a confederate who ate many crackers relative to
when with a confederate who ate few crackers (Conger et al. 1980), suggesting mimicry of quantity
eaten. Many experimental studies have replicated these findings (Herman, Roth, and Polivy 2003;
Robinson, Blissett, and Higgs 2013). Recent data have further dissected social matching behavior and
found that people tend to mimic the timing of each other’s bites of food (Hermans et al. 2012) and
that desire for social acceptance predicts the degree of mimicry when consuming alcoholic beverages
(Caudill and Kong 2001). In summary, social factors impact the amount of food people eat, suggest-
ing that improving eating behavior among some individuals holds the potential to impact many.
Stress-Induced Overeating
There is considerable overlap among the neurobiological systems that regulate food intake and those
that mediate stress responses (Dallman 2010; Sinha and Jastreboff 2013). Acute, isolated stressors
and chronic, ongoing stressors impact eating behavior in both animals and humans (Macht 2008).
Rodent studies report increased eating of highly palatable food, especially foods high in sugar,
after standard laboratory stressors (e.g., restraining; Dallman, Pecoraro, and la Fleur 2005; Foster
et al. 2009). Human studies also report increased eating of highly palatable “comfort foods” after
standard laboratory stressors (e.g., social performance tasks; Oliver, Wardle, and Gibson 2000; Epel
et al. 2001; Rutters et al. 2009; Lemmens et al. 2011). In the following text, we review neurobio-
logical mechanisms of stress-induced overeating, studies of stress-induced overeating in humans,
and the effects of stress-induced eating on cardiometabolic health. We refer the reader interested in
animal models of stress-induced overeating to reviews on the topic (Corwin and Buda-Levin 2004;
Corwin, Avena, and Boggiano 2011).
Neurobiological Mechanisms Linking Stress and Overeating

Stress responses involve a neural network comprised of neurons in the hypothalamus, brainstem,
and afferent nerves, as well as several areas within the limbic system and frontal cortex (Kaltsas and
Chrousos 2007). Stress responses operate along two interacting pathways. One pathway is the sym-
pathetic-adrenal-medullary (SAM) axis, which is a part of the autonomic nervous system (ANS) that
involves the catecholamines adrenaline and noradrenaline. A second pathway is the limbic hypotha-
lamic-pituitary-adrenocortical (L-HPA) axis, which produces a cascade of hormone secretions that
comprise an endocrine stress response. These hormones include corticotropin-releasing hormone,
adrenocorticotropic hormone, and glucocorticoids (GCs; Herman and Cullinan 1997; McEwen 2007).
Chronic arousal of the L-HPA axis leads to dysregulation in physiological satiety mechanisms, which
can result in stress-induced overeating. Specifically, although GCs increase the hormones leptin and
insulin, which signal satiety, these increases promote insulin and leptin resistance, which can blunt
satiety signaling, thereby leading to overeating (Maniam and Morris 2012). Researchers have therefore
widely examined L-HPA axis activity in the context of cardiometabolic health (e.g., visceral adiposity)
and found the L-HPA axis to play critical roles in the neural regulation of eating and peripheral energy
balance (Kyrou, Chrousos, and Tsigos 2006; Sinha and Jastreboff 2013).
In addition to disrupting the regulation of the physiological satiety mechanisms that regulate energy
homeostasis, stress-induced L-HPA activity impacts the mesolimbic reward area, which is highly inner-
vated by dopaminergic neurons. The effects of L-HPA activity in the mesolimbic reward area can render
the experience of eating highly palatable foods especially rewarding, therefore encouraging overeating
in times of stress. For example, functional magnetic resonance imaging data show that upon view-
ing images of highly palatable food, individuals who report greater chronic stress showed exaggerated
activity in regions of the brain involving reward, motivation, and habitual decision-making, as well
as reduced activity in areas linked to strategic planning and emotional control (Tryon et al. 2013).
Furthermore, these chronically stressed individuals also ate significantly more highly palatable food
from a buffet than their less-stressed counterparts after an acute laboratory stressor.
L-HPA axis activity also amplifies neural experiences of reward by stimulating the release of
endogenous opioids, which increase eating of highly palatable food and instigate further opioid
release. Specifically, stress-induced eating stimulates (1) increases in GCs and insulin that then drive
intake of palatable food, (2) increases in opioid release, which reinforce and promote intake of these
palatable foods, and (3) increases in dopamine secretion in the mesolimbic pathway, including the
ventral tegmental area and nucleus accumbens, which amplify the neural experience of reward that
follows from eating of highly palatable food (Adam and Epel 2007; Dallman and Bhatnagar 2010).
The neural stress-reward pathways promote the development of habitual behavior focused on
obtaining relief. Repeated and strong opioid responses in the neural circuitry of reward promote the
encoding of habits in the limbic system, specifically the basal ganglia, which regulates habit-based
behavior. The limbic system is especially likely to encode memories involving strong emotions
and the solutions that people use to effectively cope with them. Thus, stress-induced overeating of
hyperpalatable food, which effectively reduces stress responses, is easily learned, remembered, and
repeated (Dallman 2010; Ulrich-Lai et al. 2015).
Studies of Stress-Induced Overeating in Humans

Researchers have used experimental methods and naturalistic settings to study the biology and
behavioral patterns of stress-induced eating in humans. One laboratory study examined the impact
of repeated (daily) exogenous administration of GC or placebo on food intake over the course of
several days. Participants ate ad libitum from an automated food selection system that tracked their
intake both prior to and on the days of administration of GC or placebo. The GC participants’
increase in caloric intake from Day 0 to Day 4 (total) was >1600 calories more than the placebo
participants’ increase in caloric intake. Another laboratory study used a standard acute psychologi-
cal stressor task, the Trier Social Stress Test (TSST), to examine the extent to which acute psycho-
logical stress impacts immediate post-stress eating behavior through GC changes. Results indicated
that women with larger increases in GC (salivary cortisol levels) following the TSST ate more total
calories and more sweet foods (after the TSST) than women with smaller increases in GC (Epel
et al. 2001). In another study, participants completed a TSST task in the laboratory, and researchers
assessed their GC responses (assessed using saliva). Researchers then collected participants’ self-
reported daily hassles (real-life stressors) and snack intake for the following 2 weeks. Data showed
that among participants with larger increases in GCs following the TSST (but not among partici-
pants with smaller increases in GCs), greater frequency of daily hassles was associated with more
frequent snacking (Newman, O’Connor, and Conner 2007). Thus, data indicate that increases in a
marker of physiological stress (GC), induced either via exogenous administration or psychological
stressor techniques, are associated with increased eating.
Stress-Induced Overeating and Cardiometabolic Health
Researchers have found direct links between stress-induced overeating and cardiometabolic outcomes.
For example, in an observational cohort study, 81 of 131 (62%) medical students self-reported on
their eating behavior in times of stress. Forty-seven of these 81 students (58%) reported eating more
than usual in times of stress (“more-eaters”) and 34 students (42%) reported eating less than usual
in times of stress (“less-eaters”). Researchers assessed traditional biomarkers of students’ metabolic
health (e.g., insulin, lipids, BMI, and waist-to-hip ratio [WHR]) and nocturnal cortisol levels (assessed
using urine) during a low-stress period (summer vacation) and also during a high-stress period (final
exams). Relative to those eating less, those eating more evidenced significantly greater increases in
nocturnal cortisol, total/high-density lipoprotein (HDL) cholesterol ratio, weight (~5 lb), and WHR
(but only among women) from low- to high-stress periods (Epel et al. 2004). Population-level data
also highlight the impact of stress-induced eating on metabolic health: The Midlife in the United States
(MIDUS II) study asked participants how they usually experience a stressful event and to indicate the
extent to which they (1) tend to eat more of their favorite foods to make themselves feel better, and
(2) tend to eat more than they usually do. Higher scores on these items (i.e., endorsing eating more
of one’s favorite foods and eating more than usual) were associated with significantly higher levels of
glucose, insulin, insulin resistance, and HbA1C, as well as higher odds of developing prediabetes and
diabetes. After adjusting for waist circumference, these associations were no longer statistically signifi-
cant, suggesting that stress-induced overeating may be an important mediator of the association between
visceral adiposity and cardiometabolic risk (Tsenkova, Boylan, and Ryff 2013). Taken together, these
studies suggest that stress-induced eating is associated with elevated glucose levels and insulin resistance,
with the effects occurring largely through weight gain and central adiposity.
Compulsive Overeating
At least 30% of treatment-seeking obese individuals identify times when they feel out of control
over their eating and when they overeat highly palatable food in the absence of physical hunger,
regardless of a desire to not do so (Spitzer et al. 1992; de Zwaan 2001). Such compulsive overeat-
ing has behavioral similarities to drug addiction (e.g., Volkow et al. 2012, 2013). Researchers have
thus designed self-report measures to assess behaviors that are core features of substance misuse
disorders, but in the context of overeating. One measure, the Yale Food Addiction Scale (YFAS;
Gearhardt, Corbin, and Brownell 2016, 2009), conceptualizes problematic eating behavior simi-
larly to traditional substance-related and addictive disorders as defined by criteria in the Diagnostic
and Statistical Manuals for Mental Disorders (DSM-IV-TR, DSM-5; American Psychological
Association 2000; American Psychological Association 2013). Another measure, the Binge Eating
Scale (BES, Gormally et al. 1982), assesses compulsive overeating behavior, and scores on this
measure correlate with a DSM diagnosis of BED (Celio et al. 2004; Grupski et al. 2012). Measures
of addictive-like eating, such as the YFAS, are highly correlated with measures of binge eating, such
as the BES (Flint et al. 2014). Compulsive overeating might best be considered along a continuum,
with the most extreme end encompassing severe overeating indexed by high scores on measures of
binge and addictive-like overeating (Davis 2013a,b). In the following text, we review neurobiologi-
cal mechanisms of compulsive overeating, investigations of compulsive overeating in animals and
humans, and associations between compulsive eating and cardiometabolic health.
Neurobiological Mechanisms Underlying Compulsive Overeating

Food and drug reward activate pathways in the same neural structures in the brain, and researchers
have thus posited neurobiological models of binge eating that parallel those of addictive processes
(Volkow et al. 2012, 2013; Smith and Robbins 2013). Similar to processes of addiction, the dopamine
and opioid systems play key roles in compulsive behavior (e.g., overeating) by motivating reward-
seeking behavior and mediating the neural perception of hedonic reward, respectively (Avena, Rada,
and Hoebel 2008; Volkow et al. 2008; Davis et al. 2009). Compulsive overeating, both in animals
and humans, generally involves overeating of highly palatable foods, which exert physiologic effects
that suggest addictive potential (Gearhardt et al. 2011). For example, eating highly palatable foods
activates dopaminergic neurons and increases μ-opioid receptor binding within the nucleus accum-
bens and other reward centers in the brain (Nathan and Bullmore 2009; Volkow, Wang, and Baler
2011). Repeated stimulation of the neural reward system can alter dopamine and opioid receptor
binding in ways that promote addictive behavior (Colantuoni et al. 2001; Rada, Avena, and Hoebel
2005; Koob and Volkow 2009). The combination of genetic factors and environmental exposures
that encourage consumption of highly palatable foods may thus converge to reinforce neural cir-
cuitry that drives compulsive overeating in susceptible individuals.
One method of studying neurobiological models of compulsive overeating is to administer inter-
mittent feeding schedules to rodents to condition binge eating. These schedules involve intermittent
access to highly palatable foods, such as sugar/fat and “cafeteria diets” generally comprised of pro-
cessed snacks and desserts (Heyne et al. 2009; Corwin, Avena, and Boggiano 2011). Researchers
then examine rodents’ behavior in experimental paradigms traditionally used to test models of drug
addiction (Avena 2010). Such paradigms induce behaviors and symptoms such as bingeing and
tolerance (increases in amount and frequency of substance use over time), withdrawal (behavioral
symptoms induced by removal of substance or pharmacologic blockade of neural effects of sub-
stance), and craving (enhanced responding to cues and increased motivation to work for substance
after removal of substance), among others (Davis 2013a). Following binge eating of highly pal-
atable food, rodents administered the opioidergic antagonist naloxone show symptoms similar to
those observed during withdrawal from addictive drugs (e.g., heroin), such as aggression, anxiety,
teeth-chattering, and head-shaking (Colantuoni et al. 2002). Notably, greater self-reported com-
pulsive overeating among humans is associated with larger nausea and GC responses following
administration of naloxone (Daubenmier et al. 2014; Mason et al. 2016). Conversely, administration
of an opioid agonist (butorphanol) increases overeating of highly palatable food (Boggiano et
al. 2005), and rodents prone to binge eating tolerate significant discomfort in the pursuit of highly
palatable food, such as higher levels of foot shock in order to access Oreo cookies (Oswald et al.
2011). Similarly, rodents formerly administered an intermittent sugar-feeding schedule work harder
to obtain sugar after a two-week period of abstinence than they ever did before, and the amount of
work that they will do to obtain the sugar increases over time (Grimm, Fyall, and Osincup 2005).
Finally, there is evidence that sugar intake cross-sensitizes with addictive drugs, and vice versa
(Avena and Hoebel 2003; Avena, Rada, and Hoebel 2008). Thus, animal models have shed light
on behavioral and biological overlaps between compulsive overeating of highly palatable food and
drug addiction; clarification of these overlaps can inform intervention development.
There is evidence that compulsive overeating is a specific phenotype within human obesity that
is characterized by biological and behavioral characteristics (Davis et al. 2011; Burmeister et al.
2013). Obese individuals who meet the YFAS diagnostic criteria for food addiction (relative to
their obese counterparts who do not) self-report and show behavioral and psychological profiles
similar to those observed among individuals with drug addictions. For example, individuals meet-
ing YFAS criteria for food addiction score more highly on measures of impulsivity and emotional
reactivity. They are also significantly more likely to meet criteria for a diagnosis of BED, severe
depression, and attention-deficit/hyperactivity disorder (ADHD); to have more addictive personal-
ity traits; to experience stronger food cravings; and to more frequently snack on sweet foods and
eat in response to emotions (Davis et al. 2011). These individuals also endorse greater sensitivity
to the rewarding properties of hyperpalatable food, which aligns with research linking greater sen-
sitivity to reward with greater binge eating (Davis et al. 2007; Mathes et al. 2009). This enhanced
sensitivity to reward has been associated with genetic polymorphisms that (1) influence dopamine
receptor binding in ways that increase motivation to engage in appetitive behavior, and (2) affect
opioid receptors in ways that increase reactivity to the hedonic properties of food (Davis et al.
2009, 2012). Importantly, greater food addiction severity, as indexed by continuous scoring of the
YFAS, is associated with poorer adherence to, and effectiveness of, behavioral weight-loss inter-
ventions (Burmeister et al. 2013).
Compulsive Overeating and Cardiometabolic Health

Compulsive overeating, defined in terms of BED or food addiction, carries nontrivial cardiometa-
bolic health risks (Hudson et al. 2010; Abraham et al. 2014; Klatzkin et al. 2015). For example, obese
individuals who report compulsive overeating, relative to those who do not, experience more weight
cycling (de Zwaan, Engeli, and Müller 2015) and are at greater risk for hypertension (Schulz et al.
2005; Hudson et al. 2010), dyslipidemia (Hudson et al. 2010), poor glycemic control, and type 2
diabetes (Hudson et al. 2010; Abraham et al. 2014; Raevuori et al. 2015), all of which increase risk
for poor cardiovascular outcomes (DeFronzo and Ferrannini 1991).
BEHAVIORAL INTERVENTIONS FOR OVEREATING

Standard behavioral treatments that target reductions in overeating in the service of weight loss
typically involve dietary changes and behavioral intervention techniques to support these changes
(Bray and Bouchard 2014). Genetic predispositions, family and cultural backgrounds, and a host
of other factors impact weight status; however, the primary targets of behavioral interventions are
environmental factors and patterns of learned behavior that impact eating. In the following text, we
review behavioral (nonpharmacologic) interventions that target overeating, including environmental
approaches, approaches based on and incorporating CBT techniques (e.g., “lifestyle interventions”),
and both independent and combination mindfulness approaches.
Environmental Approaches
The proliferation of environmental barriers to healthy eating highlights the relevance of interven-
tions that directly address these barriers (Brownell and Horgen 2004; Wansink and Chandon 2014).
Environmental interventions are rooted in behavioral principles (e.g., conditioning and stimulus
control) and have been a centerpiece of interventions targeting overeating since the 1960s (e.g.,
Stuart 1967). Classical conditioning principles hold that repeatedly presenting a stimulus prior to (or
simultaneously with) a given behavior will associate that stimulus with that behavior. For example,
repeatedly purchasing chocolates from a convenience store may stoke cravings for chocolates
whenever one walks by the convenience store on the way to work. In this context, stimulus control
involves the management of cues that are associated with overeating, such as walking a different
way to and from work to avoid a cue to purchase and eat chocolates (Wadden, Crerand, and Brock
2005). Thus, behavioral principles form the basis of environmental approaches targeting reductions
in overeating.
Large-scale changes to the food environment, such as decreasing the availability and portion
sizes of highly palatable foods, and increasing the attractiveness, availability, and affordability of
healthier alternatives, will require considerable time and other resources. Researchers have therefore
emphasized methods by which (1) institutions can implement changes in financially feasible ways
(Wansink 2014) and (2) individuals can modify their personal food environments (Lowe 2003). The
food radius (Wansink 2014), defined as the five areas where people purchase or consume their food
approximately 80% of the time, includes the home, supermarkets, restaurants, work, and, if appli-
cable, school. In the following text, we review environmental interventions in the five food radius
areas within the institutional and individual levels.
Institutional-Level Environmental Interventions

One environmental approach to reducing overeating involves implementing policies or practices at
the institutional level. Such interventions strive to increase consumption of healthy foods by increas-
ing the availability, accessibility, and/or attractiveness of these foods. These interventions have been
tested across several types of institutions, including the workplace (Pratt et al. 2007), retailers
(e.g., Glanz, Bader, and Iyer 2012), schools (e.g., Hanks, Just, and Wansink 2013), and restaurants
(e.g., Wansink, van Ittersum, and Painter 2005). In the following text, we describe applications of
environmental interventions tested in these settings and data on the effects of such interventions on
cardiometabolic outcomes.
When given the option to select smaller portions of food in workplace and supermarket set-
tings, people do choose this option (Vermeer et al. 2011); thus, providing smaller-sized options can
reduce consumption. Preference for smaller portions may be fueled by widespread dissemination
of research findings (in the lay press) showing that people eat dramatically more food when eating
from larger versus smaller plates or containers (e.g., Leonhardt 2007). Indeed, workers purchase
price-adjusted smaller versions of standard dishes (approximately 2/3 of the standard option avail-
able) when offered the opportunity (Vermeer et al. 2011). Retailers have capitalized on consumer
preferences by developing single-serving packages of snack foods, such as the 100-calorie package,
which can reduce consumption by as much as 25% (Wansink, Payne, and Shimizu 2011).
Product placement in supermarkets can also dramatically impact purchasing behavior and there-
fore eating behavior. Approximately 30% of all supermarket sales result from end-aisle displays
(Cohen et al. 2014) and placing potato chips at eye level, rather than higher or lower, is associated
with more purchases (Sigurdsson, Saevarsson, and Foxall 2009). Replacing unhealthy items typically
found at checkout stations (e.g., candy) with healthier options (e.g., dried fruit and nuts) increases
purchasing of the healthy options at checkout stations (Sigurdsson, Larsen, and Gunnarsson 2014).
In response to these data and customer complaints (e.g., children asking for candy placed at their
eye level in checkout stations), some food retailers, such as Lidl and Tesco (the United Kingdom’s
largest retailer), have removed candy from checkout stations (Smithers 2014). These broad types of
structural changes can benefit customers by reducing the constant burden of coping with environ-
mental cues to purchase hyperpalatable foods at checkout stations, without compromising retailers’
sales of food items (e.g., healthier snack foods, such as nuts).
Similar environmental effects can dramatically impact children’s and adolescents’ eating behav-
ior in schools and adults’ eating behavior in restaurants. High school students given the option to
use a “convenience line” in the cafeteria that included only healthier foods (e.g., salad bar, whole
fruits and vegetables), in addition to a standard line that offered less healthy foods (e.g., tacos,
hamburgers), choose and eat different types and quantities of foods compared to when only offered
a standard line. In one study, inclusion of the option to use this type of convenience line led to a
27.3% reduction in actual eating of less healthy foods (Hanks et al. 2012). Similarly, middle school
cafeterias selling apples presliced rather than in whole form increased apple purchases by 71%, and
students offered sliced apples ate more than half of their purchase (73%), whereas students offered
whole apples ate less (48%; Wansink et al. 2013). Increasing attractiveness of healthy options also
impacts eating behavior. Elementary school cafeterias that assigned attractive, fun names to health-
ier food options (e.g., X-ray Vision Carrots) led elementary students to eat twice the percentage of
their carrots relative to when cafeterias assigned these foods generic names (e.g., Food of the Day;
Wansink et al. 2012). Additionally, people selecting food from a buffet line take more of the foods
offered earlier in the line. Thus, placing healthier options at the beginning of the line can dramati-
cally impact food selection (Wansink and Hanks 2013). Similarly, restaurants that use descriptive
menu labeling can dramatically increase sales (Wansink, Painter, and Van Ittersum 2001) as well as
patron satisfaction. Restaurant patrons who ordered and consumed meals from a menu with evoca-
tive, descriptive item descriptions (e.g., “Succulent Italian Seafood Filet”), relative to those who
ordered from a generic menu (e.g., “Seafood Filet”), reported their food to be more attractive and
tasty, and also estimated that they had eaten more calories and reported feeling more full and satis-
fied. Thus, restaurants and cafeterias can capitalize on these effects to help patrons make healthier
choices while also enhancing patron satisfaction.
Individual-Level Environmental Interventions

Interventionists have begun to test environmental adaptations as standalone interventions for weight
loss. In the literature, such interventions have been termed “small-change” interventions (e.g.,
Phillips-Caesar et al. 2015) and designed to foster small, minimally burdensome changes in one’s
environment or interaction with one’s environment. Such interventions have been administered in-
person, via telephone, and over the Internet (Carels et al. 2008; Wansink 2010; Kaipainen, Payne,
and Wansink 2012). They have also been adapted into guided self-help formats published in widely
available books, such as Mindless Overeating: Why We Eat More Than We Think (Wansink 2007)
and Slim by Design (Wansink 2014). In addition to their standalone interventions, small-change
environmental adaptations have been investigated as adjuvant techniques to promote weight-loss
maintenance following traditional cognitive-behavioral (lifestyle) interventions. In the following
text, we review investigations of environmental interventions each as standalone and adjuvant inter-
ventions and their effects on weight loss.
Trials comparing environmental interventions and traditional CBT lifestyle interventions for
weight-loss suggest that environmental interventions may be effective ways for individuals seeking
alternatives to traditional CBT-based lifestyle interventions. In one study, overweight and obese par-
ticipants were randomized to receive an in-person, 14-week environmental intervention (Carels et al.
2009) or an in-person, 14-week course of the LEARN (Lifestyle, Exercise, Attitudes, Relationships,
Nutrition) program (Brownell 2004). All participants self-monitored their dietary intake throughout
the study period. The LEARN program is a traditional CBT-based lifestyle intervention that targets
changes in the domains of eating behavior and physical activity, as well as building and strengthen-
ing psychological tools to work with one’s attitudes, goals, and emotions. The environmental inter-
vention in this study focused on adapting one’s food environments, including convenience, serving
sizes, and cue exposure, among others. Participants in both groups experienced significant weight
loss (approximately 8.6 lb [3.9 kg]) from pre- to post-intervention; however, both groups also expe-
rienced weight regain (approximately 3.3 lb [1.5 kg]) from postintervention to 6-month follow-up
(Carels et al. 2011). These data suggest that the environmental intervention was as effective as the
lifestyle intervention. Further data is needed to help identify which approach may work best for dif-
ferent people.
The incorporation of environmental interventions as maintenance-oriented interventions that fol-
low traditional CBT-based lifestyle interventions may bolster the benefits of existing behavioral
weight-loss interventions. In one study, overweight or obese participants received a 16-week course
of the LEARN program, and then were randomized to receive (or not) a 6-week intervention focused
on changing their food environments, such as the home kitchen and how they eat in restaurants.
Participants experienced similar weight loss during the LEARN program; however, participants
randomized to receive the post-LEARN 6-week intervention (relative to control) evidenced sig-
nificantly greater weight loss, percent of body weight loss, and body fat reduction from baseline
through the 6-month follow-up period (Carels et al. 2008). Researchers have yet to investigate the
effects of providing environmental interventions prior to other interventions, which may prove espe-
cially beneficial. Other investigations have found that teaching weight-loss maintenance skills (e.g.,
learning about energy balance principles and fine-tuning lifestyle habits by making quick, small, and
easy adjustments that do not require effort and attention) prior to introducing a phase of acute weight
loss is associated with better long-term weight-loss maintenance (Kiernan et al. 2013).
Studies of environmental interventions underscore that personalizing intervention components to
fit individuals’ cultural, lifestyle, and otherwise personal preferences is critical for promoting inter-
vention acceptability (e.g., Phillips-Caesar et al. 2015). For example, environmental adaptations
and techniques that participants rate as “easy” garner greater adherence, suggesting that personal-
izing such techniques is needed to maximize participant retention and long-term behavior change
(Wansink 2010; Kaipainen, Payne, and Wansink 2012).
Environmental Interventions and Cardiometabolic Outcomes

Though selected trials have examined the impact of environmental interventions on weight loss as
reviewed earlier, the majority of studies that have incorporated environmental intervention tech-
niques have done so in the context of larger institution-wide initiatives to improve employee health.
The National Heart, Lung, and Blood Institute developed the Obesity Prevention in the Worksite
initiative in 2004, which took a population-based approach to promoting behavior change through
environmental interventions targeting prevention and control of weight gain (for a review of funded
trials, see Pratt et al. 2007). The trials completed under this initiative generally included a variety
of environmental intervention techniques that targeted behavior related to both eating and physical
activity. Hence, it is difficult to parse the extent to which weight-loss outcomes are attributable to
environmental intervention components directed at eating versus other intervention components
targeting physical activity. Nevertheless, these interventions have begun to report promising results.
For example, the Images of a Healthy Worksite group-randomized trial (Fernandez et al. 2015)
reported on 3799 employees assessed at baseline and post-intervention (2 years) at 10 worksites.
There were statistically significant differences between control (3.3% increase in percentage of
overweight or obese workers) and intervention (4.5% decrease in percentage of overweight or obese
workers) worksites. Thus, intervening on the food environments in places where individuals do not
control options available to them (e.g., workplace and schools) is a promising way to impact eating
behavior.
Cognitive-Behavioral Approaches
Behavioral approaches to reduce overeating are predicated on a core assumption of social learning
theory (Bandura and McClelland 1977), which is that health behaviors such as healthy eating and
exercise constitute learned behaviors that can be modified. In the following text, we review cogni-
tive-behavioral techniques commonly packaged into “lifestyle interventions” that target weight loss
through eating behavior change.
Cognitive Restructuring
The cognitive model holds that dysfunctional or distorted thoughts are key determinants of mood
and behavior (Beck 2011). For example, the thought “I’m a failure because I ate a cookie today”
can lead to feelings of guilt and sadness, which may precipitate relief-seeking in reactive, generally
maladaptive ways (e.g., eating more cookies), thus reinstating the feedback loop. There are several
types of dysfunctional or distorted thoughts that are problematic for individuals struggling with
overeating, such as all-or-nothing and catastrophic thinking (e.g., “I’ve had one bite so I might as
well eat the entire pint of ice cream”), labeling (e.g., “I’m a failure because I ate one cookie”), and
“should” and “must” statements (e.g., “I should only eat steamed vegetables for all meals”; for a
review of thought distortions, see Greenberger and Padesky 1995). Cognitive restructuring is a core
cognitive-behavioral technique and first involves the identification of (1) a maladaptive thought, (2)
the feelings that follow from the thought, and (3) evidence for and against the thought. Individuals
then use this information to develop alternative, adaptive thoughts (Beck 2011). Cognitive restruc-
turing can therefore be used to uncover cognitive drivers of overeating by targeting maladaptive
thoughts that lead to a cascade of negative mood and maladaptive behaviors that serve to reinforce
problematic eating behavior. Indeed, in the context of treatment for overeating, interventions that
include cognitive restructuring can increase self-efficacy (e.g., Wolff and Clark 2001), which pre-
dicts greater weight loss (Warziski et al. 2007).
Self-Monitoring
Self-monitoring, or deliberately attending to some aspect of one’s behavior and recording details of
the behavior, has been examined as both a cornerstone of lifestyle interventions and a standalone
lifestyle intervention for weight loss (Burke, Wang, and Sevick 2011). The theoretical basis for
self-monitoring is rooted in self-regulation theory (Kanfer 1970; Kanfer and Gaelick-Buys 1991),
which posits that self-monitoring must occur before it is possible to evaluate progress toward a goal
(self-evaluation). Such self-evaluation is what provides the self-reinforcement needed to maintain
progress toward a goal. Truthful, consistent, and accurate self-monitoring is therefore needed for
effective self-regulation of a given behavior (e.g., weight loss; Burke et al. 2008, 2012).
Self-monitoring of diet (dietary self-monitoring) directly targets mindless eating by increasing
awareness of what, when, how much, and potentially where individuals are eating. A recent review
of 15 studies testing the effects of dietary self-monitoring on weight loss found that more frequent
or complete dietary self-monitoring records were associated with greater weight loss (Burke, Wang,
and Sevick 2011). Dietary self-monitoring practices are highly conducive to mobile platforms, which
can dramatically reduce burden associated with paper-and-pencil methods (Khaylis et al. 2010;
Raaijmakers et al. 2015). This reduced burden may promote sustained dietary self-monitoring, as
evidenced by data showing that mobile self-monitoring tools can lead to more consistent and lon-
ger-term self-monitoring than paper-and-pencil methods (Burke et al. 2012; Wharton et al. 2014).
Importantly, such longer-term dietary self-monitoring is associated with long-term weight-loss main-
tenance (Klem et al. 1997); hence, further investigation of how to optimize long-term adherence to
mobile dietary self-monitoring interventions is warranted (Turner-McGrievy et al. 2013).
Goal Setting and Goal Striving

Setting and striving toward goals, which are mental representations of desired end states to which
people are committed (Fishbach and Ferguson 2007), are common components of lifestyle interven-
tions. After setting goals related to eating behavior (e.g., eating fewer desserts) or consequences that
follow from eating behavior (e.g., weight change), individuals engage in goal striving strategies that
reduce the intention–behavior gap (Sheeran 2002) and bring them closer to their goals.
Goal setting involves determining which goal to pursue and the criteria to evaluate whether one
has achieved the goal. There are several characteristics of goals that are particularly relevant to
health behavior, including motivational orientation (approach versus avoidance), difficulty (simple
versus challenging), and type (performance versus mastery; Mann, de Ridder, and Fujita 2013).
Data on goal motivational orientation show that approach-oriented goals (e.g., “eat fruit for a snack
in the afternoon”) are more effective than avoidance-oriented goals (e.g., “do not eat donuts for
a snack in the afternoon”; e.g., Sullivan and Rothman 2008). Data on the goal difficulty suggest
that people are more likely to achieve goals that are specific, measurable, attainable, realistic, and
timely (as indicated by the SMART acronym; Latham 2003). Finally, data on goal type suggest
that performance-based goals (e.g., “lose 10 lb” [4.5 kg]) and mastery-based goals (e.g., “learn to
prepare and enjoy healthy meals”) differ in how they impact what individuals attend to, and what
they perceive as setbacks. Individuals with performance-based goals focus on documenting goal-
related abilities and therefore perceive setbacks as evidence of lacking ability to meet the goal. In
contrast, individuals with mastery-based goals focus on improving their skillfulness in the service
of a goal and therefore perceive setbacks as opportunities to learn how to become more skillful.
Mastery-based goals promote self-efficacy, which is associated with more effective self-regulation
in the context of eating behavior (Anderson, Winett, and Wojcik 2007). In sum, setting goals that are
approach-oriented, SMART, and mastery-based is more likely to lead to effective health behavior
change.
Goal striving is the process of planning and enacting behavior that enables individuals to narrow
and ultimately close the gap between their intentions and their actual behaviors (Sheeran 2002).
Teaching and building skills of effective goal striving, as done in lifestyle interventions
targeting weight loss, include instruction in several strategies that enable individuals to reduce over-
eating (Mann, de Ridder, and Fujita 2013). One strategy is prospection and planning, which allows
individuals to predict times when they will struggle to maintain behavior that will move them closer
to, rather than further from, their goals, and to plan accordingly. For example, if an individual knows
that he will find it very tempting to purchase oversized packages of unhealthy foods from conve-
nience stores while traveling, he may therefore prepare portions of healthy snacks to bring on his
travels. Another strategy is automating behavior, which involves identifying triggers and cues for
behaviors that promote progress toward (or away from) goals and developing strategies to automate
(or disrupt) these behaviors. For example, an individual may routinely overeat potato chips if he/she
eats them directly from the bag but may be able to avoid such overeating if she pours the chips into a
small bowl before eating them. She can, for example, promote her use of a small bowl by storing the
bag of potato chips in a cupboard next to a stack of small bowls. Construal is a strategy that involves
framing health behaviors to be in line with individuals’ abstract, long-term goals (e.g., weight-loss
and increased fitness) rather than their more concrete, short-term desires (e.g., enjoying a tasty cup-
cake). In sum, the cognitive-behavioral techniques of goal setting and goal striving increase aware-
ness of overeating, thereby facilitating informed decision-making about when, where, and how to
implement changes, and providing strategies to implement changes.
Stimulus Control
Stimulus control techniques provided a foundation for early treatments targeting behavioral control
of overeating in obesity (Stuart 1967) and have remained important components of more recent
interventions (Brownell 2000; Wadden, Butryn, and Wilson 2007; Wadden et al. 2012). Stimulus
control techniques are predicated on the assumption that environmental factors stimulate behavior,
and therefore altering environmental factors can change behavior. It follows that weight-loss inter-
ventions that include this technique focus on increasing exposure to cues that reduce overeating
and reducing exposure to cues that encourage overeating. For example, individuals can choose to
keep a bowl of washed and ready-to-eat fruit (rather than a cookie jar) on the kitchen counter, which
may promote eating of a fiber-filled fruit rather than a sugar-laden cookie for an afternoon snack.
Similarly, individuals can choose to dine at menu-based restaurants rather than buffet restaurants to
avoid a smorgasbord of cues to overeat a variety of foods. We further discuss applications, adapta-
tions, and extrapolations of stimulus control techniques when we review environmental interven-
tions, in the following.
Problem Solving
Problem solving techniques focus on developing adaptive solutions for difficult problems and gen-
erally comprise a five-stage process: (1) conceptualization of the experience of having problems as
normative, (2) identification of the problem that is leading to an undesired behavior, (3) generation
of potential solutions, (4) selection of a solution for initial testing, and (5) evaluation of the tested
solution as a solution to the identified problem (Perri, Nezu, and Viegener 1992; D’Zurilla and Nezu
1999, 2009). In the context of lifestyle interventions for weight loss, problem-solving techniques
are often taught in therapist-led group formats. Such formats allow for collaborative discussions
about solutions to participant-introduced barriers to weight loss, such as difficulties with overeat-
ing in particular situations (e.g., social gatherings) or coping with food cravings. Increases in self-
reported problem-solving skills following lifestyle interventions are associated with greater weight
loss (Murawski et al. 2009).
Cognitive-Behavioral Protocols for Binge Eating

CBT is a first-line treatment for BED (National Institute for Clinical Excellence 2004), particularly
in the form of CBT-based guided self-help for binge eating (Wilson et al. 2010). Individuals meeting
criteria for BED benefit from a more intensive course of cognitive-behavioral techniques in these
protocols than that traditionally included in lifestyle interventions. For example, individuals meet-
ing criteria for BED report larger reductions in binge eating when administered a guided self-help
CBT protocol (Overcoming Binge Eating; Fairburn 1995) relative to when administered a guided
self-help behavioral weight-loss (lifestyle) protocol (LEARN Program for Weight Management;
Brownell 2000; Grilo and Masheb 2005).
Clinician-administered CBT protocols for binge eating behavior include CBT for bulimia
nervosa (CBT-BN), CBT for binge eating disorder (CBT-BED), and, more recently, enhanced
cognitive behavioral therapy (CBT-E; Fairburn et al. 2008; Murphy et al. 2010). CBT-E is a
transdiagnostic treatment for eating disorders, meaning that it is based on a theoretical framework
that identifies common factors that affect the maintenance of several related eating disorders,
such as binge eating, bulimia, and anorexia (and is therefore used to treat a wide spectrum of
such disorders). CBT interventions are effective when delivered in both individual and group
formats (Brownley et al. 2007; Hay 2013), except for CBT-E, which is only administered indi-
vidually (Fairburn et al. 2008). CBT-BN, CBT-BED, and CBT-E protocols include a more inten-
sive emphasis on cognitive-behavioral techniques common to lifestyle interventions for weight
loss, including dietary self-monitoring, cognitive restructuring, and exposure-based practices.
Such techniques directly target both stress-induced and binge eating through the identification
of (1) thoughts and feelings that precipitate overeating, (2) alternatives to overeating, (3) barriers
to change in eating behavior, and (4) effective problem-solving strategies (Fairburn 1995). These
treatment protocols also include treatment components that specifically target core symptoms of
eating disorder pathology, such as dietary restraint, meal skipping, emotional and bodily avoid-
ance, and pathological preoccupation with body shape and weight. CBT-E also includes strategies
and procedures targeting barriers to change often observed in individuals with depression, such
as clinical perfectionism, low self-esteem, and interpersonal struggles (Fairburn et al. 2008). In
sum, CBT interventions have demonstrated widespread effectiveness in reducing binge eating,
and meta-analytic data suggest that CBT interventions should be recommended as first-line treat-
ments for BED (Vocks et al. 2010).
Cognitive-Behavioral Interventions and Cardiometabolic Outcomes

Lifestyle interventions that use the cognitive-behavioral techniques reviewed earlier (in combina-
tion with diet and physical activity recommendations) can significantly improve cardiometabolic
health. The Diabetes Prevention Program (DPP) is one of the most widely examined behavioral
weight-loss interventions (Diabetes Prevention Program Research Group 2002b). In the landmark
27-center (N = 3234) randomized controlled trial of people with prediabetes, the DPP achieved sig-
nificant reductions in diabetes risk (58%) compared with placebo and was significantly more effec-
tive than metformin therapy, which reduced diabetes risk by 31% compared with placebo (Diabetes
Prevention Program Research Group 2002a). Secondary analyses reported significant reductions in
triglycerides and significant increases in HDL-cholesterol in the DPP group relative to the metformin
and placebo groups (Diabetes Prevention Program Research Group 2005). A subsequent 16-center
(N = 5145) randomized controlled trial in people with type 2 diabetes, Look AHEAD (Action for
HEAlth in Diabetes), reported that a behavioral weight-loss intervention similar to the DPP led to
significant reductions in several cardiometabolic risk factors, including weight and glycemic control
with a median of 9.6 years of follow-up. This trial did not, however, find significant reductions in
cardiovascular events (Look AHEAD Research Group 2013). The effects of the DPP and other inter-
ventions on weight loss, as well as diabetes prevention and control, have been reviewed extensively
(Norris et al. 2005; Franz 2007; Franz et al. 2007). We refer the interested reader to these and other
reviews of large-scale randomized clinical trials, many of which have documented the effects of
lifestyle interventions on a broad array of cardiometabolic outcomes, including lipids (Aucott et al.
2011), weight-loss maintenance (Barte et al. 2010), and glycemic control in type 2 diabetes (Ismail,
Winkley, and Rabe-Hesketh 2004). A recent review and meta-analysis suggests that overweight and
obese adults with type 2 diabetes may require a more potent suite of diet-specific intervention com-
ponents, including nutritional therapies, to achieve cardiometabolic improvements and that focusing
on weight as a primary outcome in this population (e.g., >5% weight loss) might not be the optimal
strategy with which to improve glycemic control (Franz et al. 2015).
Independent and Combination Mindfulness Approaches

Mindfulness approaches involve noticing, becoming curious about, and accepting unpleasant
thoughts, rather than labeling such thoughts as dysfunctional and attempting to change them. These
techniques have been applied in the treatment of problematic overeating as independent interventions
(e.g., mindful eating for binge eating; Kristeller and Wolever 2010) and as combination approaches
(e.g., packaged with cognitive-behavioral approaches, as in dialectical behavioral therapy [DBT]
for binge eating; Telch, Agras, and Linehan 2001). There are several recent reviews of interven-
tions testing these approaches (Katterman et al. 2014; O’Reilly et al. 2014; Godfrey, Gallo, and
Afari 2015). The extent to which mindfulness approaches are more effective than existing cognitive-
behavioral approaches, or whether mindfulness approaches are most effective when administered
in combination protocols, remains unclear. In the following text, we review findings from studies
of independent mindfulness approaches and combination mindfulness approaches for problematic
overeating, as well as their effects on cardiometabolic outcomes.
Independent Mindfulness Approaches

Mindfulness, broadly defined as paying attention on purpose, in the present moment, and non-
judgmentally, is a core component of mindful eating and acceptance and commitment techniques
targeting problematic overeating (Kristeller and Wolever 2010; Forman and Butryn 2015). Such
approaches seek to strengthen abilities to become aware of and tolerate uncomfortable sensations
(e.g., food cravings) without reacting (e.g., indulging food cravings). Mindfulness protocols have
demonstrated some effectiveness in reducing binge eating. Katterman et al. (2014) systematically
reviewed mindfulness interventions targeting reductions in overeating (excluding acceptance and
commitment therapy and DBT, which do not include mindfulness training in all sessions). Seven
of fourteen eligible trials collected data on changes in binge eating using traditional self-report
measures (e.g., BES, Eating Disorder Examination Questionnaire; Gormally et al. 1982; Fairburn
and Beglin 1994). All seven trials reported reductions in binge eating among participants receiving
the mindfulness interventions. Of these seven trials, four trials included comparison groups. Two of
these four trials reported significant between-group differences in reductions in binge eating, with
the mindfulness groups showing greater improvements compared to waitlist control groups (Alberts,
Thewissen, and Raes 2012; Kristeller, Wolever, and Sheets 2014), and one showing a statistical
trend in this same direction (Daubenmier et al. 2011). In one of these trials, relative to active control
groups (i.e., CBT groups), the mindfulness groups showed no significant differences (Kristeller,
Wolever, and Sheets 2014). The last of these four trials reported a statistical trend toward greater
reductions in binge eating in the mindfulness group relative to the CBT group (Smith et al. 2008).
These data collectively suggest that mindfulness approaches may contribute to reductions in binge
eating; however, whether these approaches are more effective than CBT techniques remains uncer-
tain. We want to highlight the importance of the type of mindfulness training on eating behavior.
Combination Mindfulness Approaches

Mindfulness approaches for the treatment of overeating may prove more effective when combined
with additional intervention components (Forman and Butryn 2015), such as cognitive-behavioral
techniques and environmental adaptations. O’Reilly et al. (2014) reviewed mindfulness-based inter-
ventions for overeating, including those co-administered with other intervention techniques (e.g.,
cognitive-behavioral techniques). Of 12 eligible studies that targeted binge eating using mindful-
ness and/or acceptance techniques, 11 reported reductions in binge eating frequency and/or sever-
ity. These 11 studies tested combinations of mindfulness with cognitive-behavioral techniques
(Baer, Fischer, and Huss 2005; Leahey, Crowther, and Irwin 2008; Courbasson, Nishikawa, and
Shapira 2010; Woolhouse, Knowles, and Crafti 2012), mindful eating protocols (Dalen et al. 2010;
Kristeller, Wolever, and Sheets 2014), acceptance-based practices (Tapper et al. 2009), and combi-
nations of mindfulness exercises (Kristeller and Hallett 1999; Alberts et al. 2010). The only study
that did not report reduced binge eating used a general mindfulness-based stress reduction protocol
(Smith et al. 2006). Godfrey et al. (2015) recently conducted a systematic review and meta-analysis
of mindfulness-based interventions specifically for binge eating and included DBT, which is a CBT
intervention that includes mindfulness-based emotion regulation practices (e.g., DBT). Analysis of
the 19 included studies supported large or medium-large effects of interventions on binge eating
(within-group random effects mean Hedge’s g [a standardized measure of effect size] = −1.12, 95%
CI [−1.67, −0.80], k = 18; between-group mean Hedge’s g = −0.70, 95% CI [−1.16, −0.24], k = 7).
These data indicate that combination mindfulness approaches for the treatment of binge eating are
promising, and future research should clarify the optimal combination approach.
Mindfulness Approaches and Cardiometabolic Outcomes

In one of the largest trials to date testing the long-term effects of a mindfulness intervention on weight
loss, Daubenmier et al. (2016) randomized 194 obese adults to receive a 17-session program of diet
and exercise information with either (1) training in select cognitive-behavioral techniques (active
control group), or (2) training in mindfulness-based eating and stress reduction (mindfulness group).
Relative to the active control group, the mindfulness group lost more weight (though not a statistically
significant difference) and evidenced statistically significant reductions in fasting glucose at 18 months
post-baseline. Analyses also revealed that increases in self-reported mindful eating among participants
assigned to the mindfulness group, but not among participants assigned to the active control CBT
group, were associated with larger reductions in self-reported eating of sweet and dessert foods, as
well as larger reductions in fasting glucose levels, at 12 months post-baseline (Mason et al. 2016).
While this trial suggests that mindfulness training may impart some benefit in terms of cardiometa-
bolic health, it may require optimization to surpass the effectiveness of traditional CBT-based inter-
ventions. Alternatively, the benefits of mindfulness training may be most potent when combined with
other intervention techniques, such as specific dietary recommendations or goal-setting.
In their reviews of mindfulness interventions, Katterman et al. (2014) and O’Reilly et al. (2014)
each reported on 10 studies that included weight outcomes. The Katterman and O’Reilly reviews
included 5 common studies, meaning that taken together, these reviews reported on 15 analyses of
weight change following mindfulness interventions targeting eating behavior. Excepting one study
(Miller et al. 2012; Cohen's d [a standardized measure of effect size] = −3.29), Katterman and col-
leagues reported Cohen’s d’s ranging from −0.17 to +0.04, and the average intervention effect size
was small (Cohen’s d = 0.10, favoring greater weight loss in mindfulness groups). O’Reilly and col-
leagues reported a slightly larger average effect size (Cohen’s d = 0.19, favoring greater weight loss in
mindfulness groups). Notably, 3 of the 10 studies included in Katterman and colleagues’ review (2014)
included treatment components targeting weight loss as a specific goal of the intervention, and all three
of these interventions reported significantly greater weight loss in the mindfulness group (Dalen et al.
2010; Miller et al. 2012; Timmerman and Brown 2012). In summary, data suggest that mindfulness
approaches may be more effective in the treatment of binge eating when in combination with other
intervention components (e.g., cognitive-behavioral techniques); however, more definitive trials are
needed to test the effectiveness of such combination approaches on cardiometabolic outcomes.
CONCLUSION
We have reviewed three types of overeating and three broad categories of interventions for overeat-
ing. Here, we reviewed types and interventions for overeating separately, though these types may
co-occur and interventions may be most effective in combination. Indeed, we hypothesize that the
intervention components we reviewed in this chapter might be most effective in targeting several
types of overeating (and cardiometabolic health) when administered in combinations. We believe
that current data support referral of overweight and obese persons with increased cardiometabolic
risk factors to behavioral intervention programs (including programs that incorporate training in
mindful eating, where available), to promote weight loss and weight-loss maintenance, as well as
improvements in cardiometabolic risk factors. In settings without ongoing, high-quality group inter-
ventions, referral to a nutritionist with expertise in behavioral weight-loss strategies may be helpful.
Referral to the lay books mentioned in this chapter, which, in plain language, detail simple environ-
mental intervention techniques that can be adapted in ways that fit a variety of individual needs, may
also be helpful. Future research is needed to optimize behavioral interventions, including mindful-
ness interventions, and to ascertain the most effective combinations of behavioral and environmental
interventions for improving weight loss and weight-loss maintenance.
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3 Energy Balance and
Regulation of Body Weight
Are All Calories Equal?
Kevin D. Hall
CONTENTS
Introduction...................................................................................................................................... 51
Energy Balance as a Conceptual Framework................................................................................... 52
Regulation of Macronutrient Metabolism: Is a Calorie a Calorie?.................................................. 52
Models of Obesity That Implicate Individual Macronutrients......................................................... 53
The Carbohydrate–Insulin Model............................................................................................... 54
The Protein Leverage Model....................................................................................................... 55
The Dietary Fat Model................................................................................................................ 56
Body Weight Regulation.................................................................................................................. 56
Conclusion....................................................................................................................................... 57
Acknowledgment............................................................................................................................. 57
References........................................................................................................................................ 57
ABSTRACT
While the human body obeys the law of energy conservation, this alone does not imply that dietary
carbohydrate, fat, and protein have contributed equally to the rise in obesity prevalence over the past
several decades. Indeed, three scientific models of obesity have implicated each macronutrient as
being the prime culprit, but the evidence supporting these models is, at best, mixed and, at worst,
demonstrably false. This chapter reviews the concepts of energy balance and macronutrient balance
as applied to the human body and presents the evidence for and against three scientific models that
alternately place the blame for obesity on each dietary macronutrient. Finally, the chapter discusses
putative mechanisms for why obesity prevalence has been increasing despite the likelihood that
human body weight is actively regulated by a biological feedback control system.
INTRODUCTION
We live in an age where the developed world is becoming increasingly overweight and obese with
potentially dire consequences for population health and the economy (Swinburn et al. 2011). At
least a dozen putative causes of obesity have been proposed (Keith et al. 2006, McAllister et al.
2009), and media coverage of obesity has soared (Hilton, Patterson, and Teyhan 2012). Every com-
ments section following an Internet article about obesity includes opinions from people who have
avoided excess weight gain or have successfully lost weight and are convinced of their personal
superiority over those who have succumbed to obesity. They often make strong claims about what
really underlies obesity and how to solve it.
If your town still has a book shop, visit its nutrition and health section. You will find row upon
row of books suggesting that the reader forget everything they have been told about diet and nutri-
tion. The truth was somehow buried or unknown until now when the author will finally reveal that
51
there is a painless way to melt the pounds away without feeling hungry or deprived. The science is
surprisingly simple (and adequately covered in a couple of cursory chapters) as is the eating plan.
Unfortunately, the truth is more complicated.
This chapter will attempt to clarify some popular misconceptions about the relationships between
energy balance and body weight regulation as well as provide a brief overview of several models of obesity
that purport causal relationships between dietary macronutrients and the accumulation of excess body fat.
ENERGY BALANCE AS A CONCEPTUAL FRAMEWORK

Any imbalance between the energy derived from food eaten and the energy expended to maintain
life and perform physical work will be accounted for by changes in the body’s energy stores, pri-
marily body fat. This concept of energy balance as applied to body weight regulation is a useful
bookkeeping device and is a necessary consequence of the physical law of energy conservation.
However, energy balance is not a statement about causality and does not explain why some people
have obesity.
In particular, it does not follow from the energy balance principle that the obesity epidemic has
resulted from widespread moral failings or lack of willpower leading to population-wide increases
in gluttony and sloth. Prevention of obesity is often erroneously portrayed as a simple matter of bal-
ancing calories in with calories out and that people with obesity should simply eat less and exercise
more. Conversely, the fact that these failed concepts are incorrect does not imply that energy balance
is an erroneous or useless concept.
Rather, the energy balance principle provides important constraints that are useful for experi-
mental investigation of body weight regulation. For example, the complex biochemistry that derives
energy from oxidation of macronutrients obeys the law of energy conservation and when these
processes take place at constant pressure, as they do inside cells, the corresponding heat released
is quantified by the enthalpy of the reactions. Furthermore, Hess’s law of path independence dic-
tates that the same net enthalpy change occurs in both the bomb calorimeter and the human body
(although the reactions inside the calorimeter occur at constant volume and thermodynamics pro-
vides the means of accounting for this difference). In other words, “a calorie is a calorie” when it
comes to macronutrients being oxidized either in the calorimeter through combustion or via the
intricate biochemical pathways of oxidative phosphorylation inside cells. (Protein is incompletely
oxidized in the body and the measurements can be corrected via application of Hess’s law to account
for the enthalpy of formation of the nitrogenous end products.)
The fact that the energy derived from macronutrient oxidation in the body can be equated
to the energy derived from their combustion in a bomb calorimeter is a keystone for the study
of in vivo energy metabolism. However, this equivalence does not imply that altering dietary
macronutrients will necessarily have no effect on the body’s energy expenditure or composition.
Rather, manipulation of dietary macronutrients may alter overall calorie intake and/or expendi-
ture with a corresponding requisite change in the energy stores of the body, likely primarily body
fat changes.
Alternatively, changing dietary macronutrient composition may alter endocrine factors that
influence the propensity to accumulate body fat or direct the storage of fat to particular locations.
These possibilities do not violate the principle of energy balance since any changes in the body’s
overall energy stores must therefore also be accompanied by changes in calorie intake and/or
expenditure.
REGULATION OF MACRONUTRIENT METABOLISM: IS A CALORIE A CALORIE?

The human body has been analogized to an automobile that runs on arbitrary mixtures of three
different fuels (Hall 2010, 2012). Such a flex-fuel vehicle would allow the driver to fill the fuel
tank with whatever fuel was cheaper or more readily available, regardless of the mixture already
Energy Balance and Regulation of Body Weight 53
in the tank. Imagine the additional complexity if the vehicle was not allowed to have a fuel tank.
Rather, the vehicle itself is composed of its fuel and is continually breaking down and reconstruct-
ing its components. Furthermore, despite the daily fluctuations of fuel delivery, the composition of
the vehicle remains relatively stable and maintains similar performance characteristics, at least over
the short term.
Exactly this remarkable engineering feat is accomplished by the human body through its use of
the three dietary macronutrients (carbohydrate, fat, and protein) to both fuel metabolism and provide
substrates for body constituents. The ability to adapt to a wide variety of diets was an evolution-
ary necessity that has allowed humans to thrive across the globe in widely different environmental
conditions.
In the United States, roughly 50% of dietary energy is derived from carbohydrate, 35% from
fat, and 15% from protein (Austin, Ogden, and Hill 2011). However, these average diet proportions
can vary widely from person to person and also from day to day. Complex physiological control
mechanisms maintain normal functioning of the body despite marked fluctuations of diet quantity
and composition. However, these physiological adaptations may be imperfect and it is theoretically
possible that sustained long-term manipulations of diet composition might lead to substantial altera-
tions in body composition independent of calorie intake.
For example, isocaloric diets differing in macronutrient composition may result in preferential
partitioning of energy storage toward body fat and away from other pools such as body protein.
Such energy partitioning differences over the long term will thereby alter fat-free mass and influ-
ence energy expenditure. Dietary protein in particular is known to both influence body composition
(Leidy et al. 2015) and is thermogenic (Bray et al. 2012, Ebbeling et al. 2012, Thearle et al. 2013,
Wycherley et al. 2012).
Even with clamped dietary protein, varying the ratio of carbohydrate to fat between isocalo-
ric diets is predicted to have an effect on energy expenditure by altering metabolic flux patterns
through energy-requiring pathways such as gluconeogenesis (Veldhorst, Westerterp-Plantenga, and
Westerterp 2009) and the triglyceride fatty acid cycle (Elia et al. 1987). For example, computational
model simulations have found that wide variations in the proportion of carbohydrate to fat are
predicted to result in modest changes in total energy expenditure and body fat in a non-monotonic
pattern over a relatively narrow range (Hall et al. 2015). Thus, the nutritional aphorism that “a
calorie is a calorie” (Buchholz and Schoeller 2004) may be only approximately true and the body
achieves this near equivalence only as a result of major changes in the underlying metabolic flux
patterns that adapt to the diet variations.
MODELS OF OBESITY THAT IMPLICATE INDIVIDUAL MACRONUTRIENTS

Scientific models seek to integrate a variety of data and explain a set of observations about a sys-
tem within an overarching theoretical and mechanistic framework. Importantly, scientific models
go beyond providing putative explanations and make experimentally testable predictions that are
capable of falsifying the models.
Experimental confirmation of a model’s predictions thereby provides additional support and
repeated confirmations may eventually lead to widespread acceptance of the model as the scien-
tific standard. However, models cannot be proven to be true but are rather provisional representa-
tions of our understanding. Countering evidence may lead to model corrections or possibly outright
rejection.
Putative explanations of obesity abound (Keith et al. 2006, McAllister et al. 2009), but these are
typically derived from observed correlations and scientific models of obesity that are amenable to
experimental investigation in humans are rare. A few models of obesity have been proposed that
focus on the macronutrient composition of the diet as the cause of excess adiposity. Unfortunately,
while these macronutrient models are more amenable to scientific scrutiny, none appear to ade-
quately explain the observations.
The Carbohydrate–Insulin Model

An increased proportion of refined carbohydrates in the diet has been purported to be particularly
fattening due to the propensity of such diets to elevate insulin secretion (Ludwig and Friedman
2014, Lustig 2006, Taubes 2013, Wells and Siervo 2011). According to this “carbohydrate–insulin
model” of obesity, elevated insulin suppresses the release of fatty acids into the circulation and
directs circulating fat toward storage and away from oxidation by metabolically active tissues such
as heart, muscle, and liver. The altered fuel delivery to such tissues has been suggested to result in
adaptive decrease in energy expenditure and increased food intake (Astwood 1962, Ludwig and
Friedman 2014, Pennington 1952, 1953, Wells and Siervo 2011). Therefore, the positive energy
balance associated with the development of obesity is hypothesized to be a consequence of the
insulin-driven shift in fat partitioning toward storage and away from oxidation due to an increased
proportion of dietary carbohydrates.
The carbohydrate–insulin model of obesity has gained a following in recent years due to various
popular accounts (Ludwig 2016, Taubes 2007, 2011) and commentaries in influential medical jour-
nals (Ludwig and Friedman 2014, Taubes 2013). Some epidemiological evidence appears to support
the carbohydrate–insulin model since added sugars and sweeteners have increased in parallel to
the development of the obesity epidemic (Wells and Buzby 2008), and self-reported carbohydrate
intake has also increased (Austin, Ogden, and Hill 2011). However, correlations do not necessarily
imply causation and there is little evidence that the overall carbohydrate fraction of the available
food supply has substantially changed during the development of the obesity epidemic (Hiza, Bente,
and Fungwe 2008).
Experimentally testing the carbohydrate–insulin model requires devising a study that mechanisti-
cally evaluates the model’s predictions. For example, the carbohydrate–insulin model predicts that
reduction of dietary carbohydrates will result in decreased insulin secretion, increased fat oxida-
tion, increased energy expenditure, and greater body fat loss compared to an isocaloric reduction
of dietary fat. Many of these predictions were recently tested in a study that confined adults with
obesity to a metabolic ward where all food intake and physical activities were strictly monitored
and controlled (Hall et al. 2015). The subjects were fed a standard baseline diet representing a typi-
cal habitual diet that provided 35% fat, 15% protein, and 50% carbohydrate with about 20% of
total calories coming from sugar, and where calories were matched to participants’ energy expendi-
ture. In a randomized crossover design, diet calories were then cut by 30%, either entirely through
restricting carbohydrates, keeping protein and fat at baseline, or selectively restricting fat and keep-
ing protein, carbohydrates, and sugar at baseline.
The reduced-carbohydrate diet led to decreased insulin secretion and increased net fat oxida-
tion as predicted by the carbohydrate–insulin model. However, despite no significant change in
insulin secretion, the reduced-fat diet resulted in a significantly greater rate of fat loss than the
reduced-carbohydrate diet, which is contrary to the carbohydrate–insulin model predictions. The
small observed difference in body fat loss between the diets was detected using the metabolic bal-
ance method that is more precise than direct body composition assessment methods that were unable
to measure significant differences between the diets (Hall et al. 2015).
The reduced-carbohydrate diet also led to a significant decrease in energy expenditure, both dur-
ing sleep and throughout the day, whereas the reduced-fat diet had no significant effect on energy
expenditure (Hall et al. 2015). Again, these results are contrary to the carbohydrate–insulin model
but are in line with previous inpatient isocaloric diet studies employing clamped protein and varying
carbohydrates from 20% to 75% of total calories that have either found small decreases in energy
expenditure with lower-carbohydrate diets (Astrup et al. 1994, Dirlewanger et al. 2000, Horton et al. 1995,
Shepard et al. 2001) or no statistically significant difference (Davy et al. 2001, Eckel et al. 2006, Hill
et al. 1991, Rumpler et al. 1991, Schrauwen et al. 1997, Smith et al. 2000, Thearle et al. 2013, Treuth
et al. 2003, Yerboeket-van de Venne and Westerterp 1996). Dietary carbohydrate restriction has only
ever been observed to result in significantly increased energy expenditure if accompanied by an
increase in dietary protein (Ebbeling et al. 2012, Veldhorst, Westerterp-Plantenga, and Westerterp
2009), which is known to be thermogenic (Bray et al. 2012, Ebbeling et al. 2012, Thearle et al.
2013, Wycherley et al. 2012).
In sum, the predictions of the carbohydrate–insulin model have been experimentally demon-
strated to be false. However, this does not imply that dietary carbohydrates or insulin are unim-
portant for the development of obesity or its treatment. Diets with high amounts of insulinogenic
carbohydrates may result in greater overall energy intake and cause obesity by mechanisms quite
different from that proposed by the previous carbohydrate–insulin model, such as by increasing
palatability or decreasing satiety. Conversely, prescribing very-low-carbohydrate diets appears
to offer some short-term benefit for weight loss (Bueno et al. 2013), but this is unlikely due to
increased metabolic rate (Hall et al. 2015, Johnston et al. 2006). Rather, such diets likely reduce
appetite by promoting an increase in circulating ketones (Gibson et al. 2015), although the mecha-
nism for this effect is unclear (Paoli et al. 2015). Furthermore, low-carbohydrate diets often result
in comparatively greater protein intake that may increase satiety, decrease overall energy intake,
increase energy expenditure, and beneficially influence energy partitioning and body composition
(Leidy et al. 2015).
The Protein Leverage Model

The “protein leverage model” of obesity that postulates that the body seeks to consume a target
amount of dietary protein, and if the protein fraction of diet is lowered then overall energy intake
increases in an attempt to achieve the protein target, thereby resulting in positive energy balance and
obesity (Simpson and Raubenheimer 2005). The protein leverage model of obesity is consistent with
the observation that self-reported protein intake has decreased as a percentage of total calories since
the 1970s (Austin, Ogden, and Hill 2011), but there is little evidence that the protein fraction of the
available food supply has decreased (Hiza, Bente, and Fungwe 2008). Protein leverage may explain
why higher-protein diets are more satiating and why increased dietary protein results in improved
maintenance of lost weight (Leidy et al. 2015). However, the central tenet of the protein leverage
model has recently been tested in three randomized controlled studies (Gosby et al. 2011, Martens,
Lemmens, and Westerterp-Plantenga 2013, Martens et al. 2014) with the data from two of the three
studies countering the model predictions.
Using a randomized crossover design, a total of 137 adults with a wide range of ages and
degrees of adiposity were provided with ad libitum diets that varied the protein proportion of
energy: 5%, 15%, or 25% of calories with a constant 33% fat for three periods of 12 days each
(Martens, Lemmens, and Westerterp-Plantenga 2013, Martens et al. 2014). Contrary to the pro-
tein leverage model of obesity, the low-protein diet did not result in increased energy intake com-
pared to the moderate-protein diet, whereas the high-protein diet led to a significantly decreased
energy intake.
Another study was conducted by the originators of the protein leverage model who investigated
22 lean subjects in a randomized crossover fashion for three periods of 4 days each where the
diets had 10%, 15%, and 25% of their energy as protein (Gosby et al. 2011). That study provided
confirmatory evidence for the protein leverage model by demonstrating that the low-protein diet
resulted in a significant increase in energy intake amounting to ~260 kcal per day compared to the
moderate-protein diet, but the higher-protein diet did not result in a significant decrease in energy
intake. If sustained for decades, such an increase in energy intake with the low-protein diet could
fully account for the mean weight gain corresponding to the U.S. obesity epidemic (Hall et al. 2011).
But considering the lack of effect of decreased dietary protein in the longer-term studies (Martens,
Lemmens, and Westerterp-Plantenga 2013, Martens et al. 2014), the overall weight of the evidence
does not appear to support the protein leverage model of obesity. Nevertheless, higher-protein diets
may play an important role in weight loss interventions, maintenance of lost weight, and prevention
of obesity (Leidy et al. 2015).
The Dietary Fat Model

The dietary fat model of obesity postulates that an increased proportion of the diet coming from fat
results in elevated energy intake along with efficient storage of the excess energy as body fat (Astrup
et al. 2000, Bray and Popkin 1998). It has long been recognized that dietary fat contains more than
twice the calories per gram compared to carbohydrate and protein. Furthermore, naturally low-fat
foods contain dietary carbohydrate and protein that are associated with large amounts of water that
further decreases their energy density. Dietary fat is also less satiating than protein or carbohydrate
(Stubbs 1998) and results in a smaller increment in energy expenditure compared to isocaloric feed-
ing of dietary carbohydrate or protein (Westerterp 2004). Covert experimental increases in dietary
fat content have been demonstrated to result in increased energy intake, positive energy balance,
and accumulation of body fat (Stubbs et al. 1995a,b). Dietary fat does not directly promote its own
oxidation (Flatt et al. 1985, Schutz, Flatt, and Jequier 1989) and body fat accumulates efficiently
with added dietary fat and is relatively unopposed until adipose tissue has sufficiently expanded
such that daily lipolysis increases to an extent sufficient to elevate circulating fatty acids and bal-
ance fat oxidation with fat intake (Flatt 1988). Furthermore, decreasing the fat content of meals
leads to lower ad libitum energy intake (Rolls 2009, Williams, Roe, and Rolls 2013) and experi-
mental overfeeding of dietary fat results in greater increases in body fat compared to isocaloric
carbohydrate overfeeding (Horton et al. 1995). Therefore, several aspects of the dietary fat model
have been confirmed.
The dietary fat model of obesity, along with concerns about saturated fat being linked to increased
cardiovascular mortality, helped lead to public health messages to decrease our intake of dietary
fat (Walker and Parker 2014). Unfortunately, the food industry capitalized on this opportunity to
increase the production and marketing of highly processed, inexpensive, and convenient low-fat
foods with high sugar content that lacked the benefits of foods naturally low in fat (Moss 2013,
Roberts 2008, Swinburn et al. 2011). As a result, while self-reported fat intake decreased as a pro-
portion of calories, absolute fat intake was relatively unchanged (Austin, Ogden, and Hill 2011) and
added sugars, fats, and oils in the food supply have increased (Wells and Buzby 2008) in parallel
with the rise in obesity prevalence. Furthermore, prescriptions to reduce dietary fat have not resulted
in improved weight loss over the long term (Tobias et al. 2015). Thus, while the dietary fat model
of obesity appears to be supported by physiological studies, the epidemiological and weight loss
studies provide contrary evidence.
BODY WEIGHT REGULATION

Over the long term, all weight loss diets appear to be similarly ineffective for obesity treatment
regardless of the prescribed changes in macronutrient composition (Hu et al. 2012, Johnston et al.
2014, Tobias et al. 2015). This is likely because the body actively resists weight loss by increas-
ing appetite due to the action of feedback circuits influencing energy intake in response to weight
changes (Gautron, Elmquist, and Williams 2015, Woods and D’Alessio 2008). These adaptations
make it extraordinarily difficult to adhere to a diet over the long term (Hall 2015). Furthermore,
weight loss results in a suppression of energy expenditure beyond what would be expected based
on the body weight or composition changes alone. This phenomenon, called “metabolic adaptation”
or “adaptive thermogenesis,” has been repeatedly demonstrated in humans and both resting energy
expenditure and physical activity energy expenditure are altered in parallel (Westerterp 2013).
Therefore, achieving large sustained weight losses is difficult and weight regain is typical in the
absence of vigilant efforts to maintain behavior changes (Greenway 2015).
If both energy intake and expenditure are controlled by feedback systems that regulate body weight,
why have these systems been unable to prevent the obesity epidemic? A likely explanation is that the
feedback system is only one part of a complex neurobiological system including hedonic and cogni-
tive factors that act in concert to determine overall human food intake behavior (Berthoud 2012, Hall,
Hammond, and Rahmandad 2014). The feedback circuits were effective for body weight regulation in
the environments that were prevalent in the distant evolutionary past.
However, over the past several decades, the feedback system may have been overwhelmed by
the increased availability and marketing of inexpensive, convenient, energy-dense, palatable food
(Moss 2013, Roberts 2008, Swinburn et al. 2011). In addition, occupations have become more sed-
entary (Church et al. 2011), food has progressively become cheaper (Putnam 2000), fewer people
prepare meals at home (Lin and Guthrie 2012, Smith, Ng, and Popkin 2013), and more food is
consumed in restaurants (Lin and Guthrie 2012). Food availability has increased by approximately
750 kcal per day that is so much more than enough to explain the observed increase in body weight
that per capita food waste has increased by 50% (Hall et al. 2009).
Consider the analogy to a thermostat that attempts to control the temperature of a home through
operation of a heating and air-conditioning system (Hall, Hammond, and Rahmandad 2014).
A system that is adequately powered for the year-round temperate climate of Northern California
may be insufficient to maintain a comfortable indoor temperature of the same home in Minnesota
during the winter or in Florida during the summer. Nothing is inherently “wrong” with the heating
and air-conditioning system, it is merely underpowered for the new environment and the tempera-
ture will be maintained at a level that is different from the set point of the thermostat.
Similarly, the feedback circuits that evolved to regulate body weight eons ago may have been
overwhelmed in recent decades by the obesogenic environment of today. Whether such changes or
the elevation of body weight itself additionally resets the homeostatic set point of the body weight
“thermostat” upward is an interesting question.
CONCLUSION
While a multitude of environmental changes offer a plausible explanation for the rise in obesity
prevalence, causality has not been demonstrated. Indeed, it is difficult to imagine how one could
devise practical experimental tests of these putative explanations since such complex changes in the
environment are difficult to manipulate. In contrast, the simpler macronutrient models of obesity
discussed earlier have all been found to be contrary to some observations and, at best, incomplete.
Therefore, there has yet to be a model of obesity that has been adequately experimentally tested,
repeatedly confirmed, and commensurate with all of the observations. In other words, there is pres-
ently no scientifically accepted standard model of obesity. It is possible that the obesity epidemic
resulted from a complex confluence of multiple causes, or that a simple comprehensive model has
been too difficult to adequately experimentally investigate. What appears to be clear is that no single
macronutrient is the culprit or the cure for obesity.
ACKNOWLEDGMENT
This research was supported by the Intramural Research Program of the NIH, National Institute of
Diabetes and Digestive and Kidney Diseases.
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4 Diets for Weight Loss
George A. Bray and Patty W. Siri-Tarino
CONTENTS
Introduction.......................................................................................................................................61
Reducing Body Weight Is a Function of Reducing Calorie Intake...................................................62
Key Factors That Influence Weight Loss.....................................................................................63
Reduced-Calorie Regimens..........................................................................................................63
Does the Macronutrient Composition of Diets Influence Weight Loss?...........................................67
Low-Fat versus Low-Carbohydrate Diets....................................................................................67
Higher-Protein Diets................................................................................................................70
Low–Glycemic Index Diets.....................................................................................................71
The Mediterranean Diet..........................................................................................................71
Conclusions.......................................................................................................................................72
References.........................................................................................................................................73
ABSTRACT
This chapter has examined the use of weight loss diets in the treatment of patients with obesity. The
historical background for these diets is first reviewed noting that low-fat and low-carbohydrate diets
had appeared more than 100 years ago. Basic principles of energy balance are then briefly reviewed.
A discussion of very-low-energy (calorie) diets is followed by a discussion of the balanced deficit
diet, the low-calorie diet, the low-fat diet, the low glycemic index, and the Mediterranean-style diet.
Variability of response to each diet is characteristic and considerably larger than the average weight
difference between most if not all dietary comparisons. The best advice is to select a diet you can
adhere to and to follow your progress by periodic recording of your body weight.
INTRODUCTION
More than 2500 years ago, Hippocrates prescribed diet and exercise in the treatment of obesity
(Bray 2007). Diet was also used by Galen in Roman times and by Avicenna in the tenth century
AD when Arabic medicine was the dominant medical tradition. With the dawn of the eighteenth
century, we began to accumulate a scientific basis for understanding obesity. The “Oxygen Theory
of Metabolism” was formulated by Lavoisier in 1787 (1789) and this was followed 50 years later
by the First Law of Thermodynamics (von Helmholtz 1847) and the concept of “Energy Balance,”
which was shown to apply to animals and human beings during the latter part of the nineteenth
century (Pettenkoffer 1861; Atwater and Benedict 1902).
Modern diets can be dated to 1863 when William Banting (1864), an undertaker, published his
astounding 50-pound weight loss using a low-carbohydrate diet prescribed by his physician William
Harvey (1872). At around the same time, restriction of fat along with a significant reduction in calo-
ries was espoused by the teacher and scholar von Noorden (1903). Thus, variations in macronutrient
composition for weight reduction became part of medically prescribed programs for patients with
obesity more than 100 years ago. Effective treatments for obesity were particularly sought after
given the association of excess weight with shortened life expectancy (Bray 2007) and the shifts of
patterns of disease from infection and tuberculosis in the early twentieth century to chronic diseases
such as heart disease, cancer, hypertension, and diabetes by the end of World War II.
61
In the 1970s, the U.S. Senate Select Committee on Nutrition held hearings of “hunger” and pub-
lished reports that highlighted the detrimental effects of the American diet on the rising incidence
of noninfectious chronic diseases, that is, heart disease, diabetes, and cancer (U.S. Senate 1977;
Bray 2007). The first Dietary Guidelines advising low-fat diets for cardiovascular health were sub-
sequently issued in 1980. In part, the advice for low-fat diets was to reduce calorie intake given that
fat contains 9 calories per gram, whereas carbohydrates and protein provide 4 calories per gram.
Although the prevalence of cardiovascular disease (CVD) has decreased over the last decades,
there has been a concomitant increase in obesity and disorders related to obesity (Bray 2011; Bray
and Bouchard 2014). The increase in obesity and cardiometabolic abnormalities has been due to
a combination of lifestyle and environmental changes. These include decreased physical activity
and increased consumption of calories largely due to increased portion sizes (Scully 2014) and
heightened palatability, low cost, increased use of processed and convenience foods (Moss 2013),
and sugar-sweetened soft drinks (Bray and Popkin 2013) that are energy dense but nutritionally
void. Dietary approaches to combating obesity fundamentally rely on caloric restriction. Various
strategies to accomplish this goal are described in this chapter. Whether and how the macronutrient
composition of weight loss diets affects the magnitude of weight loss and cardiometabolic param-
eters is also considered.
REDUCING BODY WEIGHT IS A FUNCTION

OF REDUCING CALORIE INTAKE
Reducing calorie intake below what is needed to maintain weight leads to weight loss (Bray
2011; Hall et al. 2011; Jensen et al. 2014; Apovian et al. 2015; Thomas et al. 2015). It is clear that
Americans are eating more on average now compared to 25 years ago (Putnam et al. 2002; Scully
2014). In the Diabetes Prevention Program with over 1000 individuals randomized to a lifestyle
program and a similar number to a placebo control group, reduction in calorie intake was the major
predictor of weight loss (Diabetes Prevention Program 2004).
Effective weight loss programs can decrease body weight by 8% of baseline (Jensen et al. 2014).
The rate of weight loss is initially more rapid and then falls off in a logarithmic fashion as weight
is lost. Weight loss has been modeled in at least three publications and the expected weight loss
over time for a predicted energy deficit of 500 kcal/day is depicted in Figure 4.1 with each of these
computer-programmed weight loss patterns. Note that for the first 3–6 months, weight loss is almost
“linear” but gradually tapers off until a new plateau is reached (Thomas et al. 2014).
Weight loss by computer simulation

0
–5
Weight loss (kg)
Thomas
–10
Hall
Antonetti
–15
Wishnosky
–20
0 2 4 6 8 10 12
Months
FIGURE 4.1 Rate of weight loss predicted from Computer Models.

Diets for Weight Loss 63
Key Factors That Influence Weight Loss

There is a range of weight loss with all diets. This is shown for individuals in the POUNDS Lost
study, one of the clinical trials of diet composition for weight loss (Sacks et al. 2009). Several fac-
tors predict this difference in response. The first is the initial rate of weight loss (Unick et al. 2015).
In the Look AHEAD study, individuals in the highest tertile of weight loss at 1 and 2 months in the
program had nearly twice the weight loss at 4 and 8 years as those in the lowest tertile of initial
weight loss. Thus, the initial rate of weight loss is important.
A second factor predicting weight loss for any diet is the adherence to that diet. Several stud-
ies demonstrate this important factor, which is shown for the POUNDS Lost trial in Figure 4.2a
(Dansinger et al. 2005; Alhassan et al. 2008; Sacks et al. 2009; Wadden et al. 2009). Another ele-
ment of adherence, assessed in the Look AHEAD trial, was the degree to which individuals in the
lifestyle weight loss program trial used meal replacements offered at the beginning of the program.
Usage of meal replacements was positively associated with greater weight loss, as was attendance at
the scheduled sessions and the amount of physical activity reported by the participants.
Genetic variation can also influence weight loss and metabolic responses to a diet. This has
been shown for both the Diabetes Prevention Program (Florez et al. 2012; Papandonatos et al.
2015) and the POUNDS Lost Trial (Qi et al. 2012, 2013; Zhang 2012a,b,c; Lin et al. 2015; Xu
et al. 2013, 2015; Mirzaei et al. 2014; Zheng et al. 2015). Table 4.1 summarizes the effects of
the high- versus low-fat diets and high- versus average-protein diets in the POUNDS Lost study,
and the effect of differing alleles of the tested genes on such factors as blood pressure, change
in low-density lipoprotein–cholesterol (LDL-C), change in high-density lipoprotein–cholesterol
(HDL-C), weight loss, insulin sensitivity, and energy expenditure. Using genetic profiles may
be of value in the future in developing personalized medicine for the management of obesity.
Several examples of genetic influences on the response to diet in the POUNDS Lost study are
shown in Figure 4.3. Interestingly, both APOA5 alleles and LIPC alleles affected the changes in
LDL-C between individuals eating low- and high-fat diets (Zhang et al. 2012a; Xu et al. 2015).
The NPY gene also influenced the response of blood pressure to the diets that were eaten (Zhang
et al. 2012c).
Reduced-Calorie Regimens
Very-low-calorie diets (very-low-energy diets) have energy levels between 200 and 1000 kcal/day.
The theory behind them is that the lower the energy intake, the more rapid the weight and fat loss.
Most weight loss diets can produce a decline in energy expenditure with a drop in triiodothyronine
0
30
–1
20 65/15/20
–2
Weight change (kg)
10 55/25/20
Weight loss (kg)
–3 45/15/40
0
35/25/40
–10 –4
–20 –5
–30 –6
–40
–7
–50
0 10 20 30 40 50 60 0 6 12 18 24
(a) Number of sessions attended (b) Months
FIGURE 4.2 Effect of program attendance (a) and diet composition (b) on weight loss in the POUNDS Lost
study. (a: Sacks, F.M. et al., New Engl. J. Med., 360(9), 859, February 26, 2009, adherence data.)
TABLE 4.1
Genetic Markers in the POUND Lost Study That Modified Response to a
Low-Carbohydrate or Low-Fat Diet
Author Year Gene Changes Observed
Qi 2012 GIPR The GIPR that stimulates insulin release potently in the presence of elevated
glucose. The T allele (rs2287019) in those assigned the low-fat diet was associated
with more weight loss and greater decreases in fasting glucose, fasting insulin, and
HOMA-IR. Thus, GIPR rs2287019 T-allele carriers may obtain more weight loss
and improvement of glucose homeostasis than those without this allele by
choosing a low-fat diet.
Zhang 2012 APOA5 Apolipoprotein A5 is an important determinant of plasma triglyceride levels. In the
low-fat (20% energy as fat) diet, carriers of the G allele exhibited greater
reductions in TC and LDL-C than noncarriers. In the high-fat diet group (40% fat
energy), participants with the G allele had a greater increase in HDL-C than did
participants without this allele. Thus, there was more improvement in lipid profiles
from long-term low-fat diet intake in the APOA5 G-risk allele.
Zhang 2012 FTO The FTO is associated with obesity. There were significant modifications of 2-year
changes in fat-free mass, whole body % fat mass, total adipose tissue mass,
visceral adipose tissue mass, and superficial adipose tissue mass by FTO genotype
and dietary protein. Carriers of the risk allele had a greater reduction in weight,
greater change in body composition, and fat distribution in response to a
high-protein diet, whereas an opposite genetic effect was observed in the
low-protein diet. There were also significant interactions observed at 6 months.
Thus, a high-protein diet may be beneficial for weight loss and improvement of
body composition and fat distribution in individuals with the risk allele of the FTO
variant rs1558902 (see Figure 4.5).
Qi 2013 IRS-1 The IRS is a link between the insulin receptor and intracellular activity. Among
participants with the A allele, the reversion rates of the metabolic syndrome were
higher in the high-fat diet group than in the low-fat diet group over the 2-year
intervention (P = 0.002), while no significant difference between diet groups was
observed among those without A allele (P = 0.27). High-fat weight-loss diets
might be more effective in the management of the metabolic syndrome.
Xu 2013 PPM1K This factor is essential for the regulation and nutrient-induced activation of the
branched-chain alpha-keto acid dehydrogenase complex, which catalyzes the
breakdown of branched-chain amino acids (BCAA). In the high-fat diet group, the
C allele was related to less weight loss and a smaller decreases in serum insulin
and HOMA-IR, whereas an opposite effect of genotype on changes in insulin and
HOMA-IR was observed in the low-fat diet group. At 2 years, the gene–diet
interactions remained significant for weight loss (P = 0.008) but became null for
changes in serum insulin and HOMA-IR due to weight regain. The C allele of
BCAA/AAA ratio may benefit less in weight loss and improvement of insulin
resistance than those without this allele when eating a high-fat diet.
Mirzaei 2014 CRY2 Both CRY2 and melatonin-2 receptor 1B are involved in the circadian rhythms of
MTNR1B plants and animals. We found significant associations of the CRY2 rs11605924
genotype with changes of RQ, RMR, and RMR/Kg, and of the MTNR1B
rs10830963 genotype with RQ by the 2-year intervention. In addition, we
observed significant modification effects of dietary fat on RQ changes for both
SNPs. Our data indicate that genotype of glucose- and circadian-related loci CRY2
and MTNR1B might affect long-term change in energy expenditure, and that
dietary fat intake might modify the genetic effects (www.clinicaltrials.gov;
NCT00072995).
(Continued)
TABLE 4.1 (Continued)

Genetic Markers in the POUND Lost Study That Modified Response to a
Low-Carbohydrate or Low-Fat Diet
Author Year Gene Changes Observed
Zheng 2015 FTO FTO is associated with obesity. The A allele was associated with a greater decrease
in food cravings among the participants with high-protein intake (P = 0.027), but
not in those in the low-protein diet group (P = 0.384). Weight regain from 6 to
24 months attenuated the gene–protein interactions. Protein intakes did not modify
the FTO genotype effects on other appetite measures. Individuals with the FTO A
allele might obtain more reduction in food cravings by choosing a hypocaloric,
higher-protein weight-loss diet.
Xu 2015 LIPC Hepatic lipase (HL) plays a pivotal role in the metabolism of HDL-C and LDL-C.
Common variants in HL gene (LIPC) are associated with HDL-C. After 2 years of
dietary intervention, dietary fat modified the genetic effects of this gene on serum
TC, LDL-C, and HDL-C. In the low-fat diet group, the A allele was related to a
decrease in TC and LDL-C levels while an opposite genetic effect was found in the
high-fat diet group. Additionally, the A allele was associated with less increased
levels of HDL-C in the low-fat group but not in the high-fat group.
Lin 2015 NPY NPY is a potent stimulator of food intake. The C allele (rs16147) was associated with
a greater reduction in WC at 6 months. In addition, the genotypes showed a
statistically significant interaction with dietary fat in relation to WC and
subcutaneous adipose tissue: the association was stronger in individuals with high-fat
intake than in those with low-fat intake. At 24 months, the association remained
statistically significant for WC in the high-fat diet group (P = 0.02), although the
gene–dietary fat interaction became nonsignificant. In addition, we found statistically
significant genotype–dietary fat interaction on the change in total abdominal adipose
tissue, visceral adipose tissue, and subcutaneous adipose tissue at 24 months: the
rs16147 T allele appeared to associate with more adverse changes in the abdominal
fat deposition in the high-fat diet group than in the low-fat diet group. Our data
indicate that the NPY rs16147 genotypes that affect the change in abdominal
adiposity in response to dietary interventions were modified by dietary fat.
Notes: GIPR, glucose-dependent insulinotropic polypeptide receptor; PPM1K, protein phosphatase Mg/Mn 1K; IRS-1, insu-
lin receptor substrate-1; CRY2, cryptochrome 2; MTNR1B, melatonin receptor 1B; MC4R, melanocortin-4 receptor;
FTO, fat mass and obesity-associated gene; NPY, neuropeptide Y; TC, total cholesterol; WC, waist circumference;
SNP, single nucleotide polymorphism; HOMA-IR, homeostatic model assessment-insulin resistance.
and leptin (Sumithran et al. 2011). Treatment with leptin only partially corrects these changes
(Rosenbaum et al. 2008), unless an individual is leptin deficient. Although these regimens result in
rapid weight loss, the ability to maintain the weight loss beyond 6 months relative to counseling is
attenuated (Dansinger et al. 2007). Furthermore, very-low-calorie approaches have been associated
with an increased risk of gallstones compared to low-calorie approaches (Bray 2011), and therefore
providers have been advised to recommend these diets in limited circumstances and under close
medical supervision.
Diets that reduce carbohydrate, protein, and fat, the so-called balanced-deficit diets or prudent
diets, have been widely used in treating obesity. These diets reduce caloric intake by 500–750
kcal/day with a lower limit of energy intake usually set at 1200 kcal/day (Avenell et al. 2004).
In a meta-analysis of low-calorie diets, Avenell and colleagues (Avenell et al. 2004) found that
after 12 months the difference between control and treated groups was 5.31 kg (95% CI, −5.86 to
−4.77 kg) favoring the diets. In another systematic review of 16 studies that used diet and that had
more than 100 subjects in each group and a duration of more than 1 year, weight loss after 2–3 years
66
NPY genotype and change in BP NPY genotype and change in BP LIPC genotype and change in LDL-C
with low-fat diet with high-fat diet with low-fat diet
2 4 0
TT TC CC TT TC CC –5
0 2
–10
0 –15
–2
–2 –20
–4 –25
–4 –30 AG GG AA
Change in BP (mm Hg)
Change in SBP (mm Hg)

Change in LDL-C (mg/dL)
–6 –6 –35
0 6 12 18 24 0 6 12 18 24 0 6 12 18 24
(a) Months (b) Months (c) Months
LIPC genotype and change in LDL-C APOA5 genotype and LDL-C response APOA5 genotype and HDL-C response
with high-fat diet to low-fat diet to diet after 2 years
0
20 15
AA AG GG –10
CC CG GG
15 10
–20
10 CC CG GG
–30 5
5 –40 0
0 –50 –5

6 12 18 24 –60

Change in HDL-C (mg/dL)
–5 0 6 12 18 24 –10
Low fat High fat
(d) Months (e) Months (f )
FIGURE 4.3 Effect of genes on weight loss patterns in the POUNDS Lost trial: Neuropeptide Y, LIPC (hepatic triglyceride lipase), and APOA5 (apolipoprotein A-V).
(a and b) Effect of NPY genotypes Zhang et al. on systolic (a) and diastolic (b) BP in response to high- and low-fat diets; (c and d) Effect of LIPC Xu et al. on change
in LDL-cholesterol in response to high- and low-fat diets; (e and f) Effect of APOA5 genotypes Zhang et al. on response of HDL-cholesterol to high- and low-fat diets.
Nutrition and Cardiometabolic Health
was usually <5 kg below baseline (3.5 ± 2.4 kg; range, 0.9–10.0 kg) and after 4–7 years where there
were data, it was still 3.6 ± 2.6 kg below baseline.
Portion-controlled diets or meal replacements are one way of achieving a caloric deficit (Wadden
et al. 2011). This can be done most simply by using individually packaged foods. Frozen low-calorie
meals containing 250–350 kcal/package can be a convenient way to do this, except for the high salt
content of many of these foods. In one 4-year study, this approach resulted in early initial weight
loss, which then was maintained (Flechtner-Mors and Ditschuneit 2000; Ditschuneit and Flechtner-
Mors 2001).
Finally, intermittent or alternate-day fasting regimens (as detailed in Chapter 27) combine days
of eating at “normal” consumption levels with days of caloric restriction, so that overall calorie
consumption over a weeklong period is reduced. These dietary patterns represent an effective
weight loss strategy, although studies to date have been limited by sample size (Varady et al. 2013).
Intermittent fasting has further been shown to improve fasting triglyceride (TG) without effects
on LDL-C or HDL-C concentrations. However, the TG lowering effect may not be independent of
weight loss, and there have been no studies to date that have tested this effect. Studies evaluating
long-term effects of alternate-day fasting regimes are also lacking.
DOES THE MACRONUTRIENT COMPOSITION

OF DIETS INFLUENCE WEIGHT LOSS?
Whether the macronutrient composition of diets influences the magnitude of weight loss over a short
or long term has been the subject of many research studies. In addition to their potential impact on
weight loss, diets with different macronutrient composition in the context of weight loss and main-
tenance may also affect cardiometabolic risk parameters. This section reviews the evidence related
to these topics.
Low-Fat versus Low-Carbohydrate Diets

A rationale for the potential benefit of the low-carbohydrate diet is found in the carbohydrate–insulin
hypothesis (see Chapter 3). Several randomized clinical trials lasting 1 year (Brehm et al. 2003;
Foster et al. 2003; Samaha et al. 2003; Stern et al. 2004; Yancy et al. 2004; Dansinger et al. 2005;
Gardner et al. 2007; Brinkworth et al. 2009 [Figure 4.4a]) or 2 years (Shai et al. 2008 [Figure 4.4b];
Sacks et al. 2009 [Figure 4.3b]; Foster et al. 2010) have tested this hypothesis in head-to-head trials
0 0
–2 –2
Weight loss (kg)
Weight loss (kg)
–4 –4
–6 –6
–8 WW –8 Low fat
Zone
Atkins Mediterranean
–10 –10
Ornish Low carobhydrate
–12 –12
0 2 4 6 8 10 12 0 3 6 9 12 15 18 21 24
(a) Months (b) Months
FIGURE 4.4 Comparison of diets over 1 year and 2 years. (a) Trial conducted by Dansinger et al. comparing
the Atkins, Ornish, Weight Watchers, and Zone Diets; Participants were both male and females; (b) Trial com-
paring a low-fat diet, the Atkins diet, and a Mediterranean-style diet; The low carbohydrate diet was the Atkins
Diet; participants were predominantly males. (a: Dansinger, M.L. et al., JAMA, 293(1), 43, 2005; b: Shai, I.
et al., New Engl. J. Med., 359(3), 229, 2008.)
with low-carbohydrate or low-fat diets (Figure 4.4). In the first of these comparisons, 169 obese indi-
viduals were randomized to one of four popular diets, including the Atkins Diet (2002), The Ornish
Diet (1993), the Weight Watchers Diet, and the Zone Diet (Dansinger et al. 2005). At the end of
12 months, each diet produced weight loss of about 5 kg, but there was no difference between diets.
Adherence to the diet was the single most important criterion of success in this trial, and the Atkins
and Ornish diets were more difficult to adhere to. In a second 1-year trial, the Atkins, Zone, Ornish,
and LEARN diets were compared in a group of premenopausal women (Gardner et al. 2007). This
trial found that the Atkins diet produced more weight loss at 6 and 12 months compared to the other
three diets, which had similar results. In this study, too, a post hoc analysis showed that adherence
was the best predictor for weight loss and that the level of adherence was not very good for any diet
(Alhassan et al. 2008). Two reasons are proposed for the divergent outcomes. First, the study by
Gardner et al. (2007) had a more homogeneous population, including only premenopausal women.
Second, the Gardner study was larger and thus had more statistical power to detect differences.
The POUNDS Lost study was conducted at two sites in the United States (Sacks et al. 2009).
A total of 811 men and women were randomized to one of four diets, and 80% of them provided
weights at the end of 2 years. The two-by-two factorial comparison allowed for the assessment of
low versus high protein, low versus high fat, and lowest versus highest carbohydrate on body weight
loss. The four diets were composed as follows: (1) 20% fat, 15% protein, 65% carbohydrate;
(2) 20% fat, 25% protein, 55% carbohydrate; (3) 40% fat, 15% protein, 45%carbohydrate; or (4)
40% fat, 25%protein, 35% carbohydrate. The foods used to prepare diet plans for all four diets were
the same with variation of the specific quantities. At the end of 6 months, 12 months, or 2 years, the
weight loss was similar for all 4 diets (Figure 4.3b), supporting the concept that macronutrient com-
position is not a key determinant in weight loss success. However, two caveats should be considered
when evaluating the data from POUNDS Lost and all other diet trials. These include (1) adherence
to the dietary protocol is a key predictor of both the success and magnitude of weight loss and
(2) the consistent inability across almost all longer-term studies to achieve the protocol-specified
differential in macronutrients. The important message is that “Significant weight loss was observed
with any prescribed low-carbohydrate or low-fat diet” (Johnston et al. 2014).
The technique of meta-analysis has been used to amalgamate the findings of various clinical studies
evaluating popular diets. In a 2014 meta-analysis of 59 eligible articles reporting 48 unique randomized
trials of “named” diets (including 7286 individuals) and compared with no diet, the largest weight losses
were associated with low-carbohydrate diets (8.73 kg [95% confidence interval {CI}, 7.27–10.20 kg]
at 6-month follow-up and 7.25 kg [95% CI, 5.33–9.25 kg] at 12-month follow-up) and low-fat diets
(7.99 kg [95% CI, 6.01–9.92 kg] at 6-month follow-up and 7.27 kg [95% CI, 5.26–9.34 kg] at 12-month
follow-up) (Johnston et al. 2014). However, as the authors note, “weight loss differences between indi-
vidual diets were minimal.” We evaluated changes in lipid profiles among diets that provided data at
1-year follow-up (Table 4.2) and observed that the greater weight loss that occurred with Jenny Craig
and Weight Watchers compared to control was associated with improvements in lipids, that is, decreased
LDL-C and increased HDL-C, only in the Jenny Craig group. This may have been due to the greater
magnitude of weight lost (8% of body weight versus 4 kg in Jenny Craig versus Weight Watchers). In
studies that achieved comparable weight loss, there was either no difference in lipid profiles (Ornish
versus Zone, Weight Watchers, or Atkins; Volumetrics versus low-fat) or in a few cases, an improvement
in TG and HDL-C (with lower-carbohydrate Atkins diets versus low-fat diets) or less reduced LDL-C
and HDL-C (low glycemic Zone versus low-fat). Generally, lower-carbohydrate diets are associated
with decreased TG and increased HDL-C and lower-fat diets are associated with reduced LDL-C, but
these differences did not persist at 2 years, unless the weight differential was maintained (Bazzano
et al. 2014). How these improvements in lipids in the context of weight loss maintenance correspond to
overall improved cardiovascular health is not known. In fact, current evidence is lacking for long-term
reductions in CVD or mortality from diet-induced weight loss (Look AHEAD 2013; Kritchevsky et al.
2015; Langland 2015), and benefits to all-cause and CVD mortality have only been demonstrated with
improvements in cardiorespiratory fitness in obese patients (Lee et al. 2012).
TABLE 4.2
Changes in Plasma Lipids in Response to Several Popular Diets
Diet Comparator Achieved TC LDL-C TG HDL-C TC:HDL Non-HDL
Weight Loss
Atkins Low fat = = = ↓ ↑
(low carb)
Zone (low GI)a Low fat = Less = Less
reduced reduced
Weight Self-help ↑ = = = =
Watchers
Ornish Atkins = = = = = =
Zone
Weight Watchers
Jenny Craig Usual care ↑ = ↓ = ↑
Volumetrics Low fat = = = = = ↓
(low fat + FV)
Sources: Foster, G.D. et al., Ann. Intern. Med., 153(3), 147, 2010, Ebbeling, C.B. et al., JAMA, 97(19), 2092, 2007;
Heshka, S. et al., JAMA, 289(14), 1792, 2003; Dansinger, M.L. et al., JAMA, 293(1), 43, 2005; Rock, C.L.,
Obesity (Silver Spring), 15(4), 939, 2007; Ello-Martin, J.A., Am. J. Clin. Nutr., 85(6), 1465, 2007.
TC, total cholesterol; GI, gastrointestinal; FV, fruits and vegetables.
a 18-month intervention; data not reported colored in gray.
In another meta-analysis by Tobias et al. (2015), among weight loss interventions of similar
intensity, low-carbohydrate interventions led to significantly greater weight loss than did low-fat
interventions (18 comparisons; weighted mean difference [WMD], 1.15 kg [95% CI, 0.52 to 1.79];
I2 = 10%), in line with another meta-analysis by Bueno et al. (2013) that showed greater weight
loss with very-low-carbohydrate ketogenic diets, that is, less than 50 g/day, compared to low-fat
diets (WMD, 0.91 kg [95% CI, 0.17–1.65]). Although statistically significant, the greater weight
loss observed was quite small on an absolute scale, that is, a ~1 kg weight loss corresponds to ~1%
weight reduction in a 100 kg individual. When low-fat interventions were compared to higher-fat
interventions, this did not lead to differences in weight change (19 comparisons; WMD, 0.36 kg
[−0.66 to 1.37]). Greater weight decreases were observed only when low-fat diets were compared
with a usual diet (eight comparisons; −5.41 kg [−7.29 to −3.54]). In weight loss trials, higher-fat
weight loss interventions led to significantly greater weight loss than low-fat interventions when
groups differed by more than 5% of calories obtained from fat at follow-up (18 comparisons; WMD,
1.04 kg [95% CI, 0.06–2.03]), and when the difference in serum TGs between the two interventions
at follow-up was at least 0.06 mmol/L (~5 mg/dL) (17 comparisons; 1.38 kg [0.50–2.25]). These
findings suggest that the long-term effect of low-fat diet interventions on body weight depends on
the intensity of the intervention in the comparison group, which is probably a function of adherence.
Similarly, in the Women’s Health Initiative, a greater weight loss of ~2 kg was observed in women
who adhered to the low-fat diet compared to those who did not (Howard et al. 2006). Although these
study conclusions contrast with a systematic analysis by Hooper et al. (2012), which reported that
low-fat diets were more effective for weight loss (1.57 kg [95% CI, 1.16–1.97]) compared to other
diet interventions, the Hooper analysis included studies that were less than 1 year in duration and
excluded studies designed for weight loss; their derived risk estimate was thus overweighted with
trials that considered low-fat diets versus usual diets (Hooper et al. 2012). Furthermore, as with the
earlier meta-analyses that showed small but statistically significant weight loss with low-carbohy-
drate versus low-fat diets, the absolute amount of weight loss in the Hooper analysis showing benefit
of low-fat diets, that is, ~1.57 kg, was small.
A number of commercial weight loss programs have now published enough data to make com-
parisons possible (Gudzune et al. 2015). In a meta-analysis, Gudzune et al. reported that Weight
Watchers participants achieved at least 2.6% greater weight loss at 12 months than those assigned to
control/education. Jenny Craig resulted in at least 4.9% greater weight loss at 12 months than con-
trol/education and counseling. Nutrisystem resulted in at least 3.8% greater weight loss at 3 months
than control/education and counseling. Very-low-calorie programs (Health Management Resources,
Medifast, and OPTIFAST) resulted in at least 4.0% greater short-term weight loss than counsel-
ing, but some attenuation of effect occurred beyond 6 months when reported. Atkins resulted in
0.1%–2.9% greater weight loss at 12 months than counseling (Gudzune et al. 2015). The investiga-
tors concluded that clinicians might refer patients to Jenny Craig or Weight Watchers, the two most
efficacious programs, for weight loss. Among self-directed programs, the Atkins program was the
most effective for weight loss at 6 and 12 months compared to no dieting.
The impact of these commercial diets on CVD risk factors has also been examined by this
group (Mehta et al. 2016). They included 27 randomized controlled trials in their analysis. At
12 months, Weight Watchers and Jenny Craig showed little difference on blood pressure or lipids
as compared to control/education or counseling. In contrast, Atkins’ participants achieved the same
or better results than counseling with respect to blood pressure and TGs. This greater effect of the
Atkins group may reflect the reduction of carbohydrate intake that will lower TGs whether or not
there is weight loss and that might also reduce blood pressure through the reduction of circulating
blood volume as liver glycogen is depleted. All other programs lacked long-term studies that evalu-
ated intermediate CVD outcomes.
Nonetheless, several short-term metabolic studies lend insight into the role of dietary fat versus
carbohydrate on cardiometabolic profiles in the context of weight loss and stability. In a controlled
dietary intervention study, carbohydrate restriction from 54% to 26% of total energy in the con-
text of low-saturated-fat diets was associated with a greater improvement in lipoprotein markers of
CVD risk (Krauss et al. 2006). Thus, in persons who do not desire or are unable to achieve weight
loss, carbohydrate restriction may represent an intervention that effectively improves these lipid
parameters.
A recent randomized crossover study of 19 obese volunteers confined to a metabolic ward for
2 weeks on two occasions separated by 4–6 weeks evaluated the effects of carbohydrate versus fat
restriction on fuel oxidation and total body fat loss (Hall et al. 2015). After 6 days on a balanced
diet (35% fat, 15% protein, 50% carbohydrate), energy intake was reduced 30% by removing either
carbohydrate or fat intake. Although it has been proposed that low-carbohydrate diets result in lower
total body fat due to increased fat oxidation (Ludwig and Friedman 2014), the study by Hall et al.
did not show increased fat loss, in spite of body weight reductions, when people consumed low-
carbohydrate diets. The significant caveat of this study was its short duration, that is, 6 days; thus,
the data provided by this study cannot be used to support or refute the hypotheses related to mac-
ronutrient intake effects on fuel oxidation. Rather, this short-term metabolic ward study highlights
the energetics required to replace dietary carbohydrate with glucose and β-OH butyrate from endog-
enous protein and fat, respectively, during acute metabolic adaptations.
Higher-Protein Diets
Higher-protein diets may be beneficial in managing weight loss (Leidy et al. 2015). Figure 4.5a
shows that individuals assigned to a high-protein diet in the POUNDS Lost study and who adhered
to that diet during the 2 years of intervention lost progressively more weight (Sacks et al. 2009).
A second 2-year study compared 12% and 25% protein diets eaten as part of a 30% fat diet
(Skov et al. 1999; Due et al. 2008). Weight loss over 36 weeks was substantially greater with the
higher-protein diet and this difference was maintained at 56 weeks but not at 104 weeks (Figure
4.5b). A meta-analysis of energy-restricted, high-protein low-fat diets compared to standard-protein
low-fat diets by Wycherley et al. (2012, 2013) showed a borderline significant effect of the higher-
protein diets on body weight in trials lasting over 12 weeks (−0.97 kg [95% CI, −2.07 to 0.13]) and
90
High protein diet 12% protein
88
0 86 25% protein
Body weight (kg)

84
82
2 year weight
change (kg)
80
–5 78
76
74
–10 72
70
Protein (%) 15.3 18.2 20.3 22.0 25.6 0 12 24 36 48 60 72 84 96 108
Sessions attended 41.6 48.5 55.0 58.4 59.3 Weeks
(a) (b)
FIGURE 4.5 Effect on weight loss of adhering to a high-protein diet (a) and a high- and low-protein diet on
a low-fat diet background diet (b). (a: Sacks, F.M. et al., New Engl. J. Med., 360(9), 859, February 26, 2009;
b: Skov, A.R. et al., Int. J. Obes. Relat. Metab. Disord., 23(5), 528, 1999; Due, A. et al., Int. J. Obes. Relat.
Metab. Disord., 28(10), 1283, 2004.)
in trials lasting less than 12 weeks (−0.79 kg [95% CI, −1.34 to 0.37]). Fat mass, however, declined
significantly more in the high-protein groups in trials lasting less than 12 weeks and in those last-
ing more than 12 weeks (−0.83 kg [95% CI, −1.31 to −0.34]) favoring high protein. The decrease
in TGs also favored the higher-protein diets (0.23 mmol/L [95% CI, −0.36 to −0.11]) as did the
increase in HDL-C (0.61 mmol/L [95% CI, 0.20–1.02]). In the trials lasting less than 12 weeks,
resting energy expenditure increased more in those eating the high-protein diet (595 kJ/day [95%
CI, 66.95–1124.05]). This would be similar to the effects observed with the measurement of energy
expenditure during overfeeding in response to different levels of dietary protein (Bray et al. 2012).
In a second meta-analysis by Schwingshackl and Hoffmann comparing low versus high glycemic
index/load, fasting insulin was again the main difference in response (−0.71 µU/mL [95% CI, −1.36
to −0.05]) favoring the low–glycemic index/load diet (Schwingshackl and Hoffmann 2013a).
Low–Glycemic Index Diets

The glycemic index is based on the rise in blood glucose in response to the test food compared to
the rise after a 50-g portion of white bread; glycemic load is the product of glycemic index and
amount of carbohydrate in the food. The effect of low–glycemic index/load diets on weight loss
has been studied in a number of randomized clinical trials in adults (Schwangshakl and Hoffman
2013b). Thomas et al. (2007) identified six studies including 202 participants that met their inclu-
sion criteria. Three of these studies compared low–glycemic index/load diets with higher–glycemic
index/load diets, while the other three compared an ad-lib low–glycemic index/load diet with a
conventional energy-restricted low-fat diet, or an energy-restricted low–glycemic index/load diet
with a normal energy-restricted diet. Interventions were relatively short ranging from 5 weeks to
6 months at the longest. There was a small significant difference in body weight of 1.1 kg (95% CI,
−2.0 to −0.2) that favored the low–glycemic index/load diets. The body mass decreased by 1.1 kg
(P < 0.05) and fat mass by a similar amount compared to the change of weight in the control diet
group. Both total cholesterol and LDL-C fell more with the low–glycemic index/load diets (Sacks
et al. 2014).
The Mediterranean Diet

The Mediterranean diet—generally characterized by high intakes of fruits, nuts, vegetables, whole-
grain cereals, and olive oil, with moderate consumption of fish, poultry, and wine, and low intake of
dairy, red meats, and sweets—has been compared to low-carbohydrate and low-fat diets for weight
loss efficacy. One 2-year study was conducted at a single site in Israel with a worksite population
that was predominantly (83%) male (Shai et al. 2008). During the rapid weight loss phase, the
very-low-carbohydrate diet group lost the most weight, with the Mediterranean diet and low-fat diet
groups having similar results. In the next 6 months, there was an acceleration in weight loss in the
Mediterranean diet group to reach the very-low-carbohydrate diet group. At the end of 2 years, the
mean weight loss was 2.9 kg for the low-fat group, 4.4 kg for the Mediterranean diet group, and
4.7 kg for the low-carbohydrate group (P < 0.001 for the interaction between diet group and time);
among the 272 participants who completed the intervention, the mean weight losses were 3.3, 4.6,
and 5.5 kg, respectively.
Two meta-analyses of the Mediterranean diet and weight loss have been published (Nordmann et al.
2011; Mancini et al. 2015). In the analysis of Mancini et al. that included five randomized clinical
trials (n = 998) of at least 12 and up to 48 months in duration, greater weight loss compared to the
low-fat diet but not the other comparator diets was observed. Similar effects of all diets on lipid
levels and blood pressure were observed. The meta-analysis by Nordmann et al. (2011) identified
6 trials, including 2650 individuals (50% women). After 2 years of follow-up, individuals assigned
to a Mediterranean diet had more favorable changes in WMDs of body weight (−2.2 kg; 95% CI,
−3.9 to −0.6), body mass index (−0.6 kg/m2; 95% CI, −1 to −0.1), systolic blood pressure (−1.7
mm Hg; 95% CI, −3.3 to −0.05), diastolic blood pressure (−1.5 mm Hg; 95% CI, −2.1 to −0.8),
fasting plasma glucose (−3.8 mg/dL; 95% CI, −7 to −0.6), total cholesterol (−7.4 mg/dL; 95% CI,
−10.3 to −4.4), and high-sensitivity C-reactive protein (−1.0 mg/L; 95% CI, −1.5 to −0.5). Thus, the
Mediterranean diet is a good option available for persons seeking weight loss. Given its significant
association with CVD risk reduction (see Chapter 22), this dietary pattern may be a preferred choice
for dieters.
CONCLUSIONS
Diets varying in macronutrient composition have existed for over 100 years but have only more
recently been systematically evaluated. In light of the current obesity epidemic, identifying effective
dietary weight loss regimens is critical. Several components are key to long-term weight loss, and
these include caloric restriction and adherence to the dietary regimen. All diets, as compared to usual
diets, work when followed, and low-fat diets that have been traditionally recommended are no better
than higher-fat diets for weight loss. Recent meta-analyses evaluating weight loss studies of similar
intensity over the longer term have shown statistically significant weight reduction in persons fol-
lowing low-carbohydrate versus low-fat diets (Bueno et al. 2013), or vice versa (Hooper et al. 2012);
the magnitude of weight loss was, however, small, that is, 1–2 kg. Similarly important to effective
weight loss is the context in which it occurs; that is to say, attention to behavior modification (see
also Chapter 2) and the support and structure provided by weight loss programs can provide dieters
with important tools for successful weight management over the longer term (?).
In general, improvements in lipids and insulin resistance have been associated with the magni-
tude of weight loss. Lower-carbohydrate diets have been associated with improved TG and HDL-C,
and lower-fat diets have been associated with lower LDL-C when differences in weight loss are
maintained. The Mediterranean diet has been shown to reduce both weight and CVD risk (see Chapter 22),
and this dietary pattern may thus be recommended assuming feasibility considerations are met.
Finally, the genes expressed during weight loss may influence both the extent of weight loss
and lipid responses to diet, and knowledge of relevant genetic polymorphisms may, one day, guide
diet strategies. Thus, individual responses to diets are more important than the mean weight loss
achieved from a diet study, and personalization of nutrition and lifestyle regimens represents a key
future direction in nutritional research. Nonetheless, the most effective interventions for weight loss
and overall cardiometabolic health will incorporate diet as just one component of a multifaceted
and comprehensive lifestyle regimen that includes increased physical activity, behavioral therapies,
social support, and other parameters, including medical management as necessary. These topics are
covered in chapters throughout this textbook.
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5 Weight Loss by Surgical
Intervention
Nutritional Considerations
and Influence on Health
Karim Kheniser and Sangeeta Kashyap
CONTENTS
Introduction...................................................................................................................................... 78
Bariatric Surgeries........................................................................................................................... 79
Roux-en-Y Gastric Bypass.......................................................................................................... 79
Biliopancreatic Diversion with Duodenal Switch....................................................................... 80
Biliopancreatic Diversion............................................................................................................ 80
Laparoscopic Adjustable Gastric Banding.................................................................................. 81
Sleeve Gastrectomy..................................................................................................................... 81
Roux-en-Y Gastric Bypass vs. Laparoscopic Adjustable Gastric Banding................................ 81
Eating Behavior, Protein Consumption Post-Surgery, and Resistance Exercise............................. 83
Dietary Compliance Post-Surgery................................................................................................... 85
Initial Consultation.......................................................................................................................... 85
Preoperative Screening and Predictors of Postoperative Outcomes................................................ 85
Postoperative Screening................................................................................................................... 87
Pre- and Post-Interventions.............................................................................................................. 87
Vitamins and Minerals..................................................................................................................... 88
Vitamin D, Parathyroid Hormone, and Calcium......................................................................... 88
Iron�� 89
Zinc�� 89
Vitamin K.................................................................................................................................... 89
Vitamin A.................................................................................................................................... 90
Vitamin B12................................................................................................................................ 90
Folate�� 90
Vitamin B6.................................................................................................................................. 90
Thiamine .....................................................................................................................................91
Conclusion....................................................................................................................................... 91
Future Research............................................................................................................................... 91
References........................................................................................................................................ 92
77
ABSTRACT
Over the past several decades, the pandemic that is obesity has gone unabated and has contrib-
uted to the rise of type II diabetes and cancer. Now more than ever, therapeutic management of
obesity and its associated comorbidities is needed. Traditional methods have failed to attenuate
the incidence of obesity, which has given rise to more contemporary modalities. Particularly,
bariatric procedures have been thrusted to the forefront, to combat obesity and its concomitant
metabolic diseases. Segregated into restrictive, malabsorptive, or bypass operations, bariatric
procedures have been demonstrated to be more efficacious in inducing weight loss and type II
diabetes remission. Subsequently, obese patients and/or individuals with type II diabetes that are
refractory to more conservative therapeutic methods, such as intensive medical therapy, have
been encouraged to undergo bariatric surgery. The most popular of which is the Roux-en-y
gastric bypass procedure, which reduces gastric volume and bypasses portions of the stom-
ach, duodenum, and the initial segments of the jejunum to facilitate weight loss. Furthermore,
purely restrictive procedures such as the sleeve gastrectomy have also become widely popular.
The mechanisms by which the aforementioned procedures promote weight loss are primarily
mediated by gastric restriction and possibly by attenuations in ghrelin secretion. This fosters
early satiety and consequently reductions in food intake, which induces a negative energy bal-
ance. These procedures have shown promise, but additional research is needed to identify demo-
graphic, behavioral, and/or anthropometric characteristics that would better predict optimal
outcomes after bariatric surgery.
INTRODUCTION
With the advent of laparoscopy and the concomitant reduction in perioperative complications,
shorter hospital stay, quicker return to vocational status, and moderated blood loss, bariatric proce-
dures have been catapulted to the forefront against the war on obesity (Nguyen et al. 2001; Schauer
et al. 2000). Alternatively, bariatric procedures have been termed metabolic procedures because
they confer positive effects on a multitude of metabolic parameters. For instance, in short- and
long-term interventions, weight loss is substantial, pharmacological agents are largely discontin-
ued, euglycemia and diabetes remission are frequently achieved, and insulin sensitivity and secre-
tion are potentiated (Brethauer et al. 2013; Hofsø et al. 2011; Kashyap, Louis, and Kirwan 2011,
2013; Savassi-Rocha et al. 2008; Schauer et al. 2014; Sjöström et al. 2004). Comparatively, medi-
cal/lifestyle therapy has been demonstrated to induce only modest decreases in weight loss and is
a requisite to improvements in high-density lipoproteins and other blood biomarkers (Ryan et al.
2010), but the positive effects are nondurable and miniscule, even with adjunct pharmacological
therapy (Yanovski and Yanovski 2002), in relation to minimally invasive adjustable gastric band-
ing procedures (Dixon et al. 2008; O’Brien et al. 2006). Unlike bariatric procedures, nonsurgical
methods may be destined for failure because the homeostatic response to weight loss is dictated
by unrelenting orexigenic stimuli and reciprocal changes in energy expenditure that would pre-
cipitate recidivism (Cummings et al. 2002; Cummings and Shannon 2003; Kotidis et al. 2006;
Leibel, Rosenbaum, and Hirsch 1995). Moreover, even when medical therapy achieves equiva-
lency in weight reduction, bariatric procedures prove to be more efficacious in promoting favorable
alterations in insulin sensitivity (Plum et al. 2011). Above all else and unrivaled by medical ther-
apy, bariatric procedures drastically and consistently reduce morbidity, mortality, cancer risk, and
health care costs (Adams et al. 2007; Christou et al. 2004; Sjöström et al. 2007). Unequivocally,
lifestyle/medical therapy has become complementary to bariatric procedures, which exert a pro-
found moderating influence on massive obesity and provide therapeutic relief to their debilitating
comorbidities.
Weight Loss by Surgical Intervention 79
BARIATRIC SURGERIES
Excluding adolescents who require a more diligent screening regimen and thorough collaboration
between the medical team and family (Inge et al. 2004), bariatric surgeries are advocated when
body mass index (BMI) exceeds 40 kg/m2 or in the presence of comorbidities and a BMI of >35 kg/m2
(National Institutes of Health 1992). Although bariatric procedures have been utilized in type II
diabetics with a BMI of <30 kg/m2, the research is currently sparse and has not gained widespread
acceptance (Mechanick et al. 2013). Contemporary and efficacious surgical modalities that induce
significant weight loss (i.e., >50% of excess weight loss or a BMI < 35 kg/m2 in the super-obese)
are segregated into restrictive, malabsorptive, and bypass operations (Brolin 2002; Reinhold 1982;
Renquist et al. 1995). Even though the latter two procedures share commonalities, in that they both
bypass segments of the small intestine and facilitate malabsorption (excluding short-limb variants
of Roux-en-Y gastric bypass, which has not been demonstrated to induce malabsorption), gener-
ally, bypass procedures are made in reference to Roux-en-Y gastric bypass, while malabsorptive
procedures pertain to variants of biliopancreatic diversion. Restrictive procedures decrease daily
caloric intake via reductions in gastric volume and the associated promotion of early satiety, while
malabsorptive techniques attenuate nutrient absorption because they bypass segments of the small
intestine (e.g., duodenum) and limit contact with biliopancreatic juices (Gletsu-Miller and Wright
2013). Unique to bypass and malabsorptive procedures, the circumvention of segments of the small
intestine and gastric fundus play a crucial role in ameliorating diabetes, irrespective of weight loss
and caloric intake (Kashyap et al. 2010; Rubino et al. 2006).
Also, in many instances, the degree and type of nutritional deficiencies that are present post-
surgery are dictated by the specific surgical intervention. Generally, malabsorptive operations and
possibly long-limb variants of Roux-en-Y gastric bypass reduce the absorption of lipids and patients
are considered to be at higher risk for micronutrient deficiencies pre- and postoperatively (Gudzune
et al. 2013). Furthermore, interindividual differences in surgical technique and intestinal morphol-
ogy will also have an effect on malabsorption. In particular, the most common bariatric surgeries
include Roux-en-Y gastric bypass, laparoscopic adjustable gastric banding, sleeve gastrectomy, and
biliopancreatic diversion with or without duodenal switch.
Roux-en-Y Gastric Bypass

Suited for individuals who have a propensity to ingest sweets and patients with gastroesophageal
reflux disease (Buchwald 2002), the purpose of Roux-en-Y gastric bypass is to induce weight loss
by reducing gastric volume to <30 mL and diverting ingested foods away from the distal stomach,
duodenum, and minute (~initial 10 cm) segments of the jejunum (Elder and Wolfe 2007; Pories et al.
1995). The gastric pouch is created by segmenting the stomach into upper and lower quadrants, of
which the lower or remnant pouch is bypassed (Moize et al. 2003). After the jejunum is transected
distally from the ligament of Treitz, gastrojejunostomy and jejunojejunostomy are formed. The
Roux limb, biliopancreatic limb, and common limb are denoted as representing the increments from
the gastrojejunostomy to the jejunojejunostomy, from the ligament of Treitz to the jejunojejunos-
tomy, and from the jejunojejunostomy to the ileocecal valve, respectively (Chen et al. 2013; Elder
and Wolfe 2007; Gletsu-Miller and Wright 2013). Depending on the size of Roux and common
limbs, the procedure can be designated as a long-limb or short-limb procedure.
Super-obese subjects (BMI > 50 kg/m2) achieve pronounced weight loss with the long-limb vari-
ant (Brolin et al. 1992; MacLean, Rhode, and Nohr 2001). The mechanism by which this occurs
does not appear to be related to malabsorption, as it had a negligible contribution to the total reduc-
tion in intestinal absorption of macronutrient energy (Brolin 2002; Cummings, Overduin, and
Foster-Schubert 2004; Odstrcil et al. 2010). However, standardizing biliopancreatic or Roux limb
lengths, across patients, when assessing malabsorption may provide inconclusive results because
it fails to account for jejunoileal length, which can improve the absorptive surface area by extending
common limb length (Savassi-Rocha et al. 2008). Thus, dietary (e.g., fewer meals and less snacking)
(Halmi et al. 1981), endocrine, and restrictive mechanisms appear to be the predominant mediators
of weight loss.
Biliopancreatic Diversion with Duodenal Switch

Biliopancreatic diversion with duodenal switch is an invasive procedure that necessitates transection
of portions of the gut and bowel and frequently requires a cholecystectomy. Initially, a sleeve gas-
trectomy (partitions the majority of the stomach, which leaves a “sleeve-like” stomach) is performed
to reduce gastric volume to 80–100 mL, which, depending on the surgical technique, excludes the
ghrelin-producing fundus (Hess and Hess 1998; Kotidis et al. 2006). Then, the duodenum is tran-
sected 4–5 cm distally from the pylorus and the distal portion of the ilium is bisected (Hess and
Hess 1998). When the proximal portion of the duodenum is preserved, the incidence of ulcerations
is reduced (DeMeester et al. 1987). Thereafter, the distal portion of the ileum is anastomosed to the
duodenum to create a duodenoileostomy; similarly, the proximal ileum is anastomosed to the distal
portion of the ileum (i.e., ileoileostomy), which is located proximally to the cecum (Hess and Hess
1998). The point from the duodenoileostomy to the ileoileostomy is denoted as representing the
alimentary limb. Thus, the procedure bypasses the majority of the jejunum and duodenum and the
biliopancreatic juices flow through the biliopancreatic limb into the distal portion of the ileum where
the ingested food converges with and forms the common limb. The delayed mixing of biliopancre-
atic juices and ailments promotes malabsorption.
Its effects on weight loss are drastic and are mediated by restrictive, malabsorptive, and endocrine
mechanisms. At 8 years, the majority of type II diabetics achieved remission and excess weight loss
was maintained at 70%; favorable modifications to total cholesterol and low-density lipoproteins
were observed (Hess and Hess 1998). Marceau et al. (1998) observed similar reductions in weight
loss, as excess body weight was reduced by 73% + 21% at 51 + 25 months.
Biliopancreatic Diversion
Scopinaro et al. (1998) noted comparable excess weight loss outcomes in biliopancreatic diver-
sion patients and this procedure can be applied in the non-super-obese, without the occurrence
of persistent severe metabolic deficiencies (Skroubis et al. 2006). However, the main difference
between biliopancreatic diversion and biliopancreatic diversion with duodenal switch is that in
biliopancreatic diversion a larger gastric reservoir and the ghrelin-producing fundus are retained
(Kotidis et al. 2006). Furthermore, in biliopancreatic diversion with duodenal switch, the pylorus
and vagal integrity remain intact, which prevent rapid gastric emptying and increased motility
(Kotidis et al. 2006); concurrently, the common limb may be more protracted in length (e.g.,
100 cm), which is sensible given that it would reduce the sequela associated with malabsorption
(Marceau et al. 1998).
Herein, a summation of Dr. Scopinaro’s experience with biliopancreatic diversion will be dis-
cussed (Scopinaro 2012). In principal, biliopancreatic diversion is a malabsorptive procedure, but
it is malleable. The pendulum can meander from a restrictive to a malabsorptive procedure, which
is predicated upon the gastric volume and lengths of the alimentary and common limbs. Indeed,
if gastric volume is significantly reduced to where energy intake underlies the energy absorption
threshold of the alimentary and common limbs, it becomes a restrictive procedure, thus making the
malabsorptive component futile (Scopinaro 2012). Conversely, if the restrictive component permits
energy intake above the energy absorption threshold, then the restrictive component is undermined.
Therefore, the operation cannot have dual mechanisms for weight loss (Scopinaro 2012).
Dichotomously, with respect to their side effects and complications, the effects of a small gas-
tric volume and a large malabsorptive component become additive (Scopinaro 2012). Generally, a
common limb and alimentary limb length of 50 and 250 cm is viable, respectively; however, in obese
and overweight type II diabetics, the common limb can be 75 and 100 cm, respectively (Scopinaro
2012). Furthermore, a small gastric volume (e.g., 150 mL) should not be incorporated into a bilio-
pancreatic diversion procedure because it would exacerbate nutritional deficiencies. Consequently,
gastric volume should permit energy intake that exceeds the energy absorption threshold, but the
overall volume of the gut (400–500 mL) should be low enough to permit rapid gastric emptying and
consequently facilitate malabsorption (Scopinaro 2012; Scopinaro et al. 2011).
Laparoscopic Adjustable Gastric Banding

One of the main advantages of laparoscopic adjustable gastric banding is that normal gastrointesti-
nal continuity is preserved and it is purely restrictive in nature. Gastric capacity is reduced to <30 mL
by placing a collar 1–2 cm inferior to the gastroesophageal junction, and gradation in the degree of
constriction can be modified by adjusting the amount of saline that is infused into the subcutaneous
port, which is connected to a balloon within the collar (Elder and Wolfe 2007). The perigastric and
pars flaccida surgical techniques are primarily utilized, with the latter associated with a lower rate
of prolapse (Biagini and Karam 2008; O’Brien et al. 2005). At the outset, the collar confers minimal
gastric restriction, but it is gradually heightened to achieve sustainable weight loss (Dixon, Dixon,
and O’Brien 2005). Due to the confined gastric volume, laparoscopic adjustable gastric banding
subjects will feel satiated, but this appears to be independent to alterations in leptin and the orexi-
genic hormone, ghrelin (Dixon, Dixon, and O’Brien 2005; Faraj et al. 2003), which has been noted
to increase (Langer et al. 2005). Consequently, this may explain the myriad of excess weight loss
values and poor efficacy, in some instances (Langer et al. 2005).
Sleeve Gastrectomy
Restrictive in nature, sleeve gastrectomy reduces gastric volume by resecting the greater curvature
of the stomach and excluding the fundus. As such, due to reduced nutriment contact (Cummings et al.
2002), ghrelin secretion is reduced (Bohdjalian et al. 2010; Karamanakos et al. 2008; Langer et al.
2005). Also, similar to Roux-en-Y gastric bypass, the hindgut hormone glucagon-like peptide-1,
which has salutary effects on glycemic control (Kashyap and Schauer 2012), and peptide YY secre-
tions are potentiated (Karamanakos et al. 2008; Kashyap et al. 2013). Together, this will foster an
anorectic state that would engender more durable weight loss. Excess weight loss of 55% has been
noted at 5 years post-surgery (Bohdjalian et al. 2010). Himpens, Dobbeleir, and Peeters (2010)
found similar results at 6 years, but 3-year outcomes were 77.5%. Short-term trials have demon-
strated excess weight loss values of 69.7% + 14.6% at 1 year, which was even greater than Roux-
en-Y gastric bypass (60.5% + 10.7%) (Karamanakos et al. 2008). However, in the midst of similar
weight loss outcomes, Roux-en-Y gastric bypass has been shown to induce more striking effects on
metabolic parameters (Kashyap et al. 2013). This data explains the increased popularity of sleeve
gastrectomy as a primary therapeutic modality. Furthermore, as denoted, its efficacy has even rivaled
that of Roux-en-Y gastric bypass in some instances.
Roux-en-Y Gastric Bypass vs. Laparoscopic Adjustable Gastric Banding

Roux-en-Y gastric bypass is favored by the majority of surgeons because it incurs favorable weight-
independent changes to metabolic parameters. Although some evidence contraindicates the senti-
ment that Roux-en-Y gastric bypass has a dampening effect on ghrelin secretion (Holdstock et al.
2003; Karamanakos et al. 2008), the incurred positive effects on metabolic parameters have been
shown to be partially mediated by attenuated gastroduodenal secretion of ghrelin (i.e., override
inhibition), which may necessitate a vagotomy along with transection of the fundus (Williams
et al. 2003). The discordant results with respect to ghrelin secretion can be due to divergent surgical
techniques and/or retrograde nutriment flow through the short biliopancreatic limb (Cummings,
Overduin, and Foster-Schubert 2004; Holdstock et al. 2003). Also contributory to its positive meta-
bolic effects are increases in postprandial enteroinsular (principally glucagon-like peptide-1), pep-
tide YY (Cummings et al. 2002; Faraj et al. 2003; Kashyap et al. 2013; Le Roux et al. 2006), and
anorexigenic adipokines such as adiponectin, while the pro-lipogenic hormone, acylation-stimulating
protein, is decreased (Faraj et al. 2003; Holdstock et al. 2003). Congruently, not only has Roux-en-Y
gastric bypass been demonstrated to alter gut hormone and enteroinsular secretion, but the small
bowel microbial milieu may be altered to where it would favor enhanced motility (Zhang et al. 2009).
Roux-en-Y gastric bypass induces definite and sustained increases in excess weight loss (Christou
et al. 2004). At the 2-year mark, gastric bypass procedures were more efficacious in inducing weight
loss in super-obese patients (>50 kg/m2), in relation to laparoscopic adjustable gastric banding
(Te Riele et al. 2007). Interestingly, some reports suggest that Roux-en-Y gastric bypass is less
effective in inducing weight loss in the aforesaid population (Jan et al. 2005; Nguyen et al. 2009).
However, this may be evident in the majority of restrictive and short-limb operations. Also, in a
4-year randomized, prospective trial, Roux-en-Y gastric bypass was superior to that of laparoscopic
adjustable gastric banding in inducing weight loss, but weight loss plateaued after year 2 (Nguyen
et al. 2009). Likewise, maximal weight loss was achieved within 1 or 2 years, but at 15 years there
was a twofold difference in weight loss (Roux-en-Y gastric bypass = 27% + 12%; laparoscopic
adjustable gastric banding = 13% + 14%) (Sjöström et al. 2007). Ten-year values were 25% + 11%
and 14% + 14%, respectively. Furthermore, euglycemia was achieved in the majority of patients
and demonstrated mean decrements of 70% excess weight loss at year 2, with excess weight loss
stabilizing at ~50% thereafter, over a similar timespan (Pories et al. 1995).
However, with periodic follow-up and an experienced surgical team, laparoscopic adjustable
gastric banding has been demonstrated to successfully ameliorate morbidities (e.g., type II diabetes,
obesity, hypertension, etc.) (Biagini and Karam 2008; O’Brien et al. 2006). Furthermore, relative
to bypass procedures, laparoscopic adjustable gastric banding is reversible, less invasive, and there
is a lower prevalence of postoperative complications (Nguyen et al. 2009; Te Riele et al. 2007).
Similar to Roux-en-Y gastric bypass, it induces remission of diabetes (73% of 30 subjects) and
profound excess weight loss (62.5%) at 2 years (Dixon et al. 2008). Others obtained similar results
(O’Brien et al. 1999). Furthermore, there is a contention that although Roux-en-Y gastric bypass
induces profound weight loss initially, the distinction in excess weight loss between the bariatric
procedures appears to be lessened after 3 years (Jan et al. 2005). Although outside the guidelines
recommended by the National Institutes of Health (National Institutes of Health 1992), multiple tri-
als have utilized the laparoscopic adjustable gastric banding or other surgeries in individuals with a
BMI between 30 and 35 kg/m2, before weight gain becomes more pronounced and morbidities unre-
sponsive to therapy (Angrisani et al. 2004; Dixon et al. 2008; Mason et al. 1997; Parikh, Duncombe,
and Fielding 2006, O’Brien et al. 2006; Rubino et al. 2006). The use of laparoscopic adjustable
gastric banding in this subpopulation may abrogate the progression of obesity and undiagnosed
comorbidities, thereby possibly reducing the need for more complex procedures such as long-limb
Roux-en-Y gastric bypass and biliopancreatic diversion with duodenal switch, when the need for
immense weight loss is required.
Nevertheless, empirical evidence indicates that Roux-en-Y gastric bypass is superior to laparo-
scopic adjustable gastric banding. One of the main downfalls that contributes to its inferiority is the
fact that laparoscopic adjustable gastric banding patients must adhere to a more frequent pre- and
postoperative visit regimen and dietary and physical activity guidelines to induce successful weight
loss, in comparison to Roux-en-Y gastric bypass (Biagini and Karam 2008; El Chaar et al. 2011;
Shen et al. 2004). If they fail to, weight loss will not be pronounced and consequently comorbidi-
ties will persist. Therefore, given the additional burden placed on these patients and the likelihood
that they will fail to adhere to postoperative guidelines, additional research is required before the
use of laparoscopic adjustable gastric banding becomes more widespread, especially in the United
States where it is relatively uncommon. In conclusion, although this will need to be empirically
substantiated, laparoscopic adjustable gastric banding may be relegated to a preemptive procedure

where it is specifically employed to prevent morbid obesity, in patients with a BMI of <35kg/m2
(Buchwald 2002). This does not come without caution, as imposing laparoscopic adjustable gastric
banding and other procedures in this population could be problematic because surgeons with limited
operative proficiency may be financially motivated to impose these procedures in poor candidates
(Steinbrook 2004), which may heighten mortality risk (Flum et al. 2005). Thus, it may be advanta-
geous to conduct high-risk (e.g., super-obese) surgeries in centers of excellence.
The invasiveness of the procedures must be counterbalanced with their safety and ability to
induce remission of diabetes and successful weight loss. Ultimately, in many instances, the spe-
cific surgical intervention that is chosen is dictated by the surgeon’s experience with a specific
bariatric procedure and their consequent bias, pay status (i.e., private [health insurance] or govern-
ment pay [e.g., Medicare and Medicaid]) (Mason et al. 1997; Renquist et al. 1996), patient risk
(Jan et al. 2005), age, gender, comorbidity status, ethnicity, and the degree of weight loss that is
required (Buchwald 2002; Cummings, Overduin, and Foster-Schubert 2004). For instance, patients
who present with fewer comorbidities and a lower preoperative BMI may be more suited for purely
restrictive procedures (Buchwald 2002). As stated by Buchwald, no gold standard procedure exists
and he utilizes numerical formulas and algorithms to discern the optimal procedure for each patient
(Buchwald 2002).
EATING BEHAVIOR, PROTEIN CONSUMPTION

POST-SURGERY, AND RESISTANCE EXERCISE
Whether by choice, via influence from a dietician or surgeon, or stemming directly from the
anatomical alterations in the gastrointestinal tract, eating behavior is significantly modified
after surgery. A multitiered dietary template is employed after bariatric surgery to facilitate healing
through optimal nutritional intake, minimize loss of fat-free mass (FFM), reduce gastrointestinal
symptoms, and potentiate weight loss (Aills et al. 2008). At the outset, ingestion of liquid-based or
soft-texture foods predominates due to inflammation and edema, which impede the passage of food
(Bock 2003; Moize et al. 2003). Diets progress, in order, from a clear liquid (1–2 days [d]) to full liquid
(10–14 d) to pureed (10–14 + d) to mechanically modified soft diet (>14 d), and finally a regular diet
is recommended (Aills et al. 2008). Osmolality and caloric density gradually increase as diets change.
To avoid dumping syndrome and facilitate weight loss, avoidance of fruit juices, liquids during meals
(Halverson and Koehler 1981), sugar, and saturated fat–laden food products is recommended (Aills
et al. 2008). Dumping syndrome, especially in Roux-en-Y gastric bypass, frequently occurs acutely
post-surgery and represents a constellation of symptoms, which include diarrhea, bloating, abdomi-
nal pain, nausea, tachycardia, syncope, palpitations, and sweating (Laurenius et al. 2013).
Except for biliopancreatic diversion, where the gastric volume is larger, patients with preopera-
tive binge eating disorders will be physically unable to consume large portions during one sitting
in the postoperative period, as they will be inhibited by a small gastric pouch and because vomiting
may ensue (De Zwaan et al. 2010); emesis is rare in laparoscopic adjustable gastric banding patients
(O’Brien et al. 1999). Furthermore, manifestations of early satiety and reductions in hunger and dis-
inhibition, especially in Roux-en-Y gastric bypass, are prerequisite to the reduction in the frequency
and volume of nutriment intake (Bock 2003; Halmi et al. 1981). Preference for palatable foods is
reduced, which indicates a lower impetus to eat (Ullrich et al. 2013). Moreover, eating behavior is
further modified in that mastication will be methodical to avoid plugging (i.e., lodging of food),
which is common when ingesting dry meat, vegetables, pasta, and bread (Mitchell et al. 2001).
Dietary composition is altered in patients with the Roux-en-Y gastric bypass who should abstain
from ingesting sweets (e.g., chocolate and cake), as it may lead to dumping syndrome, calorie-dense
beverages, meat, dairy, and fatty foods, while sleeve gastrectomy patients avoid meat, vegetables,
and fruit acutely, post-surgery (Brolin et al. 1994; Ernst et al. 2009; Halmi et al. 1981; Van De
Weijgert, Ruseler, and Elte 1999). Dumping syndrome is conspicuously absent in biliopancreatic
diversion and sleeve gastrectomy patients and putatively has a modest effect on excess weight loss
(Bohdjalian et al. 2010; Cummings, Overduin, and Foster-Schubert 2004; Papadia et al. 2012).
Moreover, there will be marked reductions in total caloric intake, especially acutely, post-surgery
(excluding biliopancreatic diversion) and consequently a greater emphasis will be placed on nutri-
tional density to prevent malnutrition (Bavaresco et al. 2010). To give a general idea, patients with
a Roux-en-Y gastric bypass will consume ~1060 + 322 kcal/d at 6 months (Gobato, Seixas Chaves,
and Chaim 2014), while sleeve gastrectomy subjects ingested between ~1163 and 1625 kcal/d at
6 months and 5 years (Moizé et al. 2013), respectively. However, caloric intake gradually increases
post-surgery (Brolin et al. 1994), which may parallel weight regain. This may be a consequence to
increased food tolerance, gastric expansion (Halverson and Koehler 1981), neo-fundus (proximal
gastric pouch dilation with gastric stenosis) formation in sleeve gastrectomy (Himpens, Dobbeleir,
and Peeters 2010), anastomotic dilation, staple line disruption, formation of gastrogastric fistula in
Roux-en-Y gastric bypass, or dietary habits that emphasize calorically dense food items that do not
lead to gastrointestinal distress (Mechanick et al. 2013; Van De Weijgert, Ruseler, and Elte 1999),
thus prompting an endoscopy or additional diagnostic methodologies.
In the early postoperative period, patients with biliopancreatic diversion achieve weight loss
through early satiety and the corresponding attenuation in food intake, which are induced by rapid
gastric emptying through the expanded gastroenterostomy and distention of the post-anastomosis
bowel by undigested chime (Koopmans et al. 1982; Koopmans and Sclafani 1981). About 4–6 months
thereafter, weight loss is mediated by malabsorption even when eating behavior is retained (food
consumption can mimic or exceed presurgery), due to the presence of a limited intestinal absorptive
capacity for fat and total energy (Marceau et al. 1998; Scopinaro et al. 1998, 2000). Specifically,
fat and total energy absorption decrease as total consumption increases (negative correlation)
(Scopinaro et al. 2000). However, this may be modulated by increasing alimentary and common
limb lengths, which purportedly increases protein and decreases fat malabsorption, respectively
(Hess and Hess 1998; Scopinaro 1997). Furthermore, a positive correlation exists, with respect
to protein and calcium intake and the absolute quantity that is absorbed (Scopinaro et al. 2000).
Therefore, calcium and protein deficiencies may be amenable to increased caloric consumption of
foods enriched in these nutrients.
Derived energy from protein (>1 g/kg of current body weight) will be heightened to promote
satiety, improve quality of life, facilitate increases in % excess weight loss, incur greater deficits
in body fat percentage, and mitigate decreases in muscle mass (Raftopoulos et al. 2011). Protein
supplementation may be needed to adhere to the aforesaid recommendation (Andreu et al. 2010).
The importance of protein supplementation is further conveyed by the fact that Roux-en-Y gastric
bypass patients will have difficulty consuming protein-rich foods (e.g., meat) and meeting the
protein intake guidelines, especially acutely after surgery (Bavaresco et al. 2010; Bock 2003;
Gobato, Seixas Chaves, and Chaim 2014; Moize et al. 2003). Furthermore, even when adequate
intake is attained, diminished levels of pepsin and hypochlorhydria, which would reduce the inci-
dence of ulcers (Smith et al. 1993), can inhibit protein digestion and facilitate cobalamin (vitamin
B12) deficiency (Behrns, Smith, and Sarr 1994; Faria et al. 2011). Although additional research
needs to be conducted, a generalized recommendation for protein intake is within the realm of
60–120 g/d (Heber et al. 2010). In particular, emphasizing whey protein intake is beneficial as it
has been demonstrated to promote greater satiety and amplify protein synthesis, thereby assisting
with the preservation of FFM, especially when coupled with resistance training (Hall et al. 2003;
Tang et al. 2009).
However, this population may be relegated to utilizing dynamic external constant resistance
devices (i.e., exercise weight machines), if they are able to fit comfortably, and/or simplistic multi-
joint and single-joint free weight exercises (American College of Sports Medicine 2009). Patients
who are diabetic, have orthopedic limitations, and/or have cardiovascular disease may need to have
the program tailored to their needs and should proceed with caution. It is highly advisable to have a
qualified exercise physiologist assist them with an exercise intervention.
DIETARY COMPLIANCE POST-SURGERY

The dissemination and prescription of vitamin supplementation without inquiring as to whether or
not the patient is compliant would prove to be counterintuitive to the initial desired positive effect,
which is to reduce the magnitude and prevalence of nutritional deficiencies. Limited adherence to
vitamin supplementation may accentuate nutritional deficiencies, thereby highlighting the need for
supplementation and patient compliance. Indeed, patients are inundated by post-bariatric nutritional
deficiencies partially because they are noncompliant and due to the substandard follow-up care
(Ahmad, Esmadi, and Hammad 2012; Coupaye et al. 2009; Gudzune et al. 2013; Modi et al. 2013;
Shah et al. 2013). Additively, nutritional deficiencies are further accentuated by failing to prescribe
prophylactic supplements from the outset (Cummings and Shannon 2003).
As such, in laparoscopic adjustable gastric banding, Roux-en-Y gastric bypass, and biliopan-
creatic diversion with duodenal switch, the minimal recommended dosage level is typified by
100%, 200%, and 200% of daily value for the majority (minimum of 2/3) of nutrients, respectively
(Aills et al. 2008). Quantitatively, others suggest a minimum of two adult multivitamins and a
mineral supplement for Roux-en-Y gastric bypass and sleeve gastrectomy patients, while lapa-
roscopic adjustable gastric banding patients should receive one of each (Mechanick et al. 2013).
However, even though the prescription of long-term vitamin and mineral therapy is vital in all
bariatric patients (Donadelli et al. 2012; Heber et al. 2010), it should be emphasized that vitamin
supplementation should not replace optimal dietary habits and alone will not abolish the presence
of nutrient deficiencies (Donadelli et al. 2012).
INITIAL CONSULTATION
Prior to surgery, it would behoove the surgeon to discuss realistic weight loss options because many
candidates have preconceived and often far-fetched assumptions about the degree of excess weight
loss that they will attain (Kaly et al. 2008; Van De Weijgert, Ruseler, and Elte 1999). Even though
bariatric operations have been demonstrated to induce significant weight loss, patients will rarely,
if ever, achieve a BMI <25 kg/m2. More importantly, it is prudent to convey that they will need to
adhere to a physical activity regimen (e.g., walking, elliptical exercise machine, or cycle ergometer
for a minimum of 5–10 min interspersed throughout the day [3–6 times]) (Donnelly et al. 2009),
dietary guidelines, and routine physician visits to optimize weight loss. Bariatric dietary and physi-
cal activity interventions have even incurred energy deficits of >2000 kcal/week (Shah et al. 2011).
Although postoperative physical activity confers positive effects on weight loss and overall health
(Evans et al. 2007; Shah et al. 2011), many patients are not physically active or are noncompliant,
although they may have good intentions (Bond et al. 2013). A lack of understanding to the afore-
mentioned does not represent a contraindication to surgery, but it is advisable that the patient dem-
onstrates some competency, with regard to the expected behavioral and dietary modifications that
occur in tangent with surgery.
PREOPERATIVE SCREENING AND PREDICTORS

OF POSTOPERATIVE OUTCOMES
Postoperative weight loss success, resolution of comorbidities, and the incidence and magnitude of
perioperative complications are dictated by a plethora of variables. For example, females, especially
with a history of sexual abuse (Fujioka et al. 2008; Ray et al. 2003), are associated with poorer
weight loss outcomes (Coupaye et al. 2010; Ma et al. 2006; Tymitz et al. 2007). However, others
contraindicate this sentiment by stating that males are more prone to attaining lower excess weight
loss values (Chen et al. 2009; Melton et al. 2008; Nguyen et al. 2009). Males have more adverse
outcomes (e.g., mortality at 1 year post-surgery was twofold higher) and are more prone to intes-
tinal leaks (Fernandez et al. 2004; Flum et al. 2005; Livingston et al. 2002; Nguyen et al. 2009).
Similarly, advancing age (>65 years), comorbidity status (e.g., sleep apnea, hypertension, diabetes),
and type of procedure (revisional > laparoscopic > open surgery) are associated with a higher
mortality risk and/or intestinal leaks (Fernandez et al. 2004; Flum et al. 2005; Livingston et al.
2002). The elevated risks associated with males and advancing age may be attributed to the higher
prevalence of comorbidities and greater weight, which is also predictive of adverse outcomes and
mortality (Fernandez et al. 2004; Livingston et al. 2002; Tymitz et al. 2007). However, evidence
differs on whether preoperative weight or BMI is positively or negatively correlated with weight
loss (Chen et al. 2009; Coupaye et al. 2010; Halverson and Koehler 1981; Ma et al. 2006; Melton
et al. 2008). Moreover, although it has not yielded improved postoperative weight loss outcomes
and reductions in perioperative complications (Alami et al. 2007; Becouarn, Topart, and Ritz 2010;
Fujioka et al. 2008; Van De Weijgert, Ruseler, and Elte 1999; Van Nieuwenhove et al. 2011), advo-
cating presurgical weight loss is advantageous because it has been demonstrated to reduce operative
time, visceral fat, hepatomegaly, and surgical difficulty (Alami et al. 2007; Edholm et al. 2011; Fris
2004; Frutos et al. 2007; Van Nieuwenhove et al. 2011).
Other factors that are predictive of reduced excess weight loss outcomes include government pay
status, which is associated with higher perioperative complication rates (Flum et al. 2005; Renquist
et al. 1996). Furthermore, type II diabetes of greater than 10 years in duration and a HbA1C > 10
predict reduced remission rates (Hall et al. 2010). Likewise, weight regain and insulin-dependent
diabetics, which indicates long-standing diabetes, are associated with recurrence of type II diabetes
(Chikunguwo et al. 2010).
The research is conflicting on whether screening for binge eating disorder should be an anteced-
ent to surgery. One year post-surgery, well-conducted prospective studies that utilized interview-
based methodologies to diagnose subjects with binge eating disorder indicated that the presence
of preoperative binge eating disorder did not significantly attenuate postoperative weight loss
outcomes or improvements in cardiovascular disease risk factors (Burgmer et al. 2005; Wadden
et al. 2011). Similarly, others (Fujioka et al. 2008; Mitchell et al. 2001) and prospective trials
that elucidated the presence of eating pathologies (e.g., binge eating disorder, loss of control over
eating, etc.) via questionnaires have substantiated these findings (Bocchieri-Ricciardi et al. 2006;
Chen et al. 2009; White et al. 2010). However, preoperative binge eating disorder may manifest as
grazing (i.e., consuming small portions over extended periods) postoperatively, which may negate
excess weight loss (Colles, Dixon, and O’Brien 2008). Concurrently, preoperative eating disor-
ders are predictive of postoperative loss of control over eating, which may moderate weight loss
outcomes, bulimic episodes, and general psychopathology (De Zwaan et al. 2010). Therefore, eat-
ing pathologies do not represent an absolute contraindication to surgery, but preoperative screen-
ing and behavioral interventions are warranted because they may result in improved postoperative
weight loss outcomes by preventing the reemergence or development of postoperative aberrant
eating behaviors (Ashton et al. 2011). Moreover, it may assist with preliminary patient selection,
prevent distention of the gastric pouch, which may permit increased nutriment consumption, and
reduce postoperative nutritional deficiencies (Royal et al. 2015; Santarpia et al. 2014; Sarwer,
Dilks, and West-Smith 2011).
Especially among the morbidly obese (Aasheim et al. 2008; Gudzune et al. 2013; Kimmons
et al. 2006), the etiology of micronutrient deficiencies (e.g., vitamin C, D, E, and selenium) mani-
fests from the consumption of calorically dense and nutritionally deprived foods (Heber et al. 2010;
Jastrzębska-Mierzyńska et al. 2012). Moreover, the concomitant presence of excess adiposity fur-
ther exacerbates malnutrition by expanding the extracellular fluid and increasing markers of inflam-
mation (Aasheim et al. 2008; Waki et al. 1991; Wannamethee et al. 2006). Therefore, a rigorous
screening regimen in the presurgical setting is warranted not only because micronutrient deficien-
cies will be present, but they will be exacerbated postoperatively by the debilitating direct (e.g.,
malabsorption or restriction) and indirect (modify eating behavior) effects of surgery. Specifically,
a preoperative comprehensive examination includes a medical history, psychosocial analysis, physi-
cal examination, and laboratory testing (Gudzune et al. 2013; Mechanick et al. 2013). With respect
to the latter, clinical monitoring of complete blood counts (CBC), liver function tests, glucose,
creatinine, electrolytes, iron/ferritin, vitamin B12, folate, calcium, parathyroid hormone (PTH),
1,25 dihydroxyvitamin D, albumin/prealbumin, vitamin A (Vit A), and zinc is recommended
(Heber et al. 2010).
POSTOPERATIVE SCREENING
Reducing the prevalence chronic or occult nutritional deficiencies proves to be more difficult
when preemptive steps are not employed and may, even, prove to be a lifelong intervention,
if a screening regimen is not installed preoperatively (Santarpia et al. 2014). Indeed, in many
instances, screening and nutritional counseling do not occur preoperatively, which would exac-
erbate postoperative nutritional deficiencies. Subsequently, postsurgical assessments become
crucial to facilitate early diagnosis and treatment; assessments should not be guided by symp-
tomatology alone, as nutritional deficiencies may only become apparent when symptoms become
clinically significant. However, empirical evidence indicates that surgeons fail to adhere to the
aforesaid recommendation (Brolin and Leung 1999; Gudzune et al. 2013). As a result, nutritional
deficiencies and their symptomatology manifest, even in purely restrictive procedures (Gudzune
et al. 2013; Santarpia et al. 2014).
Therefore, measurement of CBC, liver function tests, glucose, creatinine, and electrolyte levels
are recommended at 1, 3, 6, 12, 18, and 24 months post-intervention and annually thereafter for
all bariatric procedures (Heber et al. 2010). Furthermore, at 6, 12, 18, 24 months, and annually
afterward, biochemical monitoring for iron/ferritin, vitamin B12, folate, calcium, 1,25 dihydroxyvitamin D,
albumin/prealbumin, and PTH is advised for patients with Roux-en-Y gastric bypass, bilio-
pancreatic diversion, or biliopancreatic diversion with duodenal switch (Heber et al. 2010).
Postoperative screening should not be relegated to the measurement of blood parameters alone,
as interview-based methodologies should be utilized to screen for and diagnose pathological eating
behaviors that may contribute to poor outcomes. For instance, patients with binge eating disorder
or grazing tend to regain more weight and achieve lower excess weight loss (Kofman et al. 2010;
Livhits et al. 2010; Mitchell et al. 2001). Likewise, loss of control over eating is predictive of weight
loss at 12 and 24 months and regain at 12 months post-surgery, which may provide an indication
of an imminent plateau in weight loss (White et al. 2010). Furthermore, loss of control over eat-
ing was associated with a higher prevalence of weight-related vomiting, aberrant eating behaviors,
depression, and lower self-esteem (De Zwaan et al. 2010; Kofman, Lent, and Swencionis 2010).
Other reports have substantiated these findings by stating that uncontrolled eaters attained lower
weight loss values post-surgery, ingested more calories, and a greater proportion of energy intake
was derived from fat (Colles, Dixon, and O’Brien 2008).
PRE- AND POST-INTERVENTIONS

The diminution of micro- and macronutrient deficiencies and the induction of successful excess
weight loss are facilitated by an ongoing collaboration between the patient, surgeon, and dietician.
Dietary and behavioral counseling and frequent follow-up scheduling may be a viable way to foster
more positive outcomes after surgery. However, pre-intervention trials that have aimed to facilitate
patient success have been inconclusive and scarce. In a randomized controlled trial, although 88%
of gastric bypass subjects achieved successful weight loss (>50% excess weight loss), the delivery
of preoperative behavioral treatment conferred no significant effect on weight loss, indices of eat-
ing habits, and physical exercise 1 year post-surgery (Lier et al. 2012). Dichotomously, in relation
to those that missed >25% of their pre- and postoperative multidisciplinary team consultations,
laparoscopic adjustable gastric banding patients who missed <25% lost significantly more weight
(23% vs. 32% excess weight loss at 12 months, respectively), with preoperative compliance being
the main determinant (El Chaar et al. 2011). Furthermore, preoperative mental health or substance
abuse treatment improved postoperative excess weight loss (Clark et al. 2003). However, the latter
studies were retrospective, which limits their interpretation.
A plethora of factors may confer positive effects on postoperative outcomes. In a retrospective
study, social support, surgeon follow-up within the prior year, marital status, support group meeting
attendance, and physical activity level inferred a positive effect on weight loss success (Livhits et al.
2010), while others have noted that adherence to postoperative follow-up consults improved excess
weight loss outcomes (Shen et al. 2004). Also, the provision of bimonthly in-person or telephone-
based dietary interventions (15 min) by a dietician improved short-term (6 months) weight loss
outcomes, positively modified dietary composition, increased cognitive restraint, and decreased dis-
inhibition and hunger (Sarwer et al. 2012). Similarly, adherence to a postoperative dietary program
or attendance to a dietary behavioral counseling program is associated with improved dietary habits
and excess weight loss; furthermore, they assist with decreasing the occurrence of premature gastric
emptying, dehiscence, and pouch dilation (Halverson and Koehler 1981; Shah et al. 2013). Others
have reported similar findings (Robinson et al. 2014; Win et al. 2014).
The variable effect of presurgical counseling on weight loss outcomes precludes its wide-
spread incorporation, whereas short-term postsurgical interventions have provided the greatest
benefit and warrant inclusion. Furthermore, the addition of long-term counseling during the
aftermath of the initial sharp postoperative weight decline (17.9 months), when there is a pro-
pensity for weight regain, is an avenue worth considering (Kofman, Lent, and Swencionis 2010;
Sarwer et al. 2012).
VITAMINS AND MINERALS

Vitamins and minerals assist with a myriad of vital functions that are associated with weight mainte-
nance (Schrager 2005). Furthermore, many have cardioprotective (e.g., vitamin C and possibly Ca++)
and antiobesity effects (e.g., Ca++) (Li et al. 2012; Schrager 2005). When the homeostatic milieu is
disrupted, to where ingestion becomes inadequate, symptomatology associated with debilitating
sequel becomes apparent. Thus, recognizing and understanding common nutritional deficiencies in
bariatric patients are prudent.
Vitamin D, Parathyroid Hormone, and Calcium

The prevalence of hypovitaminosis D is heightened in the bariatric population and is common pre-
surgery. Deficiencies in vitamin D reduce calcium uptake, which causes a rise in PTH (Johnson
et al. 2006; Ybarra et al. 2005). Long-standing perturbations in vitamin D may in turn lead to
metabolic bone disease, which is most common among variants of biliopancreatic diversion and
long-limb (>100 cm) Roux-en-Y gastric bypass (Johnson et al. 2006). Subsequently, bone mineral
density scans are recommended at 12 months, 24 months, and annually thereafter for Roux-en-Y
gastric bypass and biliopancreatic diversion variants (Heber et al. 2010).
As it pertains to Roux-en-Y gastric bypass, the prevalence of vitamin D deficiency has been
noted to be between 23% to 86% and 26% to 86% in the preoperative and postoperative setting,
respectively (Aasheim et al. 2009; Coupaye et al. 2014; Gehrer et al. 2010; Ybarra et al. 2005). In
relation, the observed deficiency rate in biliopancreatic diversion with duodenal switch and bilio-
pancreatic diversion subjects has been documented as being as low as 29% preoperatively and high
as 63% postoperatively (Aasheim et al. 2009; Homan et al. 2015; Newbury et al. 2003; Slater et al.
2004). Similarly, hypocalcemia and subsequent secondary hyperparathyroidism were prevalent in
aforementioned trials (Homan et al. 2015; Newbury et al. 2003; Slater et al. 2004). Interestingly,
sleeve gastrectomy patients have also been demonstrated to be deficient in vitamin D and the preop-
erative (23%–93%) and postoperative (32%–90%) prevalence has rivaled or exceeded Roux-en-Y
gastric bypass and variants of biliopancreatic diversion (Capoccia et al. 2012; Coupaye et al. 2014;
Damms-Machado et al. 2012; Gehrer et al. 2010; Moizé et al. 2013). However, the rate tended
to decrease during the aforementioned time points (Coupaye et al. 2014; Damms-Machado et al.
2012), whereas in Roux-en-Y gastric bypass and biliopancreatic diversion variants, it was largely
maintained or increased. Consequently, the incidence postoperatively is largely dictated by the pre-
operative rate and will either be worsened or maintained, depending on screening, diagnostic, surgi-
cal, and nutritional factors.
The minimal recommended daily dosage for Roux-en-Y gastric bypass, laparoscopic adjustable
gastric banding, and sleeve gastrectomy is defined as being 1200–1500 mg and 3000 international
units (IU) for calcium and vitamin D, respectively; in cases of severe malabsorption, 50,000 IU 1–3
times per week may be needed (Mechanick et al. 2013). Scopinaro recommended ingestion of 2 g/d
of calcium, along with monthly intramuscular vitamin D supplementation of 400,000 IU for bilio-
pancreatic diversion patients (Scopinaro et al. 1998). Preoperative vitamin D levels should dictate
the specific dosage as some subjects may require more aggressive prophylactic treatment. Goldner
outlined that all subjects with baseline vitamin D levels of >62.5 nmol/L achieved >75 nmol/L post-
operatively, whereas only half did with a level <62.5 nmol/L; dosage level was 800, 1200, or 5000
IU daily in Roux-en-Y gastric bypass patients (Goldner et al. 2009).
Iron
Preoperatively, subclinical inflammation and the attendant increases in hepcidin will potentiate iron
malabsorption (Aeberli, Hurrell, and Zimmermann 2009; Tussing-Humphreys et al. 2010), while
hypochlorhydria and the circumvention of the small bowel will further accentuate this occurrence
postoperatively (Von Drygalski and Andris 2009). Therefore, iron deficiency will be most prominent
in Roux-en-Y gastric bypass and malabsorptive procedures (Gehrer et al. 2010; Homan et al. 2015;
Vargas-Ruiz, Hernández-Rivera, and Herrera 2008). However, sleeve gastrectomy subjects have
also been shown to be deficient pre- (3%–29%) and post-surgery (18%–37.9%) (Damms-Machado
et al. 2012; Gehrer et al. 2010). Indeed, anemia can also manifest and a mean corpuscular volume
of <83 fentoliters is indicative of an iron-related etiology (Homan et al. 2015; Northrop-Clewes and
Thurnham 2013; Vargas-Ruiz, Hernández-Rivera, and Herrera 2008). Initial and secondary-phase
treatments include coadministration of 300 mg of ferrous sulfate 2–3 times/d, with vitamin C and
parenteral iron administration, respectively (Heber et al. 2010).
Zinc
Relative to normal-weight individuals, obesity is associated with increased urinary zinc excretion and
moderate levels of plasma and erythrocyte zinc concentrations (Marreiro, Fisberg, and Cozzolino 2004).
Even prior to surgery, occult deficiencies can be present, with incidence ranging from 14% to 55%
(Gehrer et al. 2010; Gobato, Seixas Chaves, and Chaim 2014). Postoperatively, the rate increased in
biliopancreatic diversion variants, Roux-en-Y gastric bypass, and sleeve gastrectomy. Particularly, the
prevalence ranged from 34% to 61% (Gehrer et al. 2010; Gobato, Seixas Chaves, and Chaim 2014;
Homan et al. 2015; Slater et al. 2004). With respect to Roux-en-Y gastric bypass and sleeve gastrectomy,
administration of zinc gluconate (30 mg/d) drastically attenuated the occurrence (Gehrer et al. 2010).
Vitamin K
Although rare, vitamin K deficiencies have been observed in biliopancreatic diversion with duode-
nal switch or biliopancreatic diversion patients, with the incidence and degree increasing from
1 (51%) to 4 (68%) years (Slater et al. 2004). Congruently, Homan found that 60% of the cohort
were deficient at 3.5 years (Homan et al. 2015).
Vitamin A
Fat-soluble vitamin deficiencies are prominent in biliopancreatic diversion with duodenal switch
and biliopancreatic diversion patients. As such, Vit A deficiency is as high as 69% and as low as
28% (Homan et al. 2015; Slater et al. 2004). Furthermore, with elapsing time, Vit A levels decrease
(Aasheim et al. 2009; Slater et al. 2004). Consequently, monitoring for nyctalopia and other ocular-
related symptoms of Vit A deficiency is indicated.
Vitamin B12
When co-ingested with food, cobalamin or vitamin B12 is cleaved from peptides by gastric acid and
proteases, which then bind with R proteins that are abundant in saliva. Thereafter, pancreatic secre-
tions digest the R proteins in the duodenum and allow cobalamin to bind with intrinsic factor, which
is absorbed at the distal ileum (Carmel et al. 1969; Herbert 1988). However, due to the anatomical
alterations to the gastrointestinal tract, this sequential system is disturbed in Roux-en-Y gastric
bypass. Specifically, the etiology of cobalamin deficiency is mediated by attenuated secretions of
intrinsic factor and subsequently reduced binding with cobalamin (Marcuard et al. 1989), or from
reduced gastric acid secretion (Smith et al. 1993).
Preoperatively, the prevalence of vitamin 12 deficiency (<10%) has been low in biliopancre-
atic diversion variants, sleeve gastrectomy, and Roux-en-Y gastric bypass (Coupaye et al. 2014;
Damms-Machado et al. 2012; Gehrer et al. 2010; Vargas-Ruiz, Hernández-Rivera, and Herrera
2008). Likewise, only one study with Roux-en-Y gastric bypass subjects observed a high occurrence
(58%) in the postoperative setting (Gehrer et al. 2010), while others have noted a much smaller rate
among the aggregate (<20%, absent, or vitamin excess) (Aasheim et al. 2009; Coupaye et al. 2014;
Damms-Machado et al. 2012; Gehrer et al. 2010; Gobato, Seixas Chaves, and Chaim 2014; Vargas-
Ruiz, Hernández-Rivera, and Herrera 2008). The attenuated rates are attributed to intramuscular
injections of cyanocobalamin and a lower prevalence preoperatively (Aasheim et al. 2009; Gehrer
et al. 2010; Gobato, Seixas Chaves, and Chaim 2014).
Congruently, patients respond well to large oral doses of cyanocobalamin therapy, in relation to
parenteral administration (Kuzminski et al. 1998). Even in the presence of hypochlorhydria, crystal-
line vitamin 12 is absorbed effectively (Smith et al. 1993). For example, in sleeve gastrectomy and
Roux-en-Y gastric bypass patients, administration of 350 μg/d crystalline vitamin 12 normalized
serum vitamin 12 levels (Rhode et al. 1995, 1996).
Folate
Similar to iron, screening and supplementing for folate is recommended in menstruating women
(Mechanick et al. 2013). In sleeve gastrectomy and Roux-en-Y gastric bypass subjects, folate defi-
ciencies are less prominent pre- (3%–7%) and postoperatively (up to 22%), which are most likely
attributed to dietary habits; however, the incidence was highest among sleeve gastrectomy subjects
(Coupaye et al. 2014; Damms-Machado et al. 2012; Gehrer et al. 2010). Conversely, others have
indicated that folate deficiencies were absent (Vargas-Ruiz, Hernández-Rivera, and Herrera 2008).
Multivitamin preparations may include 400 μg of folate or up to 1000 μg/d as a secondary form of
therapy (Heber et al. 2010).
Vitamin B6
Vitamin B6 deficiency may be precipitated by systemic inflammation, which is characteristic of obe-
sity (Vasilaki et al. 2008). Deficiencies have been noted in sleeve gastrectomy subjects up to 17% post-
operatively and 19% preoperatively (Coupaye et al. 2014; Damms-Machado et al. 2012). Others have
observed no such deficiency pre- and postoperatively (Gehrer et al. 2010). Similarly, in biliopancreatic
diversion with duodenal switch (28%–15%) and Roux-en-Y gastric bypass (16%–10%) subjects, the
number of deficiencies decreased from pre- to post-surgery (Aasheim et al. 2009).
Thiamine
In a retrospective analysis, 49% of Roux-en-Y gastric bypass subjects were deficient and the authors
posited that an altered microbial bowel milieu was causal, due to high serum folate levels and a posi-
tive glucose-hydrogen breath test (Lakhani et al. 2008). Dichotomously, in biliopancreatic diversion
with duodenal switch (3%–0%) and Roux-en-Y gastric bypass (0%–10%) subjects, the prevalence
of pre- and postoperative deficiencies was negligible (Aasheim et al. 2009).
CONCLUSION
Bariatric procedures are effective in inducing weight loss, but the specific procedure that is chosen
should be predicated on the patient’s goals, obesity, and comorbidity status. Alone, bariatric pro-
cedures may have limited effectiveness, especially with regard to laparoscopic adjustable gastric
banding. Thus, highlighting the need for bimodal-based therapeutic interventions such as bariatric
surgery and lifestyle modification is most prudent. Postoperative care encompasses dietary modifi-
cation, physical activity adherence, and routine follow-up visits. However, even so, many patients
will still be obese after the initial 2-year reduction in weight and should be cognizant that weight
regain is common.
FUTURE RESEARCH
With the relative novelty of bariatric procedures, further research needs to discern how nutritional
deficiencies that are present prior to surgery can be exacerbated post-surgery and ascertain how
to effectively counteract them and their debilitating sequel. Furthermore, elucidating preoperative
characteristics that predict postoperative outcomes is needed.
Differences between Bariatric Surgeries

Effects on Metabolic Diseases
Excess Nutritional (e.g., Inducing Diabetes
Surgery Type and Popularity Invasiveness Weight Loss Deficiencies Remission)
I. Roux-en-Y gastric bypass 4 4 4 4
II. Sleeve gastrectomy 3 3 3 3
III. Adjustable gastric banding 2 2 1 2
IV. Biliopancreatic diversion 5 5 5 5
Notes: Although Roux-en-Y gastric bypass (RYGB) is invasive, it frequently fosters diabetes remission, which may occur
irrespective of weight loss outcomes (e.g., incretin effect, hormonal and neural signaling). In comparison, sleeve gastrec-
tomy (SG) is less invasive and the achieved excess weight loss values are often similar to that noted in subjects who
undergo RYGB; however, its effect on metabolic diseases is not always as drastic as that seen in RYGB. In most instances,
nutritional deficiencies are equally frequent in both surgeries. Adjustable gastric banding (AGB) and biliopancreatic diver-
sion (BPD) are less popular than SG and RYGB. BPD is highly invasive and nutritional deficiencies are common, while
AGB can be described as being the antithesis (i.e., directly opposite) of BPD: although AGB is the least invasive procedure
and nutritional deficiencies are not as common, it has fallen out of favor in recent years (especially in the United States).
Patients who undergo AGB have difficulty achieving and sustaining significant weight loss; also, diabetes remission is
least frequent. However, BPD incurs positive effects on metabolic diseases and weight loss is substantial.
1, marginal effect; 5, drastic effect; I, most popular; IV, least popular.
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6 Physical Activity and
Andrea M. Brennan and Robert Ross
CONTENTS
Introduction.................................................................................................................................... 102
Physical Activity and Cardiometabolic Health...............................................................................102
Physical Activity and Abdominal Obesity.................................................................................102
Aerobic Exercise and Abdominal Obesity............................................................................103
Resistance Exercise and Abdominal Obesity........................................................................106
Physical Activity and Insulin Resistance...................................................................................108
Aerobic Exercise and Insulin Resistance..............................................................................108
Resistance Exercise and Insulin Resistance..........................................................................109
Physical Activity and Dyslipidemia...........................................................................................109
Aerobic Exercise and Dyslipidemia......................................................................................110
Resistance Exercise and Dyslipidemia..................................................................................111
Physical Activity and Hypertension...........................................................................................112
Aerobic Exercise and Hypertension......................................................................................112
Resistance Exercise and Hypertension..................................................................................113
Influence of Age, Sex, and Ethnicity on PA-Induced CVD Reduction......................................113
Efficacy and Effectiveness.........................................................................................................113
High-Intensity Interval Training and Cardiometabolic Health..................................................114
Sedentary Time as an Independent Risk Factor for Chronic Disease.............................................114
Sedentary Behavior and Health Risk.........................................................................................115
Examination of Epidemiological Evidence...........................................................................115
Examination of Intervention Studies.....................................................................................117
Conclusion......................................................................................................................................117
References.......................................................................................................................................118
ABSTRACT
The inverse dose–response association between physical activity, morbidity, and mortality has been
firmly established by over four decades of evidence gathered from large, prospective cohort studies.
Accordingly, independent of age, sex, and ethnicity, evidence from intervention trials has estab-
lished that both aerobic- and resistance-type exercises combined with a healthful diet are effective
strategies for reducing cardiometabolic risk factors (abdominal obesity, insulin resistance, dyslipid-
emia, and hypertension) that are associated with chronic disease. The primary aim of this chapter is
to review the trial evidence establishing exercise as a primary strategy for reducing cardiometabolic
risk. In doing so, we consider the acute and chronic effects of exercise as well as evidence regarding
the separate effects of the amount and intensity of exercise on cardiometabolic risk. We conclude
with a review of recent evidence that considers the effect of sedentary behavior on cardiometabolic
health that is independent of physical activity.
101
INTRODUCTION
Current physical activity (PA) guidelines worldwide recommend that adults accumulate 150 minutes/
week of moderate-to-vigorous-intensity PA, a recommendation supported by over 60 years of sci-
entific study based on prospective observational cohorts examining the impact of moderate-to-
vigorous PA on risk for chronic disease end points (Haskell et al. 2007, Tremblay et al. 2011). While
exercise guidelines stem from observational evidence, the evidence for efficacy of PA for improving
intermediate risk factors for disease is derived from a large number of intervention trials that clarify
the specific characteristics of an exercise program (e.g., mode, type, frequency, intensity, amount,
etc.) that are most strongly associated with risk reduction. This chapter will discuss the utility of PA,
including both acute and chronic effects of aerobic and resistance exercise, independent of altera-
tions in caloric intake, as a therapeutic strategy for the reduction of cardiometabolic risk in adults.
The majority of exercise interventions reviewed did not alter caloric intake. Cardiometabolic risk
factors include abdominal obesity, insulin resistance, dyslipidemia, and hypertension; all factors
associated with increased risk for type 2 diabetes and cardiovascular disease. Where possible, the
influence of age, sex, and ethnicity will be considered.
Available evidence regarding potential dose–response relationships between the amount of exer-
cise and its intensity on cardiometabolic risk reduction will be reviewed. Knowledge regarding
dose–response relationships between exercise and cardiometabolic risk has depended largely on a
comparison of groups submitted to varying doses of exercise. More recently, large-scale randomized
controlled trials (RCTs) designed specifically to determine the separate effects of exercise amount
and intensity (exercise dose) have emerged. Exercise amount can be defined both as total minutes
of exercise prescribed and by the energy expended during an exercise session, where exercise inten-
sity is defined relative to an individual’s peak oxygen consumption during exercise (VO2peak).
Manipulating either exercise amount at a fixed intensity or exercise intensity for a fixed amount of
time allows the determination of increasing exercise amount and exercise intensity on cardiometa-
bolic risk factors.
Furthermore, opportunity for time spent in a sedentary state has increased, stemming from tech-
nological advances in the twenty-first century (Colley et al. 2011, Matthews et al. 2008). This has
prompted greater scientific inquiry into the independent effects of sedentary behavior as a unique
risk factor for chronic disease. This chapter will therefore summarize the existing epidemiologi-
cal and intervention literature examining the relationship between sedentary behavior and risk of
chronic disease, independent of moderate-to-vigorous PA.
PHYSICAL ACTIVITY AND CARDIOMETABOLIC HEALTH

Physical Activity and Abdominal Obesity
Dr. Jean Vague was the first to characterize the increased risk of chronic disease among individuals
with a phenotype characterized by excess adiposity in the upper-body or abdominal region by com-
parison to those with a predominance of adipose tissue (AT) in the lower-body region (Vague 1956).
These seminal observations have since been repeatedly confirmed leading to the well-established
notion that abdominal obesity is the phenotype associated with the greatest health risk (Despres and
Lemieux 2006). Abdominal obesity has traditionally been characterized using waist circumference
(WC) alone (Pouliot et al. 1994), or the combination of waist-to-hip ratio (Ferland et al. 1989). The
introduction of radiographic imaging modalities in the early 1980s allowed investigators to further
characterize abdominal obesity by provision of direct measures of abdominal visceral and subcuta-
neous AT depots (Ross et al. 1992). This represented a major advance as it is now firmly established
that both visceral and abdominal subcutaneous AT depots predict diabetes and cardiovascular dis-
ease risk independent of commonly measured cardiometabolic risk factors, including hypertension,
dyslipidemia, and insulin resistance in addition to body mass index (BMI) (Despres and Lemieux
2006, Janiszewski, Janssen, and Ross 2007).
Physical Activity and Cardiometabolic Health 103
Aerobic Exercise and Abdominal Obesity

Evidence from RCTs has firmly established that exercise ranging from 1000 to 4000 kcal/week
energy expenditure in the absence of alterations in energy intake is associated with reductions in
WC (Irwin et al. 2003, Ready et al. 1995, Ross et al. 2000, 2004), visceral AT (Donnelly et al. 2003,
Irwin et al. 2003, Ross et al. 2000, 2004), and abdominal subcutaneous AT (Donnelly et al.
2003, Irwin et al. 2003, Ross et al. 2000, 2004), independent of age and sex. Though the majority
of participants in the preceding studies lost body weight, exercise without weight loss achieved by
consuming energy to compensate for that expended through exercise has also been shown to signifi-
cantly reduce abdominal adiposity (Ross et al. 2000, 2004). Although these observations are based
primarily on studies in white participants, limited evidence confirms similar associations between
exercise-induced reduction in components of abdominal obesity in both Hispanics and blacks
(Church et al. 2007). While exercise reduces abdominal obesity in both men and women, the magni-
tude of response may differ. The findings from a recent meta-analysis suggest that, when examining
the overall effect of aerobic exercise on abdominal obesity, the effect size for reductions in visceral
AT was greater in men compared to women (Vissers et al. 2013). This may be due to higher energy
expenditure in men for a given number of exercise minutes prescribed and/or higher baseline vis-
ceral AT in men compared to women (Vissers et al. 2013). However, few studies included in this
review studied both men and women simultaneously (Vissers et al. 2013), precluding the ability to
directly compare sex-specific responses to the exercise interventions. Further studies designed to
evaluate the impact of sex on WC, visceral AT, and abdominal subcutaneous AT changes are needed.
To isolate the role of exercise intensity on cardiometabolic risk factors, the amount of exercise
measured by energy expenditure is ideally held constant while intensity is manipulated. Similarly,
to isolate exercise amount, intensity is held constant while amount is manipulated. Examination of
Table 6.1 reveals that increasing the intensity of exercise performed does not result in further reduc-
tions in WC, visceral AT, or abdominal subcutaneous AT.
A positive dose–response relationship between the amount of exercise (energy expenditure) and
reduction in the components of abdominal obesity (WC, visceral AT, and abdominal subcutaneous AT)
is observed across studies varying in exercise-induced energy expenditure (Donnelly et al. 2003,
Irwin et al. 2003, Kohrt et al. 1991, Ohkawara et al. 2007, Ready et al. 1995, Watkins et al. 2003).
However, large discrepancies in study designs including inclusion criteria for participants and inten-
sity of exercise performed present a limitation in defining a true dose–response relationship in this
manner. Inspection of Table 6.1 reveals that in large, long-term (>6 months) RCTs specifically
designed to examine the effect of increasing the amount or intensity of exercise on abdominal obe-
sity reduction, higher exercise-induced energy expenditure does not result in greater reductions in
WC. These observations are far more compelling than those observed by comparing exercise groups
between studies, as the interventions’ rigorously controlled and randomized design mitigates the
influence of potential confounders. Thus, this evidence should be weighed more heavily and sug-
gests that exercise reduces WC independent of the amount of exercise.
Two studies have examined the impact of increasing exercise amount on visceral and abdomi-
nal subcutaneous AT, with conflicting findings. Slentz et al. randomized 175 participants to one
of four conditions: (1) low amount, moderate intensity (12 miles at 40%–55% VO2peak/week);
(2) low amount, vigorous intensity (12 miles at 65%–80% VO2peak/week); (3) high amount, vigor-
ous intensity (20 miles at 65%–80% VO2peak); and (4) control (no exercise). Slentz et al. observed
significant reductions in visceral and abdominal subcutaneous AT measured by computed tomog-
raphy scan compared to baseline in the high amount/vigorous intensity group only, suggesting
that higher amounts of exercise were necessary for reduction in visceral and abdominal subcu-
taneous AT (Slentz et al. 2005) (Table 6.1). However, in this study, participants were required to
increase energy intake to compensate for energy expended during exercise, which limits our ability
to examine effects of exercise-induced weight loss on AT depots. Keating et al. also examined the
effect of the amount and intensity of exercise on visceral AT measured by magnetic resonance
104
TABLE 6.1
RCTs Examining Effects of Exercise Amount and/or Intensity on Abdominal Obesity
Duration
Reference Participants Intervention Groups (Weeks) Change in WC Change in ASAT Change in VAT
Slentz Sedentary, middle- Control (no exercise) 32 Control: +3.44 cm 2 Control: +14.19 cm2
et al. (2005) aged, overweight men Low amount/moderate intensity (LAMI, 14 kcal/kg LAMI: −3.44 cm2 LAMI: +2.94 cm2a
and women (n = 120) at 40%–55% VO2peak) LAVI: +9.02 cm2 LAVI: +3.85 cm2a
HAVI: −19.18 cm2a HAVI: −11.59 cm2a
Low amount/vigorous intensity (LAVI, 14 kcal/kg LAMI and LAVI LAMI and LAVI
at 65%–80% VO2peak) significantly different significantly different
High amount/vigorous intensity (HAVI, 23 kcal/kg from HAVI from HAVI
at 65%–80% VO2peak)
Slentz Sedentary, middle- Control (no exercise) 32 Control: +0.8
et al. (2005) aged, overweight men Low amount/moderate intensity (LAMI, 14 kcal/kg LAMI: −1.6 cma
and women (n = 120) at 40%–55% VO2peak) LAVI: −1.4 cma
HAVI: −3.4 cma
Low amount/vigorous intensity (LAVI, 14 kcal/kg No difference between
at 65%–80% VO2peak) exercise groups
High amount/vigorous intensity (HAVI, 23 kcal/kg
at 65%–80% VO2peak)
Ross Sedentary, middle- Control (no exercise) 24 Control: Not reported
et al. (2015) aged, abdominally Low amount/low intensity (LALI; 180 and LALI: −3.9 cma
obese men and 300 kcal/session at 50% VO2peak for women and HALI: −4.6 cma
women (n = 300) men, respectively) HAHI: −4.6 cma
High amount/low intensity (HALI; 360 and No difference between
600 kcal/session at 50% VO2peak, respectively) exercise groups
High amount/high intensity (HAHI; 360 and
600 kcal/session at 75% VO2peak, respectively)
(Continued)
RCTs Examining Effects of Exercise Amount and/or Intensity on Abdominal Obesity
Duration
Reference Participants Intervention Groups (Weeks) Change in WC Change in ASAT Change in VAT
Church Sedentary, overweight/ Control (no exercise) 24 Control: −1.4 cm
et al. (2007) obese postmenopausal 4 kcal/kg per week @ 50% VO2peak 4 kcal/kg: −1.9 cma
women, 45–75 years 8 kcal/kg per week @ 50% VO2peak 8 kcal/kg: −2.9 cma
(n = 427) 12 kcal/kg per week @ 50% VO2peak 12 kcal/kg: −1.4 cma
Physical Activity and Cardiometabolic Health
No difference between
exercise groups
Keating Sedentary, overweight/ Control (no exercise) 8 Control: +0.8 cm Control: +319.7 cm3 Control: +92.67 cm3
et al. (2015) obese men and Low-to-moderate intensity, high volume (LO:HI, LO:HI: −2.5 cma LO:HI: −596.7 cm3a LO:HI: −386.60 cm3a
women (n = 48) 50% VO2peak, 60 minutes, 4 days/week) HI:LO: −2.5 cma HI:LO: −567.8 cm3a HI:LO: −258.38 cm3a
LO:LO: −0.9 cma LO:LO: −165.5 cm3a LO:LO: −212.96 cm3a
High intensity, low volume (HI:LO, 70% VO2peak, No difference between No difference between No difference between
45 minutes, 3 days/week) exercise groups exercise groups exercise groups
Low-to-moderate intensity, low volume (LO:LO,
50% VO2peak, 45 minutes, 3 days/week)
WC, waist circumference; ASAT, abdominal subcutaneous adipose tissue; VAT, visceral adipose tissue.
a Exercise group significantly different from control.
105
–6
WC (cm) Weight (kg)
–5
–4
–3
–2
–1
0
LALI HALI
FIGURE 6.1 Effect of exercise amount on the net change in waist circumference (WC) and body weight fol-
lowing 24 weeks of exercise. WC, waist circumference; LALI, low amount low intensity (180 kcal and 300 kcal/
session for women and men, respectively); HALI, high amount low intensity (360 kcal and 600 kcal/session
for women and men, respectively). (Adapted from Ross, R. et al., Ann. Intern. Med., 162(5), 325, 2015.)
imaging (Keating et al. 2015). They randomized 48 participants to one of four conditions: (1) low-

to-moderate intensity, high volume (60 minutes, 4 days/week at 50% VO2peak); (2) high intensity,
low volume (45 minutes, 3 days/week at 70% VO2peak); (3) low-to-moderate intensity, low volume
(45 minutes, 3 days/week at 50% VO2peak); and (4) control (no exercise). In contrast to Slentz
et al., the authors found that while all exercise groups reduced visceral AT, there were no differences
between groups, suggesting that reductions in visceral AT are independent of the amount of exercise.
As shown in Figure 6.1, the apparently limited impact of increasing energy expenditure on reduc-
tion of abdominal obesity is surprising and difficult to interpret, especially given the doubling of
energy expenditure between some of the exercise conditions (Church et al. 2007, Ross et al. 2015),
but suggests that individuals have a range of treatment options that are efficacious for reducing
abdominal obesity. Additional RCTs that match energy expenditure between groups are needed to
determine the dose–response relationships of exercise amount and intensity with abdominal obesity.
While chronic exercise reduces both visceral AT and total subcutaneous AT depots independent of
sex and age, it has been suggested that a preferential mobilization of lipid from visceral adipocytes
exists (Mourier et al. 1997). However, this depends on whether reductions are presented as relative
to baseline AT amount or as absolute amount lost. Most individuals have a higher absolute baseline
amount of subcutaneous AT compared to visceral AT. Thus, for a given exercise-induced negative
energy balance, the relative reduction in visceral AT is usually greater than subcutaneous AT (Ross
et al. 2000, 2004). Conversely, it is noteworthy that the absolute reduction in subcutaneous AT after
exercise training is generally greater than visceral AT in middle-aged men and women (Ross et al.
2000, 2004). Therefore, whether exercise preferentially reduces visceral AT is unclear and can be
interpreted differently depending on the metric used to interpret the exercise-induced response.
Resistance Exercise and Abdominal Obesity
Examination of the limited evidence in Table 6.2 reveals that resistance exercise training, without altera-
tions in caloric intake, is effective for reducing WC across age groups in overweight or obese adult
men and women without significant changes in body weight (Bateman et al. 2011, Ho et al. 2012,
Stensvold et al. 2010). However, reductions appear to be smaller in magnitude compared to aerobic
TABLE 6.2
Randomized Trials Examining Effect of Aerobic and Resistance Exercise on Abdominal Obesity
Duration
Reference Participants Intervention Groups (Months) Change in WC Change in ASAT Change in VAT
Davidson Sedentary, Control (no exercise) 6 Control: −0.28 kg Control: −0.04 kg Control: +0.02 kg
et al. (2009) abdominally obese, Aerobic exercise (AE): 30 minutes of moderate-intensity RE: −3.18 kga RE: −0.21 kg RE: −0.21 kg
60–80 years old, men treadmill walking (60%–75% VO2peak) 5 times/week AE: −5.08 kga AE: −0.40 kga AE: −0.43 kga
and women (n = 136) Combined: −4.61 kga Combined: −0.40 kga Combined: −0.35 kga
Resistance exercise (RE): 1 set of 9 exercises, each to AE different from RE
volitional fatigue: chest press, shoulder raise, shoulder
flexion, leg extension, leg flexion, triceps extension, biceps
curl, abdominal crunches, modified push-ups, 3 times/week
Combined: AE plus RE, 3 times/week
Bateman Sedentary, overweight Resistance exercise (RE): 3 days/week, 3 sets/day of 8 RE: +0.25 cm
Physical Activity and Cardiometabolic Health
et al. (2011) dyslipidemic men 8–12 repetitions of 8 different exercises targeting major AE: −1.12 cm
and women, aged muscle groups Combined: −2.48 cm
18–70 years Aerobic exercise (AE): ~120 minutes/week at 75% RE different from AT
(n = 196) VO2max (p < 0.10), AE + RE
Combined: AE plus RE different from RE (p < 0.05)
Stensvold Middle-aged adults Control (no exercise) 3 Control: +1.7 cm
et al. (2010) with metabolic Aerobic interval training (AIT): Interval training as AIT: −1.3 cma
syndrome (n = 43) treadmill walking/running, 3 times/week ST: −1.4 cma
Strength training (ST): 3 sets at ~80% 1-RM, target Combined: −0.7 cm
major muscle groups 3 times/week
Combined: AIT 2 times/week, ST 1 time/week
Ho Sedentary, Control (no exercise) 3 Control: −1.2 cm
et al. (2012) overweight or obese Aerobic exercise (AE): 30 minutes on a treadmill at 60% HRR AE: −2.1 cma
men and women Resistance exercise (RE): 30 minutes, 4 sets of 8–12 RE: −2.6 cma
aged 40–66 years repetitions at 10-RM for major muscle groups Combined: −2.6 cma
(n = 64) Combined: 15-minute aerobic exercise and 15-minute RE
WC, waist circumference; ASAT, abdominal subcutaneous adipose tissue; VAT, visceral adipose tissue.
a Significantly different from control.
107
exercise (Davidson et al. 2009). While there seems to be a consistent effect of resistance training on
reduction in WC, the effect of resistance exercise on visceral AT and abdominal subcutaneous AT is less
consistent. Braith et al. summarized several RCTs that demonstrate significant reductions in visceral
AT after resistance exercise training in overweight and obese middle-aged men and women (Braith and
Stewart 2006). However, in a meta-analysis of RCTs that also included overweight and obese adults,
Ismali et al. observed no pooled effect of resistance training on visceral AT reduction compared to con-
trols (Ismail et al. 2012). Davidson et al. (Table 6.2) observed that while both visceral AT and abdominal
subcutaneous AT were significantly reduced in the aerobic exercise and combined aerobic and resis-
tance exercise groups, neither AT depot was significantly different from controls in the resistance exer-
cise group (Davidson et al. 2009). The attenuated effect of resistance exercise training on abdominal
obesity reduction compared to aerobic training is likely explained by the lower energy expended during
resistance exercise. Discrepancies in study findings may be explained by differences in study design,
differences in resistance exercise training programs, and sex-specific responses to resistance exercise.
Physical Activity and Insulin Resistance

Aerobic Exercise and Insulin Resistance
It is well established that a single bout of aerobic-type exercise is associated with marked reduction
in insulin resistance, assessed by plasma glucose, oral glucose tolerance test (OGTT), and muscle
glucose transporter phosphorylation, in adult men and women with normoglycemia, impaired glu-
cose tolerance, and type 2 diabetes (Thompson et al. 2001). Improvements in insulin sensitivity
ranging from 15% to 24% (Mikines et al. 1988) are observed after 1 hour of moderate-intensity
exercise in healthy, normoglycemic adults (Burstein et al. 1990, Mikines et al. 1988) and in adults
with insulin resistance (Perseghin et al. 1996) or type 2 diabetes (Burstein et al. 1990, Devlin et al.
1987). The acute effects of exercise on insulin resistance persist for 20–48 hours (Devlin et al. 1987,
Mikines et al. 1988, Perseghin et al. 1996).
The impact of chronic aerobic exercise on insulin resistance varies depending on the method
of measurement and baseline value for insulin and glucose. Among obese individuals with normal
glucose levels, the findings from two reviews suggest that while exercise did not impact glucose
response during an OGTT, corresponding insulin levels were reduced, suggesting a decreased
insulin-to-glucose ratio and improved insulin action (Ivy 1997, Mann et al. 2014). In addition,
the summary findings of several reviews suggest that aerobic exercise training directly improves
insulin resistance by increasing insulin sensitivity by 40%–85% when measured by hyperinsulin-
emic–euglycemic clamp, independent of weight changes, in overweight or obese men and women
(Dengel et al. 1996, Ross et al. 2000, 2004, Thompson et al. 2001).
The impact of aerobic exercise on blood glucose and insulin measures is more pronounced in
individuals with impaired glucose tolerance and those with type 2 diabetes. The findings reported
in several reviews clearly demonstrate that regular exercise improves glycemic control (HbA1c) in
addition to glucose/insulin levels in individuals with type 2 diabetes (Chudyk and Petrella 2011,
Hawley 2004, Hawley and Lessard 2008, Ivy 1997). Boule et al. performed a meta-analysis includ-
ing 14 (12 aerobic, 2 resistance) exercise training studies and found that exercise training signifi-
cantly reduced HbA1c (net change = −0.66%) among men and women with diabetes independent of
weight change (Boule et al. 2005).
Overall, there appears to be a beneficial effect of increasing exercise intensity on insulin resis-
tance. Swain et al. reviewed five clinical intervention studies comparing the effect of aerobic
exercise varying in intensity on glucose control measures (Swain and Franklin 2006). These trials
generally reported greater improvements after high-intensity exercise (>60% VO2max) compared
with moderate-intensity exercise (40%–55% VO2max) in measures including fasting glucose and
the insulin response to an OGTT (Swain and Franklin 2006). More recently, Ross et al. conducted
an RCT examining the separate effects of exercise amount and intensity on glucose tolerance
in abdominally obese adults (Ross et al. 2015). The primary finding of this trial was that in
overweight or obese men and women, the benefit of reducing the 2-hour glucose response was
restricted to the high intensity (75% of VO2max) group.
The mechanisms of action by which exercise affects insulin resistance have been described in
several reviews (Goodyear and Kahn 1998, Henriksen 2002, Ivy 1997). These include increased
glycogen synthase, increased glucose transporter-4 protein and messenger RNA expression, includ-
ing increased translocation to the plasma membrane to enhance glucose uptake, and improved mus-
cle capillary density that enhances glucose delivery to the muscle during exercise. Additionally,
changes in body composition, in particular reductions in abdominal adiposity, have been postulated
to enhance exercise-related improvements in insulin sensitivity (Ross et al. 2000, 2004).
Resistance Exercise and Insulin Resistance

Evidence for an acute effect of resistance training on insulin sensitivity is scarce. However, there
is limited evidence for improvements in whole-body insulin sensitivity assessed by an intravenous
insulin tolerance test in healthy, sedentary, normal-weight young adult men that persists 24 hours
after an acute bout of resistance exercise (Koopman et al. 2005).
The effect of chronic resistance training on glucose and insulin metabolism is equivocal. The find-
ings reported in some studies suggest that there is no significant effect of resistance exercise training
on plasma measures of insulin and glucose metabolism (e.g., fasting glucose and insulin) in both
middle-aged and older adult men and women (Bateman et al. 2011, Davidson et al. 2009). However,
investigations measuring changes in insulin action at the muscle (glucose uptake) show that resis-
tance exercise training increases insulin-mediated glucose uptake in several study groups primarily
including Caucasian individuals but ranging in age and health status (Tresierras and Balady 2009).
The effect of resistance training on insulin and glucose metabolism is apparent in men and women
with type 2 diabetes (Tresierras and Balady 2009). The findings from several studies suggest that
resistance training reduces hemoglobin A1c in diabetic men (Braith and Stewart 2006, Dunstan et al.
2002, Eriksson et al. 1997, Honkola, Forsen, and Eriksson 1997) and women (Dunstan et al. 2002,
Honkola, Forsen, and Eriksson 1997), regardless of age. Two large RCTs described in Table 6.3 were
designed to determine the separate impact of aerobic, resistance, and combined aerobic and resis-
tance training on HbA1c in individuals with type 2 diabetes. The Diabetes Aerobic and Resistance
Exercise study was the first controlled study with adequate power to compare changes in HbA1c
after aerobic, resistance, or combined aerobic and resistance training (Sigal et al. 2007). The primary
finding was that a reduction in HbA1C was observed in all exercise groups compared to controls, but
the improvement was greater within the combined group compared to resistance or aerobic training
alone. A limitation of this trial was that participants in the combined exercise group were required
to perform both the aerobic and resistance exercise prescription, which doubled the total time for
exercising for that group. Therefore, it is difficult to discern the effect of the training per se, indepen-
dent of exercise time. Church et al. addressed this limitation by examining aerobic, resistance, and
combined aerobic and resistance training of similar weekly training duration (Church et al. 2010)
(Table 6.3). In contrast to Sigal et al., they found that only the combined aerobic and resistance train-
ing group significantly improved HbA1c compared to the control group (Church et al. 2010). This
recent evidence suggests that resistance training alone is not efficacious for improving glucose con-
trol in those with type 2 diabetes. That the aerobic exercise group in this trial did not improve gly-
cemic control may be due to participant characteristics, namely, longer duration of diabetes, higher
proportions of women and nonwhite individuals, 18% of participants being treated with insulin, and
the allowance for medication changes throughout the trial (Church et al. 2010).
Physical Activity and Dyslipidemia

Abnormalities in blood lipid concentrations, including hypertriglyceridemia, low high-density lipo-
protein–cholesterol (HDL-C), and elevated low-density lipoprotein–cholesterol (LDL-C), are linked
to other cardiometabolic risk factors such as abdominal obesity (Horton 2009) and insulin resistance
TABLE 6.3
RCTs Examining Effect of Aerobic and Resistance Exercise on Glycemic Control in T2D
Study Duration
Study Participants Study Design (Weeks) Change in HgA1c
Sigal et al. Adults aged 39–70 Control (no exercise) 22 Control: +0.07%
(2007) years with type 2 Aerobic training (AT): 45 minutes AT: −0.43%a
diabetes (n = 251) at 75% HRmax, 3 times/week RT: −0.30%a
Resistance training (RT): 2–3 sets Combined: −0.90%a
of 7 exercises on weight Combined different
machines, 3 times/week from both AT and RT
Combined: Full aerobic training
plus full RT program
Church et al. Sedentary men and Control (no exercise) 36 Control: +0.11%
(2010) women with type 2 Aerobic (AT): 12 kcal/kg per week AT: −0.08%
diabetes (n = 262) Resistance (RT): 2 sets of 4 upper RT: −0.09%
body exercises, 3 sets of 3 leg Combined: −0.27%a
exercises, and 2 sets each of
abdominal crunches and back
extension, 3 days/week
Combined: 10 kcal/kg per week
plus RT (reduced) 2 times/week
a Significantly different from control.
(Howard et al. 1998). Furthermore, an atherogenic lipid profile has been shown to predict cardiovas-
cular disease and cardiovascular disease mortality in middle-aged adults with type 2 diabetes, inde-
pendent of other cardiovascular disease risk factors such as fasting plasma glucose (Lehto et al. 1997).
Aerobic Exercise and Dyslipidemia

The impact of aerobic exercise on acute and chronic blood lipid responses differs. Holoszy et al.
were one of the first to suggest that a single bout of exercise can significantly reduce triglycer-
ides (TG) and increase HDL-C in hypertriglyceridemic, middle-aged sedentary men (Holoszy et al.
1965). The observations from numerous studies in both trained and untrained individuals extend
these findings by illustrating that the acute effect of exercise (24–48 hours after a single exercise
bout) ranges from a 10% to 25% decrease for TG levels, and a 7% to 15% increase in HDL-C
(Bounds et al. 2000, Crouse et al. 1997, Grandjean, Crouse, and Rohack 2000). Not surprisingly,
the greatest change is observed among individuals with higher or lower baseline levels of TG and
HDL-C, respectively. Additionally, there is a positive correlation between exercise-induced energy
expenditure and change in plasma TG and HDL-C, independent of weight change (Cullinane et al.
1982). However, increases in exercise intensity have not been shown to have an appreciable effect
on TG or HDL-C. In contrast to TG and HDL-C, we do not observe a similar effect of acute exer-
cise on changes in LDL-C (Crouse et al. 1997, Grandjean, Crouse, and Rohack 2000).
Several reviews and meta-analyses have summarized the relationship between chronic aero-
bic exercise training and blood lipid response (Durstine et al. 2001, Katzmarzyk et al. 2001,
Kelley et al. 2011, Leon and Sanchez 2001). Overall, there is evidence for a beneficial effect of
exercise on HDL-C and TG in adult men and women; however, observations are inconsistent. A
meta-analysis of exercise training studies in adult men and women ranging in body weight and
blood lipid status observed a significant 4.6% increase in HDL-C and 3.7% decrease in TG when
diet was unchanged (Kelley et al. 2011). These findings are in agreement with those examining
obese, overweight adults with and without dyslipidemia (Leon and Sanchez 2001). These studies
differ from others wherein the effect of exercise on blood lipids was small (Katzmarzyk et al. 2001).
There are several potential explanations for the disparities reported. First, there is great heterogene-
ity in response to exercise both between and within studies that influence the overall interpretation.
This variation in response is likely due to the confounding effects of heritability on change in blood
lipid levels (Katzmarzyk et al. 2001). Additionally, potential flaws including the absence of a control
group, the timing of the blood measurements in relation to the last exercise session, sex differences
in response (discussed in the following), and effects of simultaneous dietary intervention and weight
loss may have impacted overall observations (Katzmarzyk et al. 2001). When examining exercise
interventions combined with caloric restriction, stronger improvements in blood lipid levels are
seen, indicating the importance of diet in regulating blood lipids (Durstine et al. 2001).
Whether men and women experience different lipid responses to exercise training is unclear, as
some have shown that HDL-cholesterol changes are greater in men compared to women (Stefanick
1999), while others have not (Kokkinos and Myers 2010, Leon and Sanchez 2001). Furthermore,
evidence from the HERITAGE study, wherein 675 sedentary white and black men and women par-
ticipated in 20 weeks of exercise, concluded that exercise-induced changes in HDL-cholesterol,
specifically, are not related to age, ethnicity, or sex (Leon et al. 2002).
While a clear dose–response relationship between the amount of exercise and intensity on lipid
changes has yet to be established, overall, potential changes in lipid markers seem more related
to exercise amount and not intensity (Kraus et al. 2002). Swain et al. reviewed eight clinical trials
including participants ranging in age, sex, and health status that examined the impact of exercise
intensity on lipid profiles (Swain and Franklin 2006). Generally, they found no effect of exercise
intensity on improvements in lipid profile markers. However, the majority of reviewed studies had
small sample sizes and were not adequately powered to detect an effect. Kraus et al. extended pre-
vious observations in a large RCT (described in Table 6.1) examining the effect of the amount of
exercise and intensity on lipid profile markers in adult men and women with dyslipidemia (Kraus
et al. 2002). They observed that individuals who exercised at a higher weekly energy expenditure
saw greater improvements in lipoprotein variables including HDL-C and TG compared to those
at lower energy expenditures, and these changes were not related to exercise intensity. However,
this study was limited by its design, wherein the amount of exercise was not equal across low and
high intensity groups (Kraus et al. 2002). Further research is needed to clarify the effect of exercise
intensity on reduction in lipids.
While it appears that improvements in HDL-C and TG are correlated with weight loss, there is
some evidence to suggest that improvements can occur independent of body weight changes (Carroll
and Dudfield 2004, Durstine et al. 2001, Kraus et al. 2002). Exercise intervention trials designed to
maintain body weight reduced TG levels by 5%–35% (Gan et al. 2003, Leon and Sanchez 2001) and
increased HDL-C levels by 3%–5% (Leon and Sanchez 2001, Thomas et al. 2000). Many studies
included in the preceding reviews, however, incorporate some amount of weight loss, making it dif-
ficult to separate the independent effects of PA and weight loss on blood lipid levels.
Overall, there is little evidence to suggest that aerobic exercise beneficially impacts LDL-C
levels (Durstine et al. 2001). However, the benefit of PA may not be seen from the absolute improve-
ment in lipid levels, but rather on the characteristics of lipoproteins that carry cholesterol in the
blood (Kraus et al. 2002). For example, after 24 weeks of exercise in 111 sedentary overweight
men and women, aerobic exercise increased LDL particle size, with larger particles shown to be
less likely to contribute to cardiovascular disease (CVD) than smaller denser LDL (Krauss 2014,
Kraus et al. 2002).
Resistance Exercise and Dyslipidemia

The effect of resistance exercise training on lipid profile is also unclear. While findings are incon-
sistent, most intervention studies in healthy adults show no improvement in lipid profiles after resis-
tance training (Braith and Stewart 2006, Manning et al. 1991, Smutok et al. 1993). This is seen
across a range of ages in both men and women. Comparing resistance training, aerobic training, and
combination of aerobic and resistance training, Bateman et al. observed significant changes in TG
after both aerobic and aerobic plus resistance training; however, the change in TG was not greater in
aerobic plus resistance training, indicating that aerobic exercise may be the more important stimulus
for change in TG (Bateman et al. 2011). It is known that greater exercise-induced weight and fat
loss is associated with greater improvement in lipid profiles (Durstine et al. 2001). In this study, the
aerobic training group, due to increased energy expenditure, experienced greater reduction in body
weight and WC than the resistance training group, which may explain the lack of change in lipids in
the latter group. Conversely, Kelley et al. conducted a meta-analysis of RCTs using resistance exer-
cise training lasting greater than 4 weeks and found statistically significant improvement for total
cholesterol (TC), LDL, and TG, but not for HDL-C (Kelley and Kelley 2009). Many of the existing
studies were completed in younger individuals with normal baseline lipid levels, and this population
may be less likely to show a significant response.
Physical Activity and Hypertension

Hypertension is an important predictor of CVD mortality, and relatively modest reductions in blood
pressure are associated with reduced risk for cardiovascular disease morbidity and mortality (Cook
et al. 1995, He and Whelton 1999).
Aerobic Exercise and Hypertension

The acute effects of aerobic exercise on blood pressure are well established (Kenney and Seals
1993). While normotensive individuals experience postexercise hypotension, the effect is more pro-
nounced among those with hypertension, wherein reductions in systolic blood pressure (SBP) and
diastolic blood pressure (DBP) of −11 and −6 mmHg, respectively, have been observed (Pescatello
et al. 1999, Rueckert et al. 1996). Postexercise hypotension occurs immediately after exercise and
can last for 22 hours postexercise (Brandao Rondon et al. 2002).
Reductions in blood pressure in response to chronic aerobic exercise training are also well estab-
lished (Arroll and Beaglehole 1992, Cornelissen and Fagard 2005, Fagard 1999, Hagberg, Park, and
Brown 2000, Kelley 1995, Kelley and Kelley 1999, Whelton et al. 2002). A recent meta-analysis
summarizing observations from 93 RCTs lasting greater than 4 weeks in healthy adults showed
reductions in SBP (−3.5 mmHg) and DBP (−2.5 mmHg) after endurance exercise (Cornelissen and
Fagard 2005). Similar to acute exercise, BP reductions were greater in individuals with hypertension
(SBP, −8.3 mmHg; DBP, −5.2). Most of the studies included in the analysis used at least 40 minutes
of moderate-intensity PA 3 times/week. These observations extended seminal work by Cornelissen
et al. who also observed significant reductions in SBP and DBP in response to exercise in both nor-
motensive and hypertensive adults (Cornelissen and Fagard 2005). The beneficial effects of exercise
on blood pressure were observed independent of age, sex, and BMI.
Studies examining the impact of exercise amount and intensity on blood pressure are limited.
Swain et al. reviewed four aerobic exercise trials and found no overall effect of exercise intensity on
SBP; however, DBP changed to a greater extent following vigorous exercise compared to moderate-
intensity exercise (Swain and Franklin 2006). Cornelissen et al. also reviewed the impact of exercise
intensity in training studies on blood pressure response (Cornelissen et al. 2010). The authors found
no consistent effect of exercise intensity on blood pressure reduction after aerobic exercise training,
with exercise intensity ranging from 40% to 70% of maximal exercise performance (Cornelissen
et al. 2010). The amount of exercise does not appear to have a clear impact on the reduction in blood
pressure (Kelley 1995, Kelley and Kelley 1999, Whelton et al. 2002).
There is some evidence that women with hypertension experience greater reductions in blood
pressure in response to exercise training compared to men (Hagberg, Park, and Brown 2000).
A recent review observed an average weighted reduction of 14.7 and 10.5 mmHg in SBP and DBP,
respectively, in women, compared to 8.7 and 7.8 mmHg reduction in men in response to exercise
training (Hagberg, Park, and Brown 2000). Few studies have examined ethnic differences in blood
pressure response. However, a recent meta-analysis observed that, compared to Caucasian individu-
als, black participants experienced greater reductions in SBP and Asian participants experienced
greater reductions in DBP (Whelton et al. 2002).
Resistance Exercise and Hypertension
Resistance exercise training also appears to have a beneficial effect on SBP and DBP in adult
men and women (Braith and Stewart 2006, Cornelissen et al. 2011, Kelley and Kelley 2000). Two
meta-analyses have been conducted to examine this relationship (Cornelissen et al. 2011, Kelley
and Kelley 2000). Kelley observed a 3 mmHg decrease in SBP and DBP following progressive
resistance exercise training in previously inactive adult men and women ranging in age and BMI
(Kelley and Kelley 2000). Cornelissen observed similar relationships; however, while there was a
significant reduction in SBP and DBP in normotensive and pre-hypertensive study groups, the effect
was not significant in hypertensive groups. There was also a greater effect of resistance exercise on
SBP in study groups younger than 50 years compared to those older than 50 years, as well as a larger
decrease in SBP and DBP with isometric handgrip strength training compared to dynamic resistance
training study groups (Cornelissen et al. 2011).
Influence of Age, Sex, and Ethnicity on PA-Induced CVD Reduction

While evidence concerning the impact of age, sex, and ethnicity on exercise-induced changes in
individual cardiometabolic risk factors is scarce, a recent review summarized the influence of these
factors on PA-related changes in overall cardiovascular disease risk reduction (Shiroma and Lee
2010). Overall, it appears that the inverse correlation between PA and CVD risk seen in middle-aged
adults is also seen to a similar magnitude in older men and women (Shiroma and Lee 2010). Indeed,
in the Women’s Health Initiative, the authors categorized participants into three age groups (50–59,
60–69, and 70–79 years). All three age groups had comparable relative risks of CVD when compar-
ing the most active with least active participants (Manson et al. 2002).
Both men and women experience inverse associations between PA and risk of developing CVD;
evidence suggests that the most active women experience a 40% risk reduction compared to the least
active women, whereas risk reduction is 30% in active men, suggesting that the inverse association
between PA and CVD is stronger in women (Manson et al. 2002). Very few studies have examined
the ethnic differences in CVD risk response to PA, as the majority of studies were conducted in
white individuals. The few studies that have examined nonwhite populations observed no significant
interactions among races (Gregg et al. 2003).
Efficacy and Effectiveness

The majority of the evidence reviewed from RCTs derives from tightly controlled efficacy studies.
The findings from these studies support the notion that for most cardiometabolic risk factors, exer-
cise has beneficial effects. However, while the efficacy of exercise for improving cardiometabolic
health is established, the observed inability for individuals to sustain an exercise program has led
some to conclude that exercise is not an effective model for sustained change in health risk (Ross
et al. 2012, Wadden et al. 2011). The question then is not whether exercise will decrease cardiometa-
bolic risk, but rather, can these changes realistically be maintained over the long term? The answer
to this question remains elusive. The PROACTIVE trial investigated the effectiveness of a 2-year
behaviorally based lifestyle (PA and diet) program aimed at reducing obesity and metabolic risk fac-
tors in abdominally obese adults in primary care settings (Ross et al. 2012). Participants were ran-
domized to receive usual care, in which physicians were asked not to change their routine counseling
approach for obese persons, or participate in the behavioral intervention consisting of intensive indi-
vidual counseling from health educators. The authors observed significant reductions in both body
weight and WC in the intervention compared with usual care group. However, only changes in WC
were sustained at 24 months in men, but not women. There were no significant changes at 24 months
in any of the other cardiometabolic risk factors, including lipoproteins, glucose, and blood pressure
in either men or women (Ross et al. 2012). This trial and others (Wadden et al. 2011) highlight the
difficulty of maintaining long-term lifestyle changes, as well as the critical gaps in knowledge con-
cerning the most effective way to sustain behavior change.
High-Intensity Interval Training and Cardiometabolic Health

While the current national PA guidelines recommending 150 minutes of moderate-to-vigorous-intensity
PA per week are evidence based and widely promoted, the majority of the adult population does not
meet these guidelines (Colley et al. 2011, Matthews et al. 2008). One prominent reason is perceived lack
of time to perform the activity (Reichert et al. 2007). High-intensity interval training (HIIT), which con-
sists of brief periods of high-intensity exercise interspersed by short recovery periods, offers a potential
time-efficient alternative to continuous moderate endurance activity, as the time needed to expend the
same amount of calories is lower at higher intensities (Gibala, Gillen, and Percival 2014).
Mounting evidence over the last three decades has illustrated the cardiometabolic health benefits
of HIIT. Recently, Kessler et al. published a systematic review that discusses the impact of HIIT on
individual cardiometabolic risk factors (Kessler, Sisson, and Short 2012). The authors observed a sig-
nificant improvement in insulin sensitivity, as assessed by hyperinsulinemic–euglycemic clamp, the
homeostasis model assessment of fasting glucose and insulin (HOMA-IR), and an OGTT after HIIT,
in addition to a significant improvement in 2-hour glucose and glucose area under the curve in studies
lasting between 2 and 24 weeks’ duration (Kessler, Sisson, and Short 2012). In studies that compared
the efficacy of HIIT to continuous moderate-intensity aerobic exercise, the authors found similar
improvements in glucose metabolism variables. For serum lipids and lipoproteins, the authors found
a limited effect of HIIT on TC, HDL-C, LDL-C, or TG in the majority of studies reviewed. HIIT last-
ing a minimum of 12 weeks significantly improved blood pressure in those participants not currently
on antihypertensive medication, and the magnitude of reduction was similar to continuous moderate-
intensity exercise. At least 12 weeks of HIIT was also found to be as effective as continuous exercise
for improving anthropometric markers in overweight or obese individuals (Kessler, Sisson, and Short
2012). All of the preceding cardiometabolic benefits were observed with and without changes in body
weight. This suggests that HIIT is effective for improving the majority of cardiometabolic risk factors
to a similar extent as continuous exercise and offers individuals who cite lack of time as a reason for
inactivity a potential solution to achieve their cardiometabolic health goals.
However, there are gaps in knowledge that require further investigation. There is great inconsis-
tency in the protocols used in interventions studying HIIT. The mode (cycling or treadmill), duration
of intervals and breaks, as well as intensity prescribed all varied significantly across studies. The
optimal protocol has yet to be clarified. Furthermore, while proponents of HIIT cite time efficiency
as a benefit, participating in HIIT protocols can take from 20 to 60 minutes including warm-up
(Kessler, Sisson, and Short 2012). Therefore, whether performing HIIT provides a time efficiency
benefit is unclear. Furthermore, whether sedentary, commonly overweight, or obese adults will
sustain HIIT for extended periods is not known and requires further study.
SEDENTARY TIME AS AN INDEPENDENT RISK FACTOR FOR CHRONIC DISEASE

While there is strong and unequivocal evidence that higher levels of moderate-to-vigorous PA lead
to considerable improvements in cardiometabolic health, more recently, great interest has focused
on studying the health risks associated with sedentary behavior (Hamilton, Hamilton, and Zderic
2007, Spanier, Marshall, and Faulkner 2006). Sedentary behavior can be defined as “any waking
behavior characterized by an energy expenditure <1.5 metabolic equivalents (METs) while in a sit-
ting or reclining posture” (Sedentary Behaviour Research Network 2012).
With increased technological advances in the twenty-first century, opportunity to be seden-
tary has risen. Objective data show that North American adults participate in sedentary behavior
7.5–9.5 hours per day, making up 55%–69% of their waking hours (Colley et al. 2011, Matthews
et al. 2008). This has prompted greater scientific inquiry into the independent effects of sedentary
behavior as a unique risk factor for chronic disease.
Much of the evidence supporting sedentary behavior as an independent risk factor is based on epide-
miological studies with varying degrees of quality. Few well-designed intervention trials exist that show
a cause-and-effect relationship between sedentary behavior and health across the life span. Furthermore,
until recently, many of the observed relationships between health and sedentary time were based on
subjective measures of activity whose validity is often poor (Atkin et al. 2012). These methodological
limitations preclude the identification of sedentary behavior as an independent risk factor for disease.
Sedentary Behavior and Health Risk

Examination of Epidemiological Evidence
Several recent systematic reviews and meta-analyses have summarized epidemiological evidence
describing the association between self-reported sedentary behavior and risk for chronic disease,
independent of moderate-to-vigorous PA, in multiple population subgroups (Biswas et al. 2015, de
Rezende et al. 2014, Ekelund et al. 2012, Gardiner et al. 2011, Lee et al. 2012). Most studies report
that increased sedentary time in adults is associated with increased risk for morbidity, including
CVD and incidence of type 2 diabetes, and mortality (all-cause and CVD), in addition to increased
prevalence of risk factors associated with disease (Lee et al. 2012). These observations are similar
in older adults (de Rezende et al. 2014). However, in most studies, after adjusting for moderate-to-
vigorous PA, the magnitude of the association between sedentary time and health risk is reduced,
suggesting that moderate-to-vigorous PA levels are more important for determining health risk than
sedentary time (Lee et al. 2012). While evidence points to a relationship between self-reported
sedentary behavior and disease, investigations using objective measures of sedentary time are less
consistent. For example, Healy et al. studied the independent associations of time spent in moderate-
to-vigorous PA, sedentary time, and clustered metabolic risk cross-sectionally in 169 participants
from the Australian Diabetes, Obesity and Lifestyle Study (Healy et al. 2008). There were sig-
nificant independent associations between sedentary time, moderate-to-vigorous PA, and clustered
metabolic risk score; however, these associations disappeared after adjustment for WC (Healy et al.
2008). It is reasonable to hypothesize that individuals who spend more time in sedentary behaviors
are more likely to have a greater WC due to reduced energy expenditure. The preceding observations
suggest that WC explains risk to a greater degree than sedentary time.
Recent evidence has questioned traditional statistical techniques used to study sedentary behavior
and cardiometabolic health in epidemiological investigations (Chaput et al. 2014, Chastin et al. 2015,
Pedišić 2014). By simply adjusting for moderate-to-vigorous PA statistically, we imply that these
behaviors occur in isolation from one another (Pedišić 2014). However, PA, sedentary behavior, and
sleep occur along a continuum and interact with each other to influence health (Chaput et al. 2014).
For example, reducing one behavior (e.g., sedentary time) necessitates an increase in another behavior
(light-to-vigorous activity), which may interact to influence risk factors for disease. Chastin et al. used
compositional analysis to examine relationships between PA, sedentary behavior, and cardiometabolic
risk factors (Chastin et al. 2015). This type of analysis considers the interaction and co-dependence of
various components of the movement continuum. Using this type of analysis on the National Health
and Nutritional Examination Survey data (N = 1937), the authors observed that the distribution of
behaviors as a whole was significantly associated with WC, TG, plasma glucose and insulin, SBP, and
DBP. The proportion of time spent in moderate-to-vigorous-intensity PA had a stronger effect size for
the majority of cardiometabolic health markers compared to sedentary behavior or light-intensity PA.
Furthermore, the strongest adverse effects on cardiometabolic health outcomes occurred when time
spent in moderate-to-vigorous PA was replaced with sedentary time (Chastin et al. 2015). Overall,
this important study indicates that movement behaviors are dependent on each other and the overall
distribution pattern may be more important and informative than examining behaviors in isolation.
116
TABLE 6.4
Interventions Studying Sedentary Time and Cardiometabolic Risk
Study
Reference Participants Study Design Duration Outcomes Findings
Stephens Young (26 years), Three conditions completed by each participant: 24 hours Insulin action Compared to NO-SIT:
et al. (2011) nonobese, fit men (1) Active, no sitting (high energy expenditure measured by SIT: Insulin action reduced by 39% (p < 0.001)
and women, N = 14 with energy intake matched) (NO-SIT) continuous infusion of SIT-BAL: Insulin action reduced by 18% (p = 0.07)
(2) Low energy expenditure with no reduction [6,6-2H]-glucose
in energy intake (SIT)
(3) Sitting with energy intake reduced to match
low expenditure (SIT-BAL)
Lyden et al. Recreationally active, 7-day sedentary condition, instructed to sit as 7 days Fasting lipids, glucose, 2-hour plasma insulin and area under insulin curve
(2015) young (35 years), much as possible, limit standing/walking, and insulin significantly elevated
normal weight, and refrain from structured exercise OGTT at baseline and No change in lipid concentration
overweight men and immediately after
women, N = 10 sedentary condition
Kozey Keadle Overweight/obese, Participants randomly assigned to: 12 weeks Fasting lipids, BP, EX and EX-rST decreased SBP
et al. (2014) middle-aged (1) Exercise 5 days/week for 40 minutes/ BMI, 2-hour OGTT EX-rST increased insulin sensitivity index by 17.8%
(44 years) men and session at moderate intensity (EX) and decreased insulin AUC by 19.4%
women, N = 57 (2) Reduce sedentary time and increase rST reduced BP
nonexercise PA (rST)
(3) Combination of EX and rST
(4) Control
Examination of Intervention Studies

While epidemiological evidence points to a relationship between sedentary behavior and risk for
chronic disease, evidence from intervention studies that is important for uncovering cause-and-
effect relationships is lacking. Inspection of Table 6.4 reveals that studies in humans are limited
by their short duration and small sample sizes. For example, evidence suggests that one day of
prolonged sitting reduced insulin action in 14 young, nonobese fit men and women. However, when
energy intake was matched to energy expenditure, the adverse effects of sitting were attenuated,
suggesting that positive energy balance and not just sitting per se impacts insulin action (Stephens
et al. 2011). In previously active individuals, 2-hour plasma insulin and insulin area under the curve
following an OGTT were significantly elevated following a sedentary condition lasting 7 days com-
pared to baseline (Lyden et al. 2015).
Interventions of longer duration also show conflicting findings. After a 12-week intervention
examining four sedentary time conditions (Table 6.4), exercise combined with reductions in seden-
tary time significantly increased cardiorespiratory fitness, decreased SBP, increased insulin sensitiv-
ity, and decreased insulin area under the curve, while the group who only reduced sedentary time
saw improvements solely in blood pressure (Kozey Keadle et al. 2014). This suggests that reducing
sedentary time alone, without increasing moderate-to-vigorous-intensity PA, is not effective for
improving the majority of cardiometabolic risk factors.
Overall, there is a paucity of evidence from intervention studies that links increases in seden-
tary behavior to increased risk for disease, independent of moderate-to-vigorous PA. The majority
of evidence that points to a relationship between sedentary behavior and risk for disease, inde-
pendent of activity that meets the consensus guidelines, is observational in nature. This prevents
the determination of cause and effect, which can only be determined by intervention trials that
involve manipulation of sedentary behavior and measurement of subsequent health changes. The
few existing intervention trials described earlier show inconsistent results. Well-designed RCTs
testing whether reductions in sedentary time combined with exercise training improve cardio-
metabolic risk more than exercise training alone are necessary to determine cause and effect
and examine potential mechanisms that can explain any observed relationship. The study design
should be adequately powered to detect between-group differences, and study populations should
include sedentary individuals with overweight or obesity. These studies are required to determine
the strength of a relationship between sedentary behavior and risk for disease.
CONCLUSION
There is strong and unequivocal evidence that regular PA is associated with improvements in
cardiometabolic health across a wide range of individual risk factors. Responses to exercise
may differ according to sex, age, or ethnicity, and accounting for these important factors will
be a critical aspect of future research in the field. The separate effects of increasing exercise
amount or intensity on cardiometabolic risk factors are unclear. It appears that increasing exer-
cise intensity results in greater improvements in insulin resistance only. The effect of increas-
ing exercise amount for a fixed intensity is equivocal, with some studies suggesting greater
improvement in cardiometabolic risk factors, including abdominal obesity, insulin resistance,
and dyslipidemia, and others showing no effect. Additionally, while aerobic exercise results
in greater improvements in all cardiometabolic risk factors, resistance exercise appears to be
effective only for reducing abdominal obesity, insulin resistance in individuals with type 2 dia-
betes, and hypertension. Notably, current evidence points to sedentary behavior as a risk factor
for suboptimal cardiometabolic health. Future research is needed to determine the independent
effect of sedentary behavior and how the overall distribution of PA, sedentary behavior, and
sleep influence health. Notwithstanding, it remains prudent for public health practitioners to
advocate for increases in PA and reductions in sedentary time.
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7 Diet as a Potential Modulator
of Body Fat Distribution
Sofia Laforest, Geneviève B. Marchand, and André Tchernof
CONTENTS
Abbreviations................................................................................................................................. 124
Introduction.................................................................................................................................... 125
Macronutrients and Their Relevance for Body Fat Distribution.................................................... 125
Lipids........................................................................................................................................ 125
Types of Fatty Acids............................................................................................................. 125
Medium-Chain Triglycerides vs. Long-Chain Triglycerides............................................... 126
n-3 Fatty Acids..................................................................................................................... 126
Conjugated Linoleic Acid..................................................................................................... 127
Carbohydrates........................................................................................................................... 128
Percentage of Energy Intake as Carbohydrates.................................................................... 128
Glycemic Index/Load........................................................................................................... 128
Fructose and Sugar-Sweetened Beverages........................................................................... 129
Protein....................................................................................................................................... 129
Percentage of Energy Intake as Protein................................................................................ 129
Branched-Chain Amino Acids.............................................................................................. 130
Dietary Patterns.............................................................................................................................. 131
Food Item Subgroups................................................................................................................ 132
Mediterranean Diet................................................................................................................... 133
Other Nutrients or Food Items....................................................................................................... 134
Alcohol...................................................................................................................................... 134
Dairy Products, Calcium, and Vitamin D.................................................................................. 135
Soy and Isoflavones................................................................................................................... 136
Dietary Fiber and Whole Grains............................................................................................... 136
Vitamins A and C...................................................................................................................... 137
Probiotics and the Gut Microbiota............................................................................................ 137
Infant Feeding Practices................................................................................................................. 137
Conclusion..................................................................................................................................... 139
References...................................................................................................................................... 140
ABSTRACT
Abdominal obesity has been reported to be more closely related to cardiometabolic risk than
excess adiposity per se. Specifically, high abdominal fat accumulation, especially in the vis-
ceral depot, is closely linked to metabolic abnormalities such as insulin resistance and hyper-
triglyceridemia, whereas for a similar body fat mass, subcutaneous fat accretion seems to
confer a neutral/protective effect. Mechanisms regulating body fat distribution are complex
and remain poorly understood. We reviewed the potential contribution of dietary components
123
on body fat distribution independent of body weight and total adiposity. Types and amounts
of fat, carbohydrates and glycemic load, proteins, as well as specific food components with
bioactive functions that could affect fat accretion were examined in detail. According to avail-
able literature, there is no concrete evidence that a single nutrient directly modulates visceral
fat accretion and, by extension, body fat distribution. Most available studies on diet and body
fat distribution seem to point toward a nonspecific effect on total fat accumulation. Yet, some
studies identified dietary components potentially linked to a detrimental fat accretion pattern
(increased visceral/abdominal fat or waist circumference), including fructose, especially in
the form of sugar-sweetened beverages, trans fatty acids, as well as high alcohol intake and
refined/fast-food diets.
ABBREVIATIONS
25(OH)D 25-Hydroxyvitamin D
aP2 Adipocyte Protein 2
BCAA Branched-chain amino acids
BCKD Branched-chain α-keto acid dehydrogenase
BMI Body mass index
C/EBPα CCAAT/enhancer-binding protein alpha
CI Confidence interval
CLA Conjugated linoleic acid
CT Computed tomography
DHA Docosahexaenoic acid
DXA Dual-energy X-ray absorptiometry
EPA Eicosapentaenoic acid
FA Fatty acids
GI Glycemic index
GL Glycemic load
GLUT4 Glucose transporter type 4
HC Hip circumference
hsCRP High-sensitivity C-reactive protein
LCT Long-chain triglycerides
LPL Lipoprotein lipase
MCT Medium-chain triglycerides
MD Mediterranean diet
MEDS Mediterranean score
MRI Magnetic resonance imaging
MUFA Monounsaturated fatty acids
NF-κB Nuclear factor-kappa B
PPARγ Peroxisome proliferator-activated receptor gamma
PUFA Polyunsaturated fatty acids
SAT Subcutaneous adipose tissue
SFA Saturated fatty acids
SMD Standard mean difference
SSB Sugar-sweetened beverages
TNFα Tumor necrosis factor alpha
VAT Visceral adipose tissue
WC Waist circumference
WHR Waist-to-hip ratio
WMD Weighted mean difference
Diet as a Potential Modulator of Body Fat Distribution 125
INTRODUCTION
Obese individuals have an increased risk of developing coronary heart disease, hypertension, type 2
diabetes, and several types of cancers (reviewed in Haslam and James (2005)). However, the risk
of developing these conditions is closely related to body fat distribution patterns. More specifically,
numerous studies now support the notion that excess visceral adipose tissue (VAT) accumulation is
more closely related to alterations in cardiometabolic health, whereas for any given level of total
adiposity, preferential subcutaneous adipose tissue (SAT) accumulation has protective or neutral
effects (reviewed in Tchernof and Després (2013)). Molecular mechanisms affecting lipid storage
sites have not yet been completely elucidated. Hormones, genetic or epigenetic factors, and possibly
diet may all partly contribute to human body fat distribution patterns (Berry et al. 2013).
This chapter reviews scientific evidence on the modulation of body fat distribution, with
visceral fat accumulation as the key indicator, by dietary factors such as macronutrients, food
patterns, and other particular nutrients or food items. The major question to be addressed is
whether nutritional factors can specifically modulate body fat distribution patterns or visceral fat
accumulation beyond what could be accomplished solely by modulating energy intake and total
body fat stores.
We have reviewed articles found in the PubMed database with search terms related to fat accu-
mulation: body fat distribution, peripheral adiposity, visceral fat, abdominal fat, subcutaneous fat,
and body composition. They were individually combined with nutritional keywords such as lip-
ids, carbohydrates, proteins, diet, nutrients, and nutrition. A total of 224 journal articles were kept
after removing duplicates. The articles were selected depending on the following specific criteria:
(1) emphasis was placed on human studies; and (2) body fat distribution had to be measured by
computed tomography (CT), magnetic resonance imaging (MRI), dual-energy X-ray absorptiom-
etry (DXA), ultrasound, or waist and/or hip circumference (WC/HC). Reviews were excluded.
Systematic reviews and meta-analyses were included. Relevant articles from the reference list of
identified papers were added. In total, 170 studies were retained.
MACRONUTRIENTS AND THEIR RELEVANCE FOR BODY FAT DISTRIBUTION

Lipids
Types of Fatty Acids
Fatty acids (FA) are often classified into three broad groups, namely, saturated fatty acids (SFA),
monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA). In the typical
Western diet, C4 to C18 are the major SFA. They are found primarily in animal foods and oils
derived from tropical fruits. MUFA are generally associated with the Mediterranean diet (MD) (see
the section “Mediterranean Diet”) considering that oleic acid, the major component of olive oil, is
the primary MUFA in this common diet. Nuts (almonds, pecans, peanuts, cashew nuts) are also rich
in MUFA. PUFA comprise a large group, including the essential FA that are, linoleic (C18:2n-6)
and linolenic (C18:3n-3) acids. PUFA also refer to n-3 FA such as eicosapentaenoic acid (EPA) and
docosahexaenoic acid (DHA). PUFA can be found in corn oil, sunflower oil, and seafood. Trans FA,
usually formed by commercial incomplete hydrogenation of MUFA, are also a component of the
Western diet and are known to increase cardiovascular disease risk (Mozaffarian et al. 2006).
Larson et al. reported a positive association of body fat mass with total fat, SFA, MUFA, as well
as PUFA intake independent of nonfat energy intake in men and women (Larson et al. 1996). In
regression analyses, SFA intake was a better predictor of body fat mass than total fat intake. VAT
and SAT areas, adjusted for nonfat energy intake, were positively associated with total fat, SFA, and
MUFA (Larson et al. 1996). All these associations became nonsignificant when the authors adjusted
for total body fat mass (Larson et al. 1996). Their results suggest no apparent association between
the FA content of the diet and body fat distribution.
In a longitudinal study in African-Americans and Hispanic-Americans, intake of SFA, MUFA,

and PUFA was not associated with 5-year changes in VAT or SAT area (Hairston et al. 2012).
Consistent with these results, data from a Danish cohort followed for 5 years failed to show an
association between the change in WC, a surrogate measure of VAT, and total intake of fat (Halkjaer
et al. 2006). In a randomized crossover study with overweight men, fat mass in both the trunk and
limbs was increased in the SFA diet group and decreased in the MUFA diet group after 4 weeks,
without differences in total energy intake (Piers et al. 2003). Replacement of PUFA or carbohydrates
by trans FA increased WC in men during a 9-year follow-up, whereas there was no difference when
the substitution was done with SFA or MUFA (Koh-Banerjee et al. 2003). Forouhi and collaborators
found no association between intake of SFA, MUFA, or PUFA and WC (Forouhi et al. 2009). In a
randomized 1-year control trial, participants were provided low-fat dietary advice from dieticians
and half received 30 g of walnuts per day to increase their consumption of PUFA (Tapsell et al. 2009).
Compliance was monitored by a validated diet history interview, 3-day food records coupled with
an analysis of erythrocyte FA composition. The control group preferentially lost VAT at 3 months
while the walnut group lost SAT (Tapsell et al. 2009). There was a trend (p = 0.08) at baseline for a
higher SAT area in the walnut group, which can partially explain this difference (Tapsell et al. 2009).
At 12 months, the walnut group had lost more body fat than the control group, but the difference did
not reach statistical significance (Tapsell et al. 2009).
Taken together, the earlier mentioned studies suggest that the link between FA composition of
the diet and abdominal or visceral fat accretion seems to be mediated by its association with overall
adiposity levels.
Medium-Chain Triglycerides vs. Long-Chain Triglycerides

Medium-chain triglycerides (MCT) have unique properties that distinguish them from long-chain
triglycerides (LCT). These characteristics seem to confer beneficial metabolic effects. MCT, after
cleavage to glycerol and FA, are transported directly to the liver by the portal vein, bypassing incor-
poration into chylomicrons and the lymphatic system (Poppitt et al. 2010). Unlike LCT, the MCT do
not require the carnitine acyltransferase enzyme to be incorporated into the mitochondria, allowing
more rapid β-oxidation, a phenomenon that has been linked with greater energy expenditure and less
weight gain (Bach and Babayan 1982). MCT may also increase satiety after a meal (Van Wymelbeke
et al. 1998, Krotkiewski 2001, Poppitt et al. 2010). Generally, MCT comprise FA from 6 to 10 and
even 12 carbons, since lauric acid (C12:0), structurally classified as an LCT, shows properties that
are similar to caprylic (C8:0) and capric (C10:0) acids. MCT are found primarily in vegetable oils
such as coconut (71%) and palm kernel (48%) (Health Canada 2010).
Many studies have been conducted to elucidate the effect of LCT replacement by MCT on body
weight, body fat mass, WC, SAT, and VAT. Bueno and collaborators, in a recent meta-analysis, found
that replacement of LCT by MCT (at least 5 g) in six randomized control trials reduces WC in both men
and women (weighted mean difference [WMD]: −1.78 cm [95% confidence interval {CI}, −2.4 to −1.1])
(Bueno et al. 2015). In another meta-analysis, a similar result was reported for WC (WMD: −1.46 cm
[95% CI, −2.04 to −0.87]) (Mumme and Stonehouse 2015). In addition, total SAT and VAT were also
investigated. Replacement of LCT by MCT (between <1% and 24% of energy intake) in the diet led
to a moderate loss of both SAT and VAT (standard mean difference [SMD]: −0.46 [95% CI, −0.64 to
−0.27] and −0.55 [95% CI, −0.75 to −0.34], respectively), suggesting a greater loss of VAT in these
trials. Overall, these results strongly suggest that consumption of MCT may be favorable, not only for
body composition, but also toward healthier body fat distribution. Further studies are needed to confirm
the relationship between MCT and reduced VAT area independent of other adiposity measurements.
n-3 Fatty Acids

Consumption of n-3 FA, namely, EPA and DHA, found principally in fatty fish and components of
the MD (see the section “Mediterranean Diet”) has been linked with improvements in insulin resis-
tance and cardiovascular risk factors (Wang et al. 2006, Li 2015). Preliminary results from animal
studies showed possible beneficial effects, independent of body weight. In rats, 7 weeks of a diet
with 17.4% EPA and 10.1% DHA (% of total FA) reduced VAT accumulation with no difference in
body weight compared to lard-fed rats (high-fat regimen used as control) (Rokling-Andersen et al. 2009).
In human studies, some authors found that supplements of EPA/DHA reduce WC (Kunesova et al.
2006, Thorsdottir et al. 2007, Bays et al. 2009, DeFina et al. 2011, Crochemore et al. 2012, Munro
and Garg 2012, 2013, Bender et al. 2014). In a group of 30 Japanese men and women with coro-
nary heart disease, a supplement of 1800 mg administered over 6 months decreased SAT and VAT
assessed by CT, but only the latter was also associated with the expected increase of plasma EPA
level (Sato et al. 2014). However, the effect of n-3 FA was not reported to be independent of changes
in body weight and fat mass in the earlier mentioned studies. Further studies are needed to confirm
their effect on body fat distribution in humans.
Conjugated Linoleic Acid

Conjugated linoleic acid (CLA) supplements have been of interest following reports of their
anticancer and anti-inflammatory properties as well as a potential role in modulating body fat
mass (Pariza 2004, Silveira et al. 2007). CLA occurs naturally and is found primarily in rumi-
nant meat and dairy products (Steinhart, Rickert, and Winkler 2003). It is synthetically produced
from sunflower and safflower oils in supplements (Pariza, Park, and Cook 2001). The estimated
daily intake of CLA is 0.36 g/day for women and 0.43 g/day for men, according to the German
Nutrition Study (Steinhart, Rickert, and Winkler 2003). CLA comprises a group of 28 isomers
that present two conjugated cis or trans dienes, primarily on positions C9 and C11 or C10 and
C12. CLA is well absorbed in free FA form or as digested triglycerides when compared to ethyl
ester (Fernie et al. 2004). Poor palatability was reported when CLA was ingested as free FA
(Fernie et al. 2004). CLA has multiple effects on adipose tissue metabolism. It has been linked
to decreased fat cell size and preadipocyte proliferation (Tsuboyama-Kasaoka et al. 2000, Evans,
Brown, and McIntosh 2002, Brown and McIntosh 2003). Other reports showed adverse effects
of CLA such as a decrease of preadipocyte differentiation via reduced peroxisome prolifer-
ator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha activity
C/EBPα (Brown et al. 2003, Kang et al. 2003), and activation of the nuclear factor-kappa B
(NF-κB) pathway and subsequent expression of tumor necrosis factor alpha (TNFα) (Chung
et al. 2005). Impaired insulin signaling after CLA supplementation was also reported in animal
models and was linked to the impact of TNFα on the expression of key adipogenic genes such
as glucose transporter type 4 (GLUT-4), lipoprotein lipase (LPL), and adipocyte Protein 2 (aP2)
(Chung et al. 2005). Reports (Evans, Brown, and McIntosh 2002) suggested differential effects
of the two most studied CLA isomers, trans10cis12 and cis9trans11, the latter being associated
with body composition (reduction in body fat and increase of lean body mass) and the other with
anticarcinogenic properties.
Some studies reported significant weight loss with CLA supplements (range, 0.59–6.8 g/day)
and a fat-free mass increase (Blankson et al. 2000, Smedman and Vessby 2001, Gaullier et al. 2004,
2007, Syvertsen et al. 2007, Watras et al. 2007), while others found no effect (Berven et al. 2000,
Zambell et al. 2000, Benito et al. 2001, Mougios et al. 2001, Kreider et al. 2002, Malpuech-Brugere
et al. 2004, Moloney et al. 2004, Tricon et al. 2004, Whigham et al. 2004, Taylor et al. 2006, Joseph
et al. 2011). WC was not significantly reduced in a meta-analysis of randomized control trials on
CLA supplementation (including 534 subjects) over at least 6 months of treatment (Onakpoya et al.
2012). Accordingly, in a randomized control trial following women with the metabolic syndrome on
a hypocaloric diet, women taking placebo reduced their WC, whereas women receiving CLA sup-
plements did not (Carvalho, Uehara, and Rosa 2012). Earlier fat loss occurred in women taking the
CLA supplement. However, final body fat loss was similar at the end of the 14-week trial (Carvalho,
Uehara, and Rosa 2012). In obese women supplemented with 3.4 g/day of CLA, Gaullier and col-
laborators noted a decrease in waist-to-hip ratio (WHR) and observed that fat loss was primarily
due to loss of leg fat and not abdominal fat (Gaullier et al. 2007). No change in WC was observed
in another randomized control trial involving overweight and obese men and women supplemented
with 3 g of CLA during 12 weeks (Laso et al. 2007). However, the investigators reported a trend
toward reduced trunk fat mass assessed by DXA in the overweight group only (Laso et al. 2007).
In overweight and obese men, no differences were observed in CT-assessed VAT and SAT areas
after a 4-week supplementation with ~2.6 g CLA/day (Desroches et al. 2005). Similar results were
obtained in men following a resistance training program, where VAT loss was not related to CLA
supplementation (Adams et al. 2006).
Hence, some but not all studies show that CLA may induce fat loss, possibly by increasing lipoly-
sis and apoptosis in adipose tissue (Pariza 2004). However, data are lacking regarding the capacity
of naturally found CLA to modulate body fat distribution toward a healthier pattern. In fact, studies
previously discussed used supplements including 0.59 up to 6.8 g of CLA, which is 1.3–14 times
greater than general daily intake (Steinhart, Rickert, and Winkler 2003). Studies are also needed to
assess the safety of CLA supplements, particularly in individuals at risk for type 2 diabetes. Since
both major isomers appear to present extensive differences in functional properties, specific charac-
terization is also required.
Carbohydrates
Percentage of Energy Intake as Carbohydrates
No cross-sectional study has reported a positive or negative association between total carbohydrate
intake and VAT accumulation in either young or adult individuals (Larson et al. 1996, Stallmann-
Jorgensen et al. 2007, Davis et al. 2009, Bailey et al. 2010, Lagou et al. 2011).
Glycemic Index/Load
Glycemic index (GI) and glycemic load (GL) have been extensively studied. High GI and GL diets
are generally high in energy; they may be less satiating and increase insulin secretion, possibly con-
tributing to hyperinsulinemia (Esfahani et al. 2009). In a prospective cohort study of adults, Danes
Hare-Bruun and collaborators reported that individuals consuming a high GI diet had higher intakes
of fat and added sugars as well as lower protein and fiber intake compared to those with a low GI.
Those with a high GL diet consumed less fat and more carbohydrates such as added sugars and
dietary fiber than their low GL counterparts (Hare-Bruun, Flint, and Heitmann 2006). These data
suggest that high GI and GL may contribute to cardiometabolic risk.
In men, GI and GL were not associated with any of the adiposity indices measured in this pro-
spective cohort (body weight, percent body fat, WC, and HC) (Hare-Bruun, Flint, and Heitmann
2006). In women, body weight and percent body fat changes over 6 years were positively asso-
ciated with GI, whereas a trend was found with change in WC (2 cm, p = 0.07) (Hare-Bruun,
Flint, and Heitmann 2006). Conversely, there was a borderline negative association between
change in WC and GL among women (−0.5 cm for a 10% increase of GL, p = 0.06), suggesting
a favorable effect of this diet on central adiposity (Hare-Bruun, Flint, and Heitmann 2006). On
the other hand, GI and GL were not associated with WHR in either men or women in two cross-
sectional studies comprising 8703 subjects (Liese et al. 2005, Rossi et al. 2010). In two other
cross-sectional studies, WHR was positively associated with GI and GL in men only (Toeller
et al. 2001, Mosdol et al. 2007).
A recent systematic review showed that information was insufficient to ascertain the relationship
between GI, GL, and adiposity measurements in children (Rouhani et al. 2014). There is only one
prospective cohort in children on this topic (DONALD study) (Buyken et al. 2008). This study found
no association between GI and GL and body mass index (BMI) z-score when the entire study popu-
lation was considered (Buyken et al. 2008). In fact, only one cross-sectional study reported an asso-
ciation between GI and waist z-score independently of age, sex, BMI, and education of the parents
as well as residuals of diet variables (energy, protein, fat, carbohydrate, fiber, and GL) (Barba et al. 2012).
In that study, GI was the only nutritional variable to be associated with WC (Barba et al. 2012).
Overall, there is insufficient evidence at this time to conclude on the potential benefit of a low GI/GL
diet on central obesity, in either children or adults.
Discrepancy in findings among studies could be explained partially by methodology in assessing
adiposity or dietary intake. Some limitations in the use of GI and GL should also be acknowledged.
GI and GL are often used as commutable variables, whereas GL takes into account the amount of
carbohydrates in one portion while GI does not (Venn and Green 2007). The large variation in GI
measurements, individual variability, unclear impact of mixed meals, and food transformation make
difficult to draw conclusions from GI and GL studies (Venn and Green 2007).
Fructose and Sugar-Sweetened Beverages

Added sweeteners and fructose have raised particular concern for public health (Lustig, Schmidt,
and Brindis 2012). Hepatic fructose metabolism, which differs from that of glucose, leads to a host
of metabolic alterations associated with increased plasma triglycerides and glycemia, as reviewed
in (Tappy et al. 2010), and Chapters 13 through 15 of this textbook. Fructose and sugar-sweetened
beverages (SSB) have also been studied in relation to visceral fat accumulation. As described in
Chapter 14, Stanhope and collaborators characterized differences in central fat accumulation
measured by CT in men and women consuming a fructose- or glucose-sweetened beverage provid-
ing 25% of energy for 10 weeks (Stanhope et al. 2009). For a similar weight gain in both groups, con-
sumption of a fructose-sweetened beverage increased visceral and total fat accumulation, whereas
consumption of a glucose-sweetened beverage did not. The mechanisms underlying this effect are
still unclear but may involve depot-specific modulation of lipogenic enzymes (Stanhope et al. 2009).
In a cross-sectional study of 791 Caucasian men and women, VAT mass and SAT mass (measured
by MRI) were not associated with SSB consumption; however, their intake was positively correlated
with an increased ratio of VAT to total abdominal fat area after adjustment for confounding variables
(Odegaard et al. 2012). In an observational study combining the Framingham Heart Study and the
Third Generation Cohort (n = 2596), men tended to consume more SSB than women (Ma et al. 2014).
Of note, whereas mean BMI and fasting glycemia were not different across categories of SSB con-
sumption (none; >1/month and <1 per week; >1/week and <1 per day; and >1 a day), the prevalence
of dyslipidemia increased as SSB consumption increased (Ma et al. 2014). The association between
VAT area and SSB consumption only became significant after adjustment for SAT area (Ma et al.
2014). In a 6-month, randomized control intervention study, overweight or obese men and women
were given one of four drinks (sucrose-sweetened regular cola, aspartame-sweetened diet cola, 1.7%
fat milk [isocaloric to the regular cola], or water) to assess changes in VAT area (Maersk et al. 2012).
Whereas total fat mass did not differ across beverage groups, the increase in VAT area was higher in
the regular cola group when compared to the other groups; the same was observed for total choles-
terol and triglyceride levels (Maersk et al. 2012).
Davis and collaborators found no association between SSB consumption and VAT accumulation
in young overweight Latino youth followed for 2 years (Davis et al. 2009). However, added sugar
and SSB intakes were similar at baseline and follow-up. In a cross-sectional study in teenagers, VAT
but not SAT area was associated with total fructose consumption (Pollock et al. 2012).
Available studies suggest that SSB and/or fructose may modulate body fat distribution
and promote accumulation of VAT. Additional studies are required to further document this
phenomenon.
Protein
Percentage of Energy Intake as Protein
Even though standard nutritional guidelines promote lower percentage of energy from protein
than from fat and carbohydrate, many weight loss programs promote adherence to a moderate–
high-protein intake diet (Abete et al. 2010). There is growing evidence that higher intake of
protein may increase weight loss through increased satiety and thermogenesis, as reviewed in
Halton and Hu (2004) and in Chapters 4, 17, and 18 of this textbook. High-protein diets have
been proposed to counteract the reduction in fat-free mass often observed in weight loss trials
(Farnsworth et al. 2003, Leidy et al. 2007). Whether protein intake modulates abdominal fat
accumulation will be discussed in this section.
Several randomized weight loss trials found favorable outcomes with increases in total protein
intake; however, these studies often included an exercise program, which made it difficult to assess
the specific nutritional impact. Arciero et al. investigated the effect of exercise and percentage of
energy as protein on body composition in overweight/obese men and women (2006). They found
that a 40% carbohydrate and 40% protein diet decreased abdominal fat measured by DXA by more
than 25% over 3 months, whereas a standard diet (50%–55% carbohydrates, 15%–20% proteins)
reduced abdominal fat by only 7.5%. Of note, the exercise training program was more vigorous in
the high-protein group and included high-intensity resistance and cardiovascular training, whereas
the standard diet group performed moderate cardiovascular training (Arciero et al. 2006). In a
subgroup of individuals followed for 1 year, abdominal fat was not different from baseline in the
high-protein group and was reduced in the standard diet group, which could reflect the difficulty
of maintaining long-term lifestyle modifications such as adherence to a high-protein diet (Arciero
et al. 2006). In another randomized control trial, there was no difference in abdominal fat measured
by DXA in groups assigned either to high (40%–46%) or standard (15%–20%) protein diets for
12 weeks (Clifton, Bastiaans, and Keogh 2009). Interestingly, improvements in cardiometabolic
risk factors were greater in those with high circulating triglyceride concentrations at baseline on
the high-protein diet when compared to those on the standard-protein diet (Clifton, Bastiaans,
and Keogh 2009). Similar results (no difference in fat mass measured by DXA) were obtained
in obese women assigned to diets of various macronutrient distributions (15%, 50%, or 63% of
energy as protein) and a training program over 14 weeks (Kerksick et al. 2010). In a recent meta-
analysis comparing low/standard (<20%/20%–30%) vs. high (>30%) protein randomized weight
loss clinical trials, the mean decrease in WC was 1.66 cm greater following the high-protein diets
than after the low/standard-protein diets (n = 1214) (Santesso et al. 2012). Cardiometabolic risk
factors such as systolic and diastolic blood pressure, total triglycerides, and fasting insulin were
also significantly lower, and HDL-cholesterol levels were higher, as expected with the decrease in
WC (Santesso et al. 2012).
In sum, there is little evidence to date that high protein intake is associated with reduced VAT
accumulation, independent of exercise. In fact, results from six observational studies including 2393
individuals found no association between overall protein intake and VAT area measured by CT scan
or MRI after adjustment for age, race, sex, baseline VAT and SAT, and energy intake (Stallmann-
Jorgensen et al. 2007, Davis et al. 2009, Bailey et al. 2010, Lagou et al. 2011, Hairston et al. 2012,
Kondoh et al. 2014).
Branched-Chain Amino Acids

There is growing evidence that obese individuals have a deteriorated plasma amino acid profile,
including elevated levels of branched-chain amino acids (BCAA). This increase could reflect
dysfunctional basic metabolism and altered catabolic pathways in adipose tissue and other sites.
Newgard and collaborators reported that rats fed a protein-enriched diet had higher levels of plasma
BCAA metabolites (Newgard et al. 2009). However, in a study from our group conducted on women,
no direct correlation was found between dietary amino acid intake assessed by dietary records, and
circulating amino acid levels (Boulet et al. 2015). There was also no association between plasma
BCAA and dietary BCAA (documented by food records or as part of an intervention) in other studies
(Tai et al. 2010, Piccolo et al. 2015). Available data suggest that in humans, relationships between
circulating BCAA and obesity, body fat distribution, or insulin resistance are more closely related
to lower activity of branched-chain α-keto acid dehydrogenase (BCKD) enzyme complex such as
BCKDE1α leading to diminished tissue BCAA catabolism independent of dietary BCAA intake
(Lynch and Adams 2014, Boulet et al. 2015).
DIETARY PATTERNS
Interest on diet patterns has grown over the past decades (Van Horn 2011). Dietary behavior as a
whole has been suggested to contribute to a larger extent to chronic diseases than could single nutri-
ents (Kant 2010). Grouping food into categories allows the assessment of various eating behaviors,
or dietary patterns (see description in Table 7.1), which may differ according to gender, education
level, ethnicity, and culture (Newby et al. 2003, McNaughton et al. 2007).This section synthesizes
information on the relevance of specific dietary patterns or diets, types of foods, or other dietary
indices for abdominal fat accumulation and cardiometabolic risk factors.
Villegas et al. reported that the number of men and women from south Ireland (n = 1473) with a
high WC and WHR was higher in the “alcohol and convenience foods” dietary pattern than in the
“prudent diet” (Villegas et al. 2004). Unexpectedly, WC and WHR were lower among those adopt-
ing the “traditional diet” (participants with the highest intake of nonalcoholic beverages, refined
cereals, butter, whole milk, and sweets) than those following the “prudent diet” (Villegas et al.
2004). These associations were not significant for BMI, suggesting that being part of the “alcohol
and convenience food” group relates more closely to central adiposity than to overall obesity level.
In another study, African-American men with a higher “southern pattern diet” score had a higher
WC and higher CT-measured VAT area; similar trends were observed between WC and the “fast-
food pattern” (Liu et al. 2013). No significant association was detected between these adiposity
parameters and the “prudent pattern diet.” In a Brazilian study (Vilela et al. 2014), consuming a
“western diet” was positively associated with WHR and WC after adjustment for BMI in women,
while a positive association between WHR and a trend with WC was also observed in the “regional
TABLE 7.1
Characteristics of the Main Diet Patterns Reported
Diet Pattern Descriptiona Studies
Prudent or healthy High in fruits, vegetables, white meats or fish, Villegas et al. (2004)
nuts, vegetable oils, and whole grains Esmaillzadeh and Azadbakht (2008)
Denova-Gutierrez et al. (2011)
Amini et al. (2012)
Liu et al. (2013)
Vilela et al. (2014)
Western High in refined grains, red meats, butter, eggs, Esmaillzadeh and Azadbakht (2008)
hydrogenated fats, soft drinks, and sweets Denova-Gutierrez et al. (2011)
Amini et al. (2012)
Vilela et al. (2014)
Alcohol and convenience foods High intake in alcohol and meats, low Villegas et al. (2004)
consumption of fruits, vegetables, and whole
grains
Southern Traditional rural southern U.S. foods: high Liu et al. (2013)
consumption of beans, legumes, corn
products, fried fish and chicken, margarine,
and butter
Fast food High in sugar, fast foods, and salty snacks Liu et al. (2013)
Traditional (Iranian) High consumption of grains, potato, tea, Esmaillzadeh and Azadbakht (2008)
hydrogenated fats, and legumes
Mixed diet High consumption of fruits, vegetables, low-fat McNaughton et al. (2007)
yogurt, and soy milk, but high in sweets
a Description can vary according to the study and population.
traditional diet” group (high in rice, beans, tubers, meat, eggs, coffee, and sugar). In female Iranian
teachers, lower total and central obesity was observed in the higher quintile of the “healthy pattern
diet” while the opposite relationship was found in the “western and Iranian diet” (Esmaillzadeh and
Azadbakht 2008).
Data from Iranian participants with abnormal glucose homeostasis suggest that the “west-
ern diet” is associated with central and total obesity, whereas the “high-fat dairy pattern” is
associated only with total obesity level (Amini et al. 2012). A cross-sectional study in Mexican
individuals identified three general dietary patterns: “westernized,” “high animal protein/fat,”
and “prudent.” The westernized and high animal protein/fat patterns were positively associ-
ated with percent total fat body and percent abdominal body fat measured by DXA while the
prudent diet was related to a lower percent body fat (Denova-Gutierrez et al. 2011). Taken
together, the earlier mentioned studies suggest that specific “unfavorable” dietary patterns
(such as the “fast-food pattern”) may influence abdominal fat but the effect seems dependent
on concurrent gain in body weight or fat mass.
A small number of longitudinal studies investigated the long-term impact of various dietary
patterns on body fat distribution. One study reported an inverse association between a “mixed
diet pattern” (Table 7.1) and WC, but not BMI after adjustment for confounders in men, while
the “fruit, vegetables and dairy pattern” (which shares similarities with the “prudent pat-
tern” used in other studies) was associated with a decrease in both BMI and WC in women
(McNaughton et al. 2007). In healthy men and women participating in the Baltimore longitu-
dinal study (n = 459), subjects in the “white bread pattern” subgroup had a mean annual WC
change of +1.32 cm, which was three times greater than that of participants in the “healthy
pattern” subgroup (Newby et al. 2003).
Although many studies have reported associations between dietary patterns and obesity level,
few convincingly demonstrated that they may be linked to body fat distribution profiles. Several fea-
tures of these studies may underlie this conclusion. First, there are numerous differences in popula-
tion characteristics consuming a particular diet (e.g., smoking status, ethnicity, sex, education level,
and physical activity), some of which are strong modulators of body fat distribution (reviewed in
Tchernof and Després (2013)). Moreover, the frequent use of cross-sectional designs, the variability
in the populations examined, inconsistencies in the dietary patterns identified, as well as limitations
and variation in the methodology used for dietary assessment make direct comparisons of available
studies difficult.
Food Item Subgroups

Another method to assess the impact of diet is to segregate food items in various subgroups
according to their nature or composition. The association between the relative weight of food sub-
groups in the diet and physiological variables can be subsequently analyzed. Several large cross-
sectional studies (>1000 participants) have examined the relative distribution of food groups as
a function of WC. One of these studies (McCarthy et al. 2006) including adults from Northern
Ireland observed a significant increase in the risk for high WC, but not BMI, and food categories
like pastries and cake, whole milk, cream and desserts (which included cream, puddings, chilled
desserts, and ice cream), meat, as well as alcoholic beverages. A lower risk for high WC was also
reported with higher intake of rice and pasta. Data from 1519 adults participating in the National
Diet and Nutrition Survey of British adults showed positive associations between WC and portion
sizes of various food groups, including whole milk, chips and processed potatoes, sweets, as well
as soft drinks (Kelly et al. 2009). Portion sizes of these food groups were not associated with
BMI (Kelly et al. 2009). A Swedish study including 6069 men and women examined the associa-
tion between food types and WC or HC (Krachler et al. 2006). In women, a preferential fat accu-
mulation in the hips and thighs was associated with increased consumption of vegetable oil, pasta,
and 1.5% fat milk, whereas central fat accumulation was associated with higher consumption of
hamburgers, potatoes, French fries, and soft drinks. In men, vegetable oil, pasta, and 1.5%–3%
milk was associated with gluteofemoral fat accumulation, while central fat accumulation was pos-
itively associated with increased beer, but not wine, consumption. Results of the 12-year follow-
up of the Framingham Offspring/Spouse cohort study showed that obesity-specific nutritional risk
score (based on 11 components, namely total energy, energy density, carbohydrate, protein, fiber,
calcium, alcohol, and total, MUFA, PUFA, and SFA) was related to abdominal obesity in women
(Wolongevicz et al. 2010).
A Danish monozygotic co-twin case–control study evaluated the impact of numerous diet com-
ponents on body fat distribution (Hasselbalch et al. 2010). A negative association between WC and
vegetable oil intake was found in men (Hasselbalch et al. 2010). The prospective EPIC study evalu-
ated annual WC changes in relation with food groups (Romaguera et al. 2011). A high consumption
of fruits and dairy products (including milk, yogurt, and cheese; irrespective of fat content) coupled
with low intake of white bread, processed meat, margarine, and soft drinks was associated with
lower increases in WC for a given BMI (Romaguera et al. 2011).
Although available studies could suggest detrimental effects of a more refined or fast-food type
of diet on surrogate measures of VAT, no firm conclusion can be reached about specific food items
and body fat distribution. This conclusion takes into account the relative paucity of data, the het-
erogeneity of food patterns and populations examined, the disparities in the results, and the various
adjustments that were performed (or not performed).
Mediterranean Diet
As discussed in Chapter 22, MD are characterized mainly by high consumption of vegetables, fruits,
whole grains, olive oil, nuts, a moderate intake of fatty fish, dairy, and alcohol—mostly wine—as
well as low consumption of red meat and sweets. This pattern received increasing scientific atten-
tion with the development of a Mediterranean score (MEDS) by Trichopoulou and collaborators,
in which a diet including these components was shown to positively affect life expectancy in a pro-
spective cohort study (Trichopoulou et al. 1995). In the growing literature of the past decades, many
studies have addressed adherence to the MD and body fat distribution.
In the EPIC-PANACEA PROJECT, a cross-sectional study including 497,308 individuals from
10 European countries, a higher MEDS was associated with lower WC for a given BMI in both
women and men (Romaguera et al. 2009). No significant association was found between the
MEDS and BMI (Romaguera et al. 2009). In a large prospective case-cohort study in five European
countries, a higher MEDS was associated with a reduction in WC, adjusted for BMI, at a 6.8-year
median follow-up (Roswall et al. 2014). A longitudinal study with a mean follow-up of 7 years
from the Framingham Offspring Cohort showed MEDS-related improvement in several features of
the metabolic syndrome, including WC, after adjustment for BMI and change in BMI (Rumawas
et al. 2009). These results are consistent with those of the SU.VI.MAX (Supplémentation en
Vitamines et Minéraux AntioXydants) study cohort (Kesse-Guyot et al. 2013). Similarly, a 9-year
follow-up of 3058 Spanish men and women showed that adherence to the MD was negatively
associated with increases in WC (Funtikova et al. 2014).
In contrast to the reports mentioned earlier, a cross-sectional Lebanese study found that
although the classical MEDS was associated with lower WC independent of BMI, these results
were not replicated with a customized MEDS adapted for the Lebanese population. Both BMI
and WC were lower with increasing values of the custom score, suggesting that the MD is
associated with lower total and central adiposity (Issa et al. 2011). This result is consistent
with the findings of Boghossian et al. on 258 premenopausal women (Boghossian et al. 2013).
A high MEDS was, indeed, significantly associated with lower BMI, lower WC and HC, and
lower body fat mass assessed by DXA (Boghossian et al. 2013). Negative associations between
a higher MEDS and overall adiposity were replicated in other studies (Panagiotakos et al. 2006,
Schroder et al. 2010).
Another study including 23,597 participants from the EPIC cohort showed that when energy
intake was not controlled for, the MEDS was associated with a marginal increase in BMI and WC
(Trichopoulou et al. 2005). For example, in the Greek cohort of the EPIC study, the MD was directly
linked to increased energy intake (Ferro-Luzzi, James, and Kafatos 2002). Conversely, an Italian
study reported no association between BMI or WHR and adherence to the major characteristics of
the MD (Rossi et al. 2008). In a recent nutritional intervention study in a non-Mediterranean popu-
lation at risk for cardiovascular diseases, both men and women significantly increased their MEDS
and, as a result, decreased the energy density of their diet and thus reduced their energy intake
(Leblanc et al. 2015). At the end of the intervention period (12 weeks), both genders had signifi-
cantly lower WC and BMI, along with other improvements in metabolic indicators such as HDL-C
(in men only) (Leblanc et al. 2015). Reports of nutritional data may need to be standardized across
population studies to assess the apparent favorable impact of the MD on body fat distribution and/
or overall adiposity.
One study reported lower weight gain with a higher MEDS for participants with 1 or 2 minor
alleles of the TCF7L2 gene, which has been related to diabetes (Roswall et al. 2014). In the
Prevención con Dieta Mediterránea clinical trial, participants with the minor 12Ala allele of the
PPARγ gene increased their WC significantly more than noncarriers of this allele, while no differ-
ence in BMI was noted. Yet, this result was only observed in the control group advised to follow
a low-fat diet, not in the two Mediterranean groups. Therefore, the MD appears to protect against
the detrimental effects of the 12A1a PPARγ minor allele (Razquin et al. 2009). It has been pro-
posed that the highly concentrated MUFA and PUFA of the MD could modulate PPARγ activation
(Xu et al. 1999).
While available data point toward a possible specific reduction of central adiposity with the MD,
this issue is still controversial in the literature. Adaptation of the original MEDS to local populations
may also complicate study comparisons. WC was generally used in large cohort studies, but additional
imaging studies would be required to better assess the impact of MD on abdominal fat compartments.
OTHER NUTRIENTS OR FOOD ITEMS

A wide selection of specific nutrients or food items has been studied in relation to body fat distribu-
tion and will be briefly addressed in this section. Extensive discussion of the mechanisms underlying
their possible impact on abdominal obesity is beyond the scope of this chapter.
Alcohol
Data on alcohol consumption and obesity are conflicting. Alcohol provides 7.1 kcal/g, yet some
beverages contain active biomolecules such as polyphenols (e.g., red wine), which can positively
impact cardiometabolic risk factors. Existence of the “beer belly” is a widely spread popular notion,
and heavy alcohol use also leads to liver disease and other metabolic alterations. Studies on alcohol
consumption and abdominal obesity are briefly reviewed here.
In Japanese men, only a trend between VAT area and alcohol intake was noted (Kondoh et al.
2014). However, a significant inverse relationship was found with abdominal subcutaneous fat
along with a positive association with the VAT/SAT ratio. In age-adjusted regression analyses,
alcohol intake was strongly and positively associated with VAT area (Kondoh et al. 2014). Similar
results were reported by Larson and collaborators in a cohort of men and women from the United
States. VAT adjusted for SAT was positively associated with alcohol intake (as a binary variable),
whereas SAT adjusted for VAT was negatively associated with alcohol consumption (Larson
et al. 1996). In the Framingham Heart Study, relationships between alcohol intake and VAT or
SAT were studied separately in both sexes (Molenaar et al. 2009). Men whose intake was greater
than 14 drinks per week (equivalent to >24 g ethanol/day) and women with intake greater than
7 drinks per week (>12 g ethanol/day) were identified as heavy drinkers, whereas the remaining
individuals were classified as light and moderate drinkers (Molenaar et al. 2009). In women but
not in men, SAT area was higher in light-to-moderate drinkers, whereas only in men VAT area
was higher among the heavy drinkers (Molenaar et al. 2009). Another study reported that central
obesity in women, assessed by DXA, was associated with low levels of alcohol consumption,
which is consistent with the notion that SAT is increased preferentially in women gaining weight
(Greenfield et al. 2003). In men from the Normative Aging Study, there was a trend toward a
positive association between the WHR and alcohol consumption (Troisi et al. 1991). Brandhagen
and collaborators reported associations between types of alcoholic beverages and measures of
central adiposity in both men and women of the Swedish Obese Subjects study (Brandhagen
et al. 2012). With their fully adjusted model in men, consumption of spirits was positively asso-
ciated with percentage body fat, sagittal diameter, and WC, whereas beer consumption was not
associated with any of these measurements (Brandhagen et al. 2012). In contrast, in women,
wine and total alcohol intake were negatively associated with percent body fat (Brandhagen
et al. 2012).
In overweight young adults, alcohol consumption in both sexes was not associated with, nor
predictive of, VAT or SAT area (Bailey et al. 2010). However, as reported by the authors, cafeteria-
based diet assessment may not reflect usual alcohol consumption in that cohort. In abdominally
obese men, consumption of 40 g of ethanol (450 mL of red wine) daily over a 4-week trial did not
result in increased body weight, abdominal or subcutaneous fat contents as determined by ultra-
sound (Beulens et al. 2006), but led to increased adiponectin secretion. Data from the EPIC study
showed a 0.02 cm increase in WC per 5% increase of alcohol intake (as a proportion of total energy
intake) for a given BMI only in women (Romaguera et al. 2010).
In sum, most studies show that alcohol consumption may be linked to increases in total fat mass
as well as depot-specific abdominal fat storage that may be a reflection of the propensity of each sex
to store fat either in central (men) or peripheral (women) compartments.
Dairy Products, Calcium, and Vitamin D

Many epidemiological findings suggest a favorable antiobesogenic effect of dairy products, likely
related to high calcium and vitamin D intake (Zemel et al. 2000, Pereira et al. 2002, Jacqmain
et al. 2003, Loos et al. 2004). A recent meta-analysis found that daily increase in dairy intake
(550–1000 mg of additional calcium) was associated with a lower WC compared to control groups
in energy restriction studies (−2.43 cm, 95% CI: −3.42, −1.44) but not in studies without energy
restriction (WC: −2.19 cm, 95% CI: −8.02, 2.66) (Abargouei et al. 2012). In a randomized weight
loss trial where obese men and women consumed yogurt or placebo in the form of a sugar-free
gelatin snack, there was a more pronounced weight loss in the yogurt group. Furthermore, trunk
fat loss was 81% higher than that of the control group, and there was a 4 cm reduction in WC. The
authors proposed increased fat cell lipolysis as a potential mechanism, as supported by an increase
in plasma glycerol observed only in participants who consumed yogurt (Zemel et al. 2005). In a
retrospective study including overweight and obese Australians, there was an inverse relationship
between total dairy food intake and WC after adjustment for age, sex, and total energy intake
(Murphy et al. 2013). After adjustment for total dairy food intake, dairy protein and dairy calcium
were still negatively associated with WC and DXA-measured abdominal fat (Murphy et al. 2013).
In a recent randomized control trial, men and women taking calcium and vitamin D supplemen-
tation in orange juice had greater decreases in VAT area than controls treated with orange juice
without supplementation after adjustment for baseline total abdominal area (SAT+VAT) to con-
sider for baseline group differences (Rosenblum et al. 2012). Consistent with these results, Bush
et al. reported that premenopausal women gained 2.7 cm2 less visceral fat assessed by CT scan
for each 100 mg/day increase of total dietary calcium over a 1-year period (Bush et al. 2010). In
postmenopausal women, total dietary calcium intake was negatively associated with abdominal
fat mass and percent body fat, but when adjusted for total energy intake only percent body fat
was still associated with calcium intake. Nonsignificant associations were also reported between
calcium intake and BMI, WHR, and WC (Heiss, Shaw, and Carothers 2008).
Vitamin D has been proposed to play a role in adipocyte differentiation, but its effects
remain unclear (Kong and Li 2006). Our team reported that women with higher plasma levels of
25-Hydroxyvitamin D (25(OH)D) had smaller omental adipocytes, and lower VAT and SAT areas
determined by CT (Caron-Jobin et al. 2011). In another study, overweight and obese women supple-
mented with 25 μg of vitamin D3 for 3 months showed no difference in WC and weight compared
to controls (Salehpour et al. 2012). Wamberg and collaborators reported no difference in SAT and
VAT areas (determined by MRI) in obese men and women after 26 weeks of supplementation with
175 μg of vitamin D3 compared to controls, and no difference in the cardiometabolic risk factors
including the homeostasis model assessment of insulin resistance, high-sensitivity C-reactive pro-
tein (hsCRP), plasma lipids, and blood pressure (Wamberg et al. 2013). In chronic kidney disease
patients, levels of 25(OH)D decreased as BMI increased and were also lower in diabetic patients.
However, there was no association between vitamin D status and SAT or VAT areas as well as
body fat mass (Figuiredo-Dias et al. 2012). The apparent null effect of vitamin D supplementa-
tion alone may point toward a combined action of dairy protein and/or calcium in the modulation
of body fat distribution.
Soy and Isoflavones

In the Soy Health Effects study, intake of the most common isoflavone subtypes (genistein and
daidzein) was assessed with a food-frequency questionnaire in 208 postmenopausal women
(Goodman-Gruen and Kritz-Silverstein 2003). Women with higher genistein intake had lower
BMI, fat mass, and WC than women who reported no intake of isoflavones but they were also
generally more active (Goodman-Gruen and Kritz-Silverstein 2003). In two randomized control
trials, postmenopausal women taking a daily shake with added soy protein and isoflavones for
3 months gained less total and SAT area than women in the casein placebo group (Sites et al.
2007, Christie et al. 2010). VAT gain was not different between the two groups (Sites et al. 2007,
Christie et al. 2010). Different results were obtained in randomized control trials on exercise,
isoflavones, and weight loss in postmenopausal women (Maesta et al. 2007, Choquette et al.
2011). While exercise had a favorable effect on WC, HC, and body fat mass, neither apparent
additive nor synergetic effect of isoflavones was detected (Maesta et al. 2007, Choquette et al.
2011). In a weight loss trial comparing the effect of soy vs. milk protein, only those in the milk
protein group experienced a reduction in BMI and body weight (Takahira et al. 2011). Decreased
WC was observed in both groups but only the milk group had significant decreases in both
abdominal SAT and VAT areas (Takahira et al. 2011). Overall, a specific effect of soy or isofla-
vones on visceral fat accumulation is improbable.
Dietary Fiber and Whole Grains

Dietary fiber and whole grain intake has been associated with body composition in many stud-
ies. McKeown and collaborators reported inverse relationships between intake of whole grain
cereal fiber and percent trunk fat mass as well as percent body fat in an elderly population
(60–80 years old) (McKeown et al. 2009). Of note, they did not find an association between
these measurements and total fiber intake (McKeown et al. 2009). Interestingly, those in the
highest quartile category of dietary fiber and/or whole grain intake were also the group with
the higher energy intake (McKeown et al. 2009). Two other observational studies found no
association between whole grain intake and VAT or SAT areas (Stallmann-Jorgensen et al.
2007, Davis et al. 2009).
Total fiber intake was negatively associated with VAT measured by MRI in two other stud-
ies (n = 644), but SAT was not measured (Davis et al. 2009, Parikh et al. 2012). Results from
CT scans in overweight young adults and middle-aged men and women from the United States
showed no association between VAT or SAT and total fiber intake (Larson et al. 1996, Bailey
et al. 2010). In U.S. Latino teenagers, VAT area was associated with insoluble fiber but not
soluble fiber intake (Davis et al. 2009). On the other hand, in overweight African-American and
Latino adults, soluble fiber intake was negatively associated with VAT area, whereas insoluble
fiber intake was not (Hairston et al. 2012). The effects of high fiber/whole grain diet on body
fat distribution do not seem to be mediated by reduced energy intake as the vast majority of the
earlier mentioned studies adjusted for energy intake.
Vitamins A and C
Excess storage of fat is associated with increased FA oxidation leading to the production of larger
amounts of free radicals, thus inducing a greater use of antioxidant molecules such as vitamins A
and C. Therefore, low plasma levels of vitamin A and C could be associated with low-grade chronic
inflammation related to obesity, especially in the visceral depot (Zulet et al. 2008).
In healthy young adults, high vitamin A intake was negatively associated with WC and WHR as
well as numerous cardiometabolic risk variables such as fasting glycemia, insulin, and blood pres-
sure (Zulet et al. 2008). However, a relationship between vitamin A intake and VAT or SAT accumu-
lation was not found in overweight young adults (Bailey et al. 2010).
In women, higher intake of ascorbic acid was associated with lower WC (Choi et al. 2013) and
lower odds ratio of having a WHR ≥ 0.84 (Azadbakht and Esmaillzadeh 2008). In men from the
same study, no association was found between WC and vitamin C intake (Choi et al. 2013).
Probiotics and the Gut Microbiota

There is increasing evidence linking the gut microbiota to total adiposity (Rosenbaum, Knight, and
Leibel 2015). A complete review of such evidence is beyond the scope of this chapter. Of note, a pro-
biotic bacterium (LG2055) naturally present in human gut microbiota led to significant decreases of
adipocyte hypertrophy in rats (Sato et al. 2008). More recently, the same team showed a reduction in
VAT and SAT areas as well as WC, BMI, and body fat mass in a randomized control trial of healthy
men and women supplementing their habitual diet with fermented milk containing LG2055 vs. pla-
cebo (fermented milk without LG2055) (Kadooka et al. 2010). Emerging studies on the human gut
microbiota will eventually allow assessing its potential role in body fat distribution.
INFANT FEEDING PRACTICES

Other possible dietary influences on body fat distribution patterns may include early-life nutrition.
Many studies have linked infant feeding practices or even maternal diet during pregnancy with the
onset of obesity in the offspring, and several mechanisms were proposed to explain this association
(reviewed in Lecoutre and Breton (2014, 2015) and Chapter 28 of this textbook). However, only a
few studies have directly investigated infant feeding practice or maternal diet vs. body fat distri-
bution patterns of the offspring independently of maternal BMI, socioeconomic status, and other
confounding variables.
Whether breastfeeding is related to offspring adiposity later in life is still a matter of debate.
Systematic reviews have concluded that exclusive breastfeeding for at least 6 months is protec-
tive against obesity (Kramer and Kakuma 2012). After adjustment for confounding variables,
this relation was attenuated or abolished (Kramer and Kakuma 2012). Only a few studies have
investigated duration of breastfeeding as a function of offspring body fat distribution. In a retro-
spective study of 442 children (EPOCH study), breastfeeding for less than 6 months or for more
than 6 months had no differential impact on the accumulation of MRI-assessed VAT or SAT,
sum of skinfolds, and BMI of the offspring (Crume et al. 2012). However, a protective effect of
breastfeeding was apparent for those in the upper percentiles of BMI, VAT, and SAT after adjust-
ment for confounding variables (Crume et al. 2012).
In the Generation R prospective study, children who were breastfed exclusively for at least
4 months had lower total body fat mass and peripheral fat compared to no breastfeeding at 6 months
(Durmus et al. 2012). Children who were never or nonexclusively breastfed until 4 months had
higher subcutaneous central fat at 24 months of age compared to exclusive breastfeeding (Durmus
et al. 2012). However, there was no difference in body fat distribution related to timing of the intro-
duction of solid foods (Durmus et al. 2012). Similarly, no difference in body fat distribution was
found in the same children at 6 years of age, after adjustment for confounding variables (Durmus
et al. 2014). In fact, neither timing of solid food introduction nor use of formula was associated with
total skinfolds in children between 4 and 5 years of age in two other independent studies (Zive et al.
1992, Caleyachetty et al. 2013). Similar observations were made in the HELENA cohort includ-
ing 3528 adolescents (Rousseaux et al. 2014). Of note, investigators of the HELENA cohort
showed a trend for a protective effect of breastfeeding for those in the upper percentile of skinfold
thickness and waist-to-height ratio, in line with previous results (Crume et al. 2012).
Opposite results were obtained in a Brazilian study of 185 children between 4 and 7 years.
Breastfeeding duration was positively associated with percent body fat assessed by DXA, but not
with WC or fat mass localized in the abdominal region (Magalhaes et al. 2012). Much like the pre-
viously cited study (Durmus et al. 2014), timing of solid food introduction was not associated with
BMI, WC, total body fat mass, or central adiposity (Magalhaes et al. 2012). In young infants,
introduction of cereals or meat at 5 months of age did not modulate adiposity at 9 months of age
(Tang and Krebs 2014). Regarding maternal macronutrient intake, a study in a large cohort showed
that fetal adiposity was highest in the abdominal region of infants from mothers with low dietary
intake of protein (<16%), irrespective of carbohydrate or fat intakes, and this was caused by an
increase of SAT accumulation measured by ultrasound at 19, 25, 30, and 36 weeks (Blumfield et al.
2012). Maternal diet was assessed by a validated 74-item food-frequency questionnaire during the
early and late pregnancy periods. Changes in the diet was then compared to Changes in fetal adi-
posity. On the other hand, VAT accumulation (assessed by ultrasound) was higher when maternal
calories from protein represented more than 20% energy and this was linked with carbohydrate
intake, which was diminished, thereby increasing the protein-to-carbohydrate ratio. Ultrasound-
measured mid-thigh fat in these fetuses was related to maternal intakes that were high in fat, low in
carbohydrates, and intermediary in protein. These studies identify a plausible mechanism by which
maternal protein intake can module infant body fat distribution in utero (Blumfield et al. 2012).
In another study of pregnant adolescents, intake of total and added sugar was the best predictor of
ultrasound-measured fetal abdominal fat thickness during the last trimester of pregnancy. In this
cohort, a U-shaped curve was observed between energy-adjusted carbohydrate intake and abdomi-
nal fat, pointing again to an ideal maternal carbohydrate intake. Of note, gestational weight gain
was not related to fat accretion in the fetus for these women (Whisner et al. 2015). A Danish study
examined maternal intake of animal and vegetable protein in relation to BMI and WC of offspring
at 20 years of age (Maslova et al. 2014). Offspring BMI was related to maternal protein intake
during pregnancy but WC showed no significant association (Maslova et al. 2014). In the ROLO
study, maternal protein intake was not associated with newborn anthropometric measurements, but
SFA intake in the second and third trimesters was related to offspring WC and the waist-to-length
ratio, whereas there was a negative trend between maternal PUFA intake in the third trimester and
abdominal circumference at birth (Horan et al. 2014). Consistent with these results, low level of
circulating PUFA in newborns was positively associated with higher central fat (Sanz et al. 2014).
Finally, in a randomized control trial, modulating the ratio of n-6 to n-3 FA in the diet of pregnant
women had no effect on body fat distribution in infants at birth, 6 weeks, and 4 and 12 months
(Hauner et al. 2012).
Overall, there is growing evidence that maternal diet modulates body fat distribution in the off-
spring independently of a number of confounding factors. A moderate-protein (>16% to <20% energy)
diet, and PUFA intake, appears to limit abdominal fat accretion, whereas SFA intake seems linked to
detrimental adiposity profiles. However, some important facts must be acknowledged. Investigators
often make statistical adjustment for energy intake in children, obliterating the fact that prenatal and
postnatal nutrition may impact appetite and the desire for particular foods. Age of adiposity rebound,
that is, the age between 3 and 7 years where BMI-for-age starts to increase after it reaches its low-
est point, is not always considered in available studies, which could account for some differences in
adiposity between groups.
CONCLUSION
Most available studies on diet and body fat distribution seem to point toward a nonspecific effect
on, or protection from, fat accumulation (VAT, SAT, both, or total adiposity). Nutrients potentially
linked to a detrimental fat accretion pattern (increased VAT, abdominal fat, or WC) include fruc-
tose, especially in the form of SSB, trans FA, as well as high alcohol consumption and refined/
fast-food diets. On the other hand, some nutrients (MCT, calcium, fiber, whole grains) and dietary
patterns (MD, prudent diet) have been found to reduce adiposity in epidemiological studies and
there is growing evidence supporting these notions through randomized control trials. Whether
these effects emerge from depot-specific impact in adipose tissue compartments remains unclear.
Therefore, there is no clear evidence that a single nutrient directly modulates visceral fat accre-
tion and, by extension, body fat distribution. However, some nutrients and/or diet patterns may
affect energy balance and subsequent weight gain or loss, which will be reflected in changes in
regional fat accumulation as a function of the prevailing genetic, epigenetic, and hormonal milieu
(Figure 7.1).
In this context, responses to nutrients or diets present remarkable interindividual variability most
likely resulting from interactions with a plethora of physiological factors. Future studies on this
topic should aim to identify subpopulations of responders and nonresponders with the goal of unrav-
eling biological modulators of the response. Considering the emerging impact of early-life nutrition,
prevention strategies may also have to focus on maternal nutrition and perinatal feeding.
Dietary composition
Macronutrients
Micronutrients
Patterns
Various putative
Energy balance mechanisms
?
Epigenetics
Genetics Body weight Hormones
gain or loss
Abdominal (visceral)
fat gain or loss
FIGURE 7.1 Potential impact of dietary factors on abdominal (visceral) fat gain or loss.
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8 Nutritional Considerations
for Cardiometabolic
Health in Childhood and
Adolescent Obesity
Elizabeth Prout Parks, Jennifer Panganiban,
Stephen R. Daniels, and Julie Brothers
CONTENTS
Abbreviations..................................................................................................................................150
Definition of Obesity in Childhood and Adolescence.....................................................................150
Prevalence and Trends of Childhood Obesity.................................................................................151
Critical Periods for the Onset of Childhood Obesity......................................................................151
Adiposity Rebound....................................................................................................................152
Causes of Childhood Obesity..........................................................................................................152
Treatment of Child and Adolescent Obesity...................................................................................153
Weight Management Goals in Children and Adolescents..........................................................153
Dietary Modification..................................................................................................................153
Semi-Structured Diet Regimens............................................................................................154
Meal Replacements...............................................................................................................155
Macronutrient Composition..................................................................................................155
Fruits and Vegetables.............................................................................................................155
Sweetened Beverages............................................................................................................155
Physical and Sedentary Activity.................................................................................................156
Screen Time...........................................................................................................................156
Medications for Obesity Treatment............................................................................................156
Metabolic and Bariatric Surgery................................................................................................156
Surgical Criteria....................................................................................................................157
Cardiometabolic Comorbidities in Childhood Obesity..................................................................158
Hypertension..............................................................................................................................158
Cardiovascular Risk...................................................................................................................161
Dyslipidemia of Obesity............................................................................................................161
Metabolic Syndrome..................................................................................................................162
Insulin Resistance and Type 2 Diabetes Mellitus.......................................................................163
Nonalcoholic Fatty Liver Disease..............................................................................................164
Dietary Intervention and Physical Activity...........................................................................164
Pharmacotherapy and Surgery...............................................................................................165
Summary.........................................................................................................................................165
References.......................................................................................................................................165
149
ABSTRACT
Childhood obesity develops through an interplay of genetics, environment, and behavior. Treatment
of childhood obesity includes dietary modification, increasing physical activity, and, at times,
medication and surgery. Obesity can lead to several comorbidities and chronic diseases, the most
common include hypertension, cardiac changes, dyslipidemia, metabolic syndrome, type 2 diabe-
tes mellitus, and nonalcoholic fatty liver disease. These diseases can serve to shorten the child’s
life. Our efforts should be focused on primarily reducing obesity and, when necessary, adequately
treating the secondary outcomes in an effort to reduce future morbidity and mortality.
ABBREVIATIONS
ALT Alanine aminotransferase
AST Aspartate aminotransferase
BMI Body mass index
CDC Centers for Disease Control
CDO Combined dyslipidemia of obesity
CMI Comprehensive multidisciplinary intervention
CVD Cardiovascular disease
DASH Dietary approaches to stop hypertension
FDA Food and Drug Administration
FRAGILE Low-fructose/low glycemic index/load
GB Gastric bypass
GI Glycemic index
GL Glycemic load
HbA1C Hemoglobin A1c
HDL-C High-density lipoprotein cholesterol
HOMA Homeostatic Model Assessment
HOMA-IR Homeostatic Model Assessment-Estimated Insulin Resistance
IDF International Diabetes Federation
LDL-C Low-density lipoprotein cholesterol
LDL-P Low-density lipoprotein particle
LSG Laparoscopic sleeve Gastrectomy
LVH Left ventricular hypertrophy
METSYN Metabolic syndrome
MC4R Melanocortin 4 receptor
MSD Mediterranean-style diet
NAFLD Nonalcoholic fatty liver disease
NASH Nonalcoholic steatohepatitis
NHANES National Health and Nutrition Examination Survey
SWM Structured weight management
T2DM Type 2 diabetes mellitus
TCI Tertiary care intervention
TG Triglycerides
USDA United States Department of Agriculture
DEFINITION OF OBESITY IN CHILDHOOD AND ADOLESCENCE

Obesity is caused by an energy imbalance in which individuals expend less energy than they
consume. Body mass index (BMI) is an indirect measure of body fat and provides a guideline
for weight in relation to height. BMI is measured as weight in kilograms divided by height in
Nutrition and Pediatric Cardiometabolic Health 151
meters squared. This has become an accepted standard of measurement to classify children 2 years
and older into weight categories (Deurenberg et al. 1991). Based on the Centers for Disease Control
(CDC) Growth Charts, children between the ages of 2 and 20 years can be categorized as under-
weight, normal weight, overweight, and obese (Flegal et al. 2011). More recently, formal definitions
for severe obesity with new growth charts have been developed (Gulati et al. 2012). The following
are the current weight classifications for children, based on age and sex:
• Underweight: BMI percentile < 5th percentile

• Normal weight: BMI between the 5th and 85th percentiles
• Overweight: BMI between the 85th and 95th percentiles
• Obesity: BMI ≥ 95th percentile
• Severe obesity: BMI ≥ 120% of the 95th percentile or a BMI ≥ 35 kg/m2, whichever
is lower (Flegal et al. 2011, Gulati et al. 2012), which corresponds to approximately the
99th percentile or BMI z-score (standard deviations change) ≥ 2.33 (Ogden et al. 2014)
Severe obesity that exceeds the 99th percentile is tracked on a specialized percentile curve for obe-
sity. Adult classification is used for BMI ≥ 27 kg/m2 in adolescents over age 18 years for consider-
ation of medication and bariatric surgery.
In adults, the classification of overweight and obesity is as follows (National Institutes of
Health 1998):
• Overweight: BMI ≥ 25 kg/m2

• Obesity: BMI ≥ 30 kg/m2, further classified into
• Class 1 (BMI 30–34 kg/m2)
• Class 2 (BMI 35–39 kg/m2)
• Class 3 (BMI ≥ 40 kg/m2)
PREVALENCE AND TRENDS OF CHILDHOOD OBESITY

Based on the National Health and Nutrition Examination Survey (NHANES) analysis performed from
2011 to 2012, one-third of the U.S. childhood population is either overweight or obese (Ogden et al. 2014).
The good news is that there have been.no significant changes in child overweight and obesity prevalence
between 2003–2004 and 2011–2012. However, the prevalence of severe obesity among children aged
2–19 years has continued to increase from 1.2% (1976–1980) to 3% (1988–1994) to 4.9% (1999–2004)
and, most recently, 5.9% in 2012 (Ogden et al. 2014). Additionally, there are racial and ethnic differences
in childhood obesity, with obesity being more common among American Indians, non-Hispanic blacks,
and Mexican Americans when compared to non-Hispanic whites (Wang 2011, Wang et al. 2011).
CRITICAL PERIODS FOR THE ONSET OF CHILDHOOD OBESITY

Early-life risk factors for childhood obesity include the following maternal factors: gestational dia-
betes, depression, smoking during pregnancy, and low income (Rudolf 2011). The following factors
have also been associated with the development of childhood obesity: large or small for gestational
age at birth, rapid infant weight gain during the first 6 months of life, poor infant sleep, television
viewing under age 24 months, more than 2 hours per day of television viewing over the age of
24 months, and early adiposity rebound, defined as follows (Blair et al. 2007).
Risk factors for an obese child becoming an obese adult include child age, parental obesity, sever-
ity of child obesity, and low-income household status (Whitaker et al. 1997, Parsons et al. 1999,
CDC 2009). Longitudinal studies reveal that a substantial component of adolescent obesity is estab-
lished before 5 years of age (Cunningham et al. 2014). A child with severe obesity over 12 years of
age has a 75%–80% chance of becoming a Class 3 obese adult (The et al. 2010). One parent with
obesity increases the risk for adult obesity in the child by 50%–80% (Whitaker et al. 1997).
Adiposity Rebound
Adiposity rebound is the period, between 3 and 6 years of age, when a child begins to lose excess
infant adiposity and the BMI declines. With the decline, the BMI reaches a minimum and then
begins increasing until adulthood. The earlier the age at which this minimum BMI value occurs, the
more likely a child is to be overweight or obese as an adult (Williams and Goulding 2009).
CAUSES OF CHILDHOOD OBESITY

When a child or adolescent is found to be overweight or obese, it is important to differentiate between a
primary cause, which is usually multifactorial, and a much rarer secondary cause. Secondary causes of
obesity may be due to or associated with genetic syndromes, endocrine disorders, neurologic disorders,
or medications. Table 8.1 demonstrates many of the different causes and conditions that may be associ-
ated with obesity in childhood (Gunay-Aygun et al. 1997, Speiser et al. 2005). Recently, melanocortin
TABLE 8.1
Causes of Childhood Obesity
Possible Causes Diagnosis
Primary/simple Multifactorial
Environment
Psychosocial
Lifestyle
Genetic syndromes Melanocortin 4 receptor (MC4R) deficiency
Leptin deficiency
Prader–Willi
Turner syndrome
Trisomy 21
Albright hereditary osteodystrophy
Bardet–Biedl syndrome
Cohen syndrome
Borjeson–Forssman–Lehmann syndrome
Wilson–Turner syndrome
Alstrom syndrome
Carpenter syndrome
Endocrine disorders Hypothyroidism
Growth hormone deficiency
Cortisol excess
Hyperinsulinemia
Neurologic Injury to the pituitary or the hypothalamus
Brain tumor
Cranial irradiation
Infection
Medication High dose and chronic glucocorticoids
Certain antipsychotic medications
Certain antidepressant medications
Certain antiseizure medications
Insulin
Growth hormone
Psychiatric Depression
Eating disorder
Sources: Speiser, P.W. et al., J. Clin. Endocrinol. Metab., 90, 1871, 2005;
Gunay-Aygun, M. et al., Behav. Genet., 27, 307, 1997.
4 receptor deficiency has been identified as the most common monogenetic cause of childhood obesity
and is associated with increased linear growth and hyperinsulinism during childhood (Martinelli et al.
2011). While distinguishing between primary and secondary causes of obesity may be difficult in
some instances, usually a detailed medical history, including timing of weight gain onset, developmen-
tal delays, linear growth, and diet and exercise habits; a complete review of systems; a full physical
examination, notably looking for location of adipose deposition, dysmorphic facies, enlarged tonsils,
undescended testis, acanthosis nigricans, striae, and/or hirsutism; and focused laboratory testing will
help determine the cause (Barlow and Dietz 1998, Speiser et al. 2005). Delayed linear growth in the face
of developing obesity is a major clue that there may be a secondary cause for the obesity. In addition,
there may be more than one reason for the obesity, such as a medication that may cause weight gain;
consuming excess calories and not exercising may also contribute to the weight gain.
TREATMENT OF CHILD AND ADOLESCENT OBESITY

Weight Management Goals in Children and Adolescents
In order to be successful, weight loss through lifestyle intervention in children requires behavioral
treatment for not only the child as an individual but also the whole family as a unit (Spear et al. 2007).
Target weight loss goals should be realistic and discussed at baseline and after each follow-up session
as shown in Table 8.2. Epstein et al. (1994) reported that behavioral treatments that included both the
parent and the child were more effective at reducing the percentage of overweight children compared
to those that focused only on the child or those that had a nonspecific target. Data in adults have shown
that an overall weight loss of 5%–10% reduces cardiovascular and metabolic risk. As children are still
growing, and weight is based on the growth of muscle, bone, and fat mass, BMI is commonly used to
estimate weight status. In children, a BMI z-score of 0.5 (reduction in the standard deviation from the
norm BMI z-score = 0) reduces cardiovascular risk and is equivalent to maintaining BMI for 1 year.
As shown in Table 8.2, Barlow et al. (2007) proposed a comprehensive stepwise approach for the
prevention and treatment of obesity. This includes the following stages:
• Stage 1 (prevention plus): Lifestyle intervention provided by a primary care provider

• Stage 2 (structured weight management): Monthly visits with a primary care physician and
support from a registered dietitian
• Stage 3 (comprehensive multidisciplinary intervention): Intensive weight loss program com-
posed of weekly visits for a minimum of 8–12 weeks at a pediatric weight management center
• Stage 4 (tertiary care intervention): Use of medical diets, medications, and surgery in addi-
tion to the interventions provided in Stage 3 (the approach may differ based on individual
hospital protocol)
These comprehensive interventions employ a multidisciplinary team (medical provider, registered

dietician, exercise specialist, mental health professional, nurse, and social worker) who focuses on
behavioral therapy, dietary modification, and physical activity (Chen et al. 1997). This seems to
be the most successful approach to decreasing long-term weight and risk for future comorbidities
(Levine et al. 2001). The recommendations put forth that are utilized in the community focus on
children receiving 5 fruits or vegetables per day, 2 hours or less of screen time, 1 hour or more of
physical activity, and 0 sweetened drinks (Barlow et al. 2007).
Dietary Modification
Limited research exists for evaluating dietary treatment programs in isolation. Although the outcomes
are mixed, there is evidence to support the use of a reduced energy diet as an effective component
of a weight management program typically in children over age 5 years (Epstein et al. 1990, 1994).
TABLE 8.2
Weight Goals and Intervention Stages, according to Age and BMI Categories
Age BMI Category Weight Goal Initial Intervention Highest
(Year) Stage Intervention Stage
<2 Weight for height NA Prevention counseling Prevention
counseling
2–5 5th–84th percentile or Weight velocity maintenance Prevention counseling Prevention
85th –94th percentile counseling
with no health risks
85th–94th percentile Weight maintenance or slow Prevention plus (stage 1) SWM (stage 2)
with health risks weight gain
≥95th percentile Weight maintenance (weight loss of Prevention plus (stage 1) CMI (stage 3)
up to 1 lb/month may be acceptable
if BMI is >21 or 22 kg/m2)
6–11 5th–84th percentile or Weight velocity maintenance Prevention counseling Prevention
85th–94th percentile counseling
with no health risks
85th–94th percentile Weight maintenance Prevention plus (stage 1) SWM (stage 2)
with health risks
95th–99th percentile Gradual weight loss (1 lb/month or Prevention plus (stage 1) CMI (stage 3)
0.5 kg/month)
≥99th percentile Weight loss Prevention plus (stage 1) TCI (stage 4), if
(maximum is 2 lb/week) or stage 2 or 3 if family appropriate
is motivated
12–18 5th–84th percentile or Weight velocity maintenance; after Prevention counseling Prevention
85th–94th percentile linear growth is complete, weight counseling
with no health risks maintenance
85th–94th percentile Weight maintenance or gradual Prevention plus (stage 1) SWM (stage 2)
with health risks weight loss
95th–99th percentile Weight loss Prevention plus (stage 1) TCI (stage 4), if
(maximum is 2 lb/week) appropriate
≥99th percentile Weight loss Prevention plus (stage 1) TCI (stage 4), if
(maximum is 2 lb/week) or stage 2 or 3 if patient appropriate
and family are motivated
Source: Adapted from Barlow, S.E. and Expert Committee, Pediatrics, 120, S164, 2007.
SWM, structured weight management; CMI, comprehensive multidisciplinary intervention; TCI, tertiary care intervention.
Semi-Structured Diet Regimens

Semi-structured diet regimens focused on family selection of higher nutrient quality and low energy
density have shown promising results (Epstein et al. 1990, 1994). Families are taught how to make
healthy choices using the “traffic light” format. Foods classified as “Red” should be eaten rarely,
“Yellow” eaten less often, and “Green” eaten most often (Epstein et al. 1990, 1994). The United States
Department of Agriculture (USDA) MyPlate is based on the Dietary Guidelines for Americans 2010 and
has replaced MyPyramid. MyPlate is a guideline for building an optimal diet for children and is aimed at
the general public to provide a visual representation of the different food groups and their portion sizes.
Recommendations emphasize filling half of the plate with vegetables and fruits and the other half with
protein and grains, with protein having the smallest section. Protein replaces the meat category, as many
protein sources are not from animals. Additionally, a separate dairy section is included. MyPlate has
removed foods that have low nutritional value, such as sugar-sweetened beverages and bakery products
(U.S. Department of Agriculture and U.S. Department of Health and Human Services 2011).
Meal Replacements
Meal replacements are typically composed of products such as liquid shakes, meal bars, and frozen food
entrees, which provide a fixed amount of food with a known calorie content. When added to a lifestyle
modification program, meal replacements have reliably increased weight losses by 2.5 and 2.4 kg, at
3 and 12 months, respectively. Berkowitz et al. (2011) studied 113 obese adolescents and demonstrated
a 6.3% decrease in BMI compared with a 3.8% decrease in patients placed on a conventional diet for
4 months; however, both dietary intervention groups regained their weight at 12 months of follow-up.
Macronutrient Composition
The evidence for children and adolescents does not support any specific macronutrient or dietary
strategy for BMI reduction at this time. A low-glycemic-index (GI) diet has been proposed to have
beneficial metabolic effects in treatment of obesity by decreasing the postprandial rise in glucose
and insulin. The GI describes how a controlled portion of carbohydrate affects blood glucose in the
postprandial period. A study evaluating 22 obese children placed on a low-GI diet of 60 for 6 months
(the GI for glucose = 100) found a significant decrease in BMI z-score, improved insulin resistance,
and lowered triglyceride concentrations in comparison to their counterparts placed on a hypocaloric
diet with a glycemic index of 90 (Parillo et al. 2012). The Mediterranean-style diet (MSD) has been
shown to decrease cardiovascular events and increase life expectancy in adult populations (Grosso
et al. 2014). Similarly, it has been shown to efficiently decrease metabolic syndrome (MetSyn) by
20%–43%, regardless of age, sex, physical activity, lipid levels, and blood pressure (Babio et al.
2009). This type of diet characteristically uses extra virgin olive oil, fish, wheat, olives, and grapes.
A study evaluating 49 obese children with MetSyn placed on a 16-week dietary intervention found
that children on the MSD had a significant decrease in BMI, lean mass, fat mass, glucose, total
cholesterol, triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipo-
protein cholesterol (LDL-C) in comparison to those placed on a standard diet, which comprised
55%–60% carbohydrates (45%–50% complex and no more than 10% refined and processed sugars),
25%–30% lipids, and 15% proteins (Velázquez-López et al. 2014).
A low-fructose diet has been shown to improve LDL-C and nonalcoholic fatty liver disease
(NAFLD) but does not have a significant impact on childhood BMI values (Mager et al. 2015).
Fructose is a monosaccharide typically consumed in sweeteners with sucrose (50% fructose, 50%
glucose) and high-fructose corn syrup (42% or 55% fructose, with the remainder glucose). Beverages
with added sugar and fruit juice are the most common fructose sources in children.
Finally, low-carbohydrate diets have resulted in a significant reduction in BMI, liver enzymes,
and triglyceride levels in adults with NAFLD and obesity (Foster et al. 2003, Brehm et al. 2003).
These diets seem to be more efficacious than energy-restricted low-fat diets over the short term for
weight loss, although there seems to be no significant difference in maintenance of decreased BMI
between the diets over 1 year (Baron et al. 1986, Harvey-Berino 1998, 1999, Foster et al. 2003).
Fruits and Vegetables

Research studies have shown that children are least likely to consume adequate amounts of foods
from the fruit and vegetable group. Evidence, although from cross-sectional studies, indicates that
greater fruit and vegetable intake may provide modest protection against increased adiposity (Lin
and Morrison 2002).
Sweetened Beverages
The consumption of sugar-sweetened beverages is a significant contributor to the development of
obesity (Malik et al. 2013, DeBoer et al. 2013). Energy consumed in liquid form seems to be less reg-
ulated compared to energy consumed in solid form. According to a national survey, sugar-sweetened
beverages were the sixth leading food source of energy among children, constituting 10%–15% of
total caloric intake (Murphy et al. 2005). Reducing the consumption of sugar-sweetened beverages
among overweight and obese adolescents is associated with a decrease in BMI (Ebbeling 2012a).
Physical and Sedentary Activity

Although energy intake is a significant portion of the energy equation, the role of energy expenditure
is also important. Physical activity is the only modifiable component of the energy expenditure por-
tion of the energy balance equation. An increase in sedentary activity along with an overall decrease
in physical activity are major contributing factors to the increased prevalence of overweight and
obesity in children and adolescents (Dowda et al. 2001, Berkey et al. 2003). The American Academy
of Pediatrics recommends that children and adolescents participate in at least 60 minutes of mod-
erate-intensity physical activity most days of the week, preferably daily (American Academy of
Pediatrics 2000). Strategies to increase physical activity should include increases in structured and
nonstructured physical activity and reduction in the amount of time spent in sedentary activities
(Goran and Treuth 2001).
Screen Time
Sedentary behavior is usually in the form of screen time, which includes television, video games, com-
puters, tablets, phones, and other media that are not used for educational activities (Falbe et al. 2013).
Substantial evidence supports the importance of reducing sedentary behavior as a means of prevent-
ing and treating obesity in children (Waters et al. 2011, Braithwaite et al. 2013, Falbe et al. 2013).
Reducing sedentary behavior may be more effective than increasing structured physical activity
with the secondary benefit of reducing caloric intake (Epstein et al. 2008, Epstein et al. 1995).
Experts recommend screen time to be limited to less than 2 hours a day, and children under the
age of 2 should have no screen time (Barlow et al. 2007). Television viewing is the most estab-
lished environmental influence on the development of obesity during childhood Dietz et al. 1985,
Robinson et al. 1999. The amount of time spent watching television or the presence of a television
in a child’s bedroom is directly related to the prevalence of obesity in children and adolescents
(Gilbert-Diamond et al. 2014, Gortmaker et al. 1996, Kaur et al. 2003).
Medications for Obesity Treatment

Currently, the only medication that is approved for the treatment of obesity in adolescents under
the age of 18 years is Orlistat (Yanovski and Yanovski 2014). Orlistat is a lipase inhibitor that pre-
vents the absorption of fat and on average results in a 3% weight loss over time. It is rarely used
in adolescents as it causes flatulence, abdominal pain, smelly stools, malabsorption of fat-soluble
vitamins, and alteration in liver enzymes. Thus, adherence to this medication is understandably
challenging for this age group. Other medications that are approved for patients over age 18 years
include phentermine, phentermine plus topiramate (Qsymia), lorcaserin, and bupropion plus nal-
trexone (Contrave) (Yanovski and Yanovski 2014). Expected weight loss from these medications
is between 5% and 10%. Topiramate is approved by the Food and Drug Administration (FDA) in
children over age 10 years for seizures and migraine headaches, but not specifically for weight
loss alone. The medication, however, is often used off-label for this purpose. Topiramate is a car-
bonic anhydrase inhibitor and binds to the GABA receptors in the brain and appears to decrease
appetite, as well as alter the taste of food, resulting in decreased food consumption (Yanovski and
Yanovski 2014).
Metabolic and Bariatric Surgery

Bariatric surgery combined with lifestyle management currently results in greater sustained weight
loss and improvement and/or resolution of comorbidities compared with lifestyle management alone
(Alqahtani and Elahmedi 2015, McGinty et al. 2015). Surgeries that are currently FDA approved for
adolescents above age 13 years include the Roux-en-Y gastric bypass (GB), and the laparoscopic
sleeve gastrectomy (LSG) (see criteria for surgery here). Although still available for adults, the
laparoscopic adjustable band, secondary to complications in adults, was removed from FDA trials
in adolescents in 2012. Both GB and LSG have shown significant improvement in obesity-related
comorbidities, with 70%–100% resolution of type 2 diabetes, prediabetes (impaired glucose tol-
erance), insulin resistance, hypertension, dyslipidemia, and nonalcoholic steatohepatitis (NASH)
(Bondada et al. 2011, Alqahtani and Elahmedi 2015). Improvements in insulin sensitivity are seen
quickly, usually prior to discharge of the patient from the hospital, which implies that they are not
due solely to weight loss. These changes are believed to be caused by alteration of gut hormone
interactions (GLP-1, PPY, and ghrelin) with the pancreas and the brain (Lee et al. 2011). Because
of these metabolic changes, the terminology “metabolic and bariatric surgery” is a more accurate
description of the surgery. The LSG has very similar effects on weight loss and comorbidities to the
GB but has fewer nutritional and surgical complications. Thus, the LSG is currently the most widely
performed procedure in both adults and adolescents and is the surgery of choice for adolescents.
The GB is the oldest of the surgeries and is therefore considered to be the gold standard.
Expected weight loss is 70%–80% of excess weight loss (weight loss over ideal body weight
minus weight loss × 100%). The duodenum is bypassed and reconnected to the lower part of the
jejunum, and the stomach is reduced to pouch that is able to hold 15 mm and is approximately
the size of a chicken nugget. The amount of food that can be ingested is limited due to the size
of the stomach. Further, because the majority of the stomach and small intestine are bypassed,
the fats and simple sugars are malabsorbed, thus limiting the consumption of these foods. For the
first 6 months after surgery, consuming foods high in fat or sugar results in diarrhea, a drop in
blood pressure, lightheadedness, sweating, and nausea; this is called the “dumping syndrome.”
These symptoms may result in behavior change regarding the types of food eaten, but then
decrease and often go away completely with time as the body adjusts. Unfortunately, fat-solu-
ble vitamins (A, D, E, and K), water-soluble vitamins (B12, thiamin, and folate), and minerals
(calcium, iron, zinc, and copper) are also not absorbed. Patients therefore require vitamin and
mineral supplementation for life (Xanthakos 2008). Patients are also at increased risk for other
surgical complications (see Chapter 5).
The LSG procedure removes the greater curve of the stomach or 80% of the stomach, leaving a
narrowed gastric tube, which forms the shape of a sleeve. The reduction in the size of the stomach
limits the amount of food consumed. Additionally, the hormone ghrelin, which stimulates hunger and
appetite, is normally produced in the greater curvature of the stomach; removal of this section of the
stomach eliminates the stimulation of hunger for at least the first 9–12 months after surgery. Nutritional
complications directly related to the surgery include vitamin B12 deficiency due to the lack of the
production of intrinsic factor and fluoride deficiency. Due to concerns for limited intake of food and
possible rapid transit of food during digestion, additional vitamin supplementation is recommended.
Further, severely obese adolescents have demonstrated an increased risk of vitamin D deficiency as
well as iron-deficiency anemia prior to surgery (Xanthakos 2008, Aarts et al. 2011).
Surgical Criteria
The criteria for an adolescent to undergo metabolic and bariatric surgery are more stringent than
for an adult. This surgery is considered elective with permanent and serious long-term conse-
quences extending into adulthood. As well, there is a lack of long-term outcome data in the pediatric
population.
According to Michalsky et al. (2011), the medical criteria for metabolic and bariatric surgery for
an adolescent should include
• Age ≥ 13, although most facilities do not perform surgery under age 14 in the United States
• BMI ≥ 50 kg/m2 without any comorbidities
• BMI ≥ 40 kg/m2 with 1 serious or 2 minor comorbidities: NASH, severe psychological
distress, arthropathies related to weight, hypertension, dyslipidemia, chronic venous insuf-
ficiency, panniculitis
• BMI ≥ 35 kg/m2 with type 2 diabetes mellitus (T2DM), moderate-to-severe obstructive

sleep apnea, pseudotumor cerebri, NASH with advanced fibrosis (Nobili et al. 2015)
• Tanner stage 4 or 5 and 95% growth completion as indicated with bone age
Additional criteria include
• Demonstrates maturity to understand risks as assessed by multiple members of the bariatric

team, including the psychologist
• Attends a medical weight management program for at least 6 months
• Takes required medications, vitamins, and supplements
• Attends appointments with specialists
• Agrees not to get pregnant for at least 18 months after surgery
• Has family who will support the adolescent by providing the necessary food and materials,
and supports attendance to visits and changes in the household
• Absolute contraindications include a medically correctable cause of obesity; active substance
abuse problem; medical, psychiatric, or cognitive disability that impairs ability for adherence
Prior to surgery, patients are asked to demonstrate the ability to comply with behaviors that will
decrease surgical complications and will allow them to be successful with surgery (Nogueira and
Hrovat 2014). Patients are asked to separate eating and drinking by 20–30 minutes, which decreases
the risk of vomiting early after surgery. Additionally, liquids allow for the passage of more food at
a time through the sleeve. Patients must drink 64–90 fluid ounces of water daily and take sips as
gulping liquid will cause chest pain. Adequate fluid intake is important in that the most common
reason for hospital readmission is dehydration (Nogueira and Hrovat 2014). Patients are required to
eliminate carbonated beverages to prevent bloating and gas pains with the surgery. They are asked
to only drink water and soy, skim, or almond milk, because sugar-sweetened beverages have no
nutritional value but have excess calories that can pass through the sleeve easily. Patients must track
their protein intake to make sure they are consuming a minimum of 60 g/day to allow for sufficient
wound healing, prevent hair loss, and prevent loss of muscle mass. Prior to metabolic and bariatric
surgery, patients are put on a 2-week modified meal replacement diet to reduce the size of the liver.
The components of this diet are not standardized but generally are comprised of 1000–1300 calories
and are high in protein (Nogueira and Hrovat 2014).
CARDIOMETABOLIC COMORBIDITIES IN CHILDHOOD OBESITY

A child with a BMI at the 85th percentile or higher is at risk for medical problems affecting mul-
tiple organ systems. More than half of obese adolescents have at least one risk factor for premature
cardiovascular disease (CVD) and 10% have at least three CVD risk factors, such as hypertension,
dyslipidemia, and insulin resistance (Freedman et al. 1999, Daniels 2009, Steinberger et al. 2009,
Power et al. 1997). This section will focus on the cardiometabolic health risks associated with child-
hood obesity and nutritional considerations with these risks.
Hypertension
Maintaining normal blood pressure throughout childhood and adolescence is an important aspect of
pediatric cardiometabolic health. During childhood, blood pressure is strongly related to age, sex,
and body size. Thus, blood pressure percentiles for children and adolescents are standardized for
age, sex, and the height percentile. Normal blood pressure is defined as systolic and diastolic blood
pressure below the 90th percentile (Daniels et al. 2011). Prehypertension is defined as either systolic
or diastolic blood pressure between the 90th and 95th percentile or, for teenagers, above 120/80,
which has been used as the definition of prehypertension in adults, but below the 95th percentile.
Stage 1 hypertension is blood pressure above the 95th percentile, but below the 99th percentile
plus 5 mmHg. Stage 2 hypertension is systolic or diastolic blood pressure persistently above the
99th percentile, plus 5 mmHg. This value is generally about 12 mmHg above the 95th percentile.
For a patient to have hypertension, the blood pressure must be persistently elevated on three or more
occasions, days, weeks, or months apart.
Blood pressure should be measured routinely at all health maintenance visits and other health
care visits after the age of 3 years. When an elevated blood pressure is detected, repeat measure-
ments should be made within 3–6 months for prehypertension; within 1 month for stage 1; and within
1 week or sooner for Stage 2 hypertension, or if there are signs or symptoms related to hypertension.
As in adults, childhood blood pressure has many different determinants, including genetic and
environmental factors. Important environmental factors include obesity, as well as diet (discussed in
the following text) and physical activity. The mechanisms by which these factors raise blood pres-
sure are often not clearly understood. However, such mechanistic knowledge is usually not neces-
sary to implement appropriate preventive or treatment strategies.
Obesity in childhood is associated with blood pressure elevation along with a variety of other car-
diometabolic alterations, as discussed elsewhere in this chapter. Weight management strategies that
result in reduction of the BMI percentile have been clearly shown to reduce blood pressure, particu-
larly when elevated blood pressure was present prior to the intervention. Haynes (1986) reviewed
early studies of the impact of weight reduction on blood pressure in adults and found evidence that
weight reduction resulted in lower blood pressure. Rocchini et al. (1988) demonstrated in a clinical
trial that weight loss is associated with blood pressure reduction in obese adolescents. One question
has been whether weight loss may be effective for blood pressure reduction because weight loss diets
are often also low in sodium. Reisin et al. (1978) reported that a diet designed to reduce weight, but
without restriction in sodium, results in lower blood pressure. However, the relationships may be more
complex. Rocchini et al. (1989) found that adolescents with obesity were quite sensitive to sodium in
their diet, such that increased sodium results in a rise in blood pressure. However, after a weight loss
intervention, obese adolescents were significantly less sensitive to sodium in their diet.
For patients with severe obesity, lifestyle intervention may not be sufficient to produce adequate
weight loss. For these adolescents, metabolic and bariatric surgery may be needed, as discussed
earlier in this chapter. Ippisch et al. (2008) found that weight loss surgery can result in lower blood
pressure, as well as decreased left ventricular mass index.
A number of dietary factors have been implicated in blood pressure elevation. These include
sodium, caffeine, potassium, calcium, magnesium, folic acid, and fat content (Simons-Morton
et al. 1997, Dwyer et al. 1998, Knuiman et al. 1988, Sinaiko et al. 1993, Falkner et al. 2000,
He and MacGregor 2006). Sodium, caffeine, and increased fat in the diet are associated with
increased blood pressure, while potassium, calcium, and magnesium have been associated with
decreased blood pressure. Some evidence for these associations comes from animal studies while
other evidence is derived from studies of adults, children, and adolescents. Clinical evidence may
come from observational epidemiologic studies and from clinical trials. Clinical trials generally
provide the best evidence, but these studies are most often focused on treatment of individuals
who have already developed high blood pressure as opposed to those who are well and need
preventive intervention.
Dietary sodium has attracted the most attention related to blood pressure elevation across the age
span. Many studies, particularly in adults, have found a positive association between sodium intake
and blood pressure. Also, some individuals are more sensitive to sodium in their diet than others
(Appel et al. 2006). Unfortunately, there is no simple clinical evaluation to determine if someone
has heightened sensitivity to salt, a phenomenon that may underlie the finding that the association
between dietary salt and blood pressure is more evident in some groups compared to others (Falkner
et al. 1986). These groups would include African Americans and those with a family history of
hypertension. It is believed that sensitivity to sodium is in large part genetically determined, but
specific salt sensitivity genes or polymorphisms have yet to be identified.
Most of the sodium in the diet comes from salt that is added in processing of food (canned,
boxed, or frozen), not from salt that is added with cooking or at the table. This makes it more
difficult for an individual or a family to lower sodium in their diet because the consumption
of preprepared and packaged foods is so high compared with home-prepared foods. Most
American children have a sodium intake that is well above the defined adequate intake levels,
which are 1.2 g/day for children aged 4–8 years and 1.5 g/day for older children and adolescents
(Appel et al. 2006, CDC 2011). This means that children and adolescents could reduce their
sodium intake substantially and would have the potential benefit of lower blood pressure with no
potential for adverse effects.
Studies in adults that focused on potassium supplementation have demonstrated a blood p ressure–
lowering effect (Weaver 2013), but there have been few studies in children. Sinaiko et al. (1993)
found lower blood pressure with age in children supplemented with potassium compared to those
who were not supplemented. Calcium supplementation may also be beneficial, particularly for those
with a combination of elevated blood pressure and low baseline calcium intake (Dwyer et al. 1998).
Calcium supplementation is associated with increased sodium excretion in the urine, which may be
a potential mechanism of action related to its blood pressure–lowering effect (Lasaridis et al. 1989).
Currently, there is insufficient evidence to recommend either potassium or calcium supplementation
in the clinical setting.
While it is important to study dietary micro- and macronutrients, it is more useful from a clini-
cal perspective to evaluate dietary patterns. This is because individuals and families develop their
diet and purchase food related to their dietary patterns. The diet pattern that is most widely studied
in adults and has emerging evidence in children is the Dietary Approaches to Stop Hypertension
(DASH) diet pattern. The DASH diet has been studied extensively in adults. The DASH dietary pat-
tern is high in fruits and vegetables and emphasizes low-fat dairy and whole grain products, while
being low in simple carbohydrates, red meats, and other foods high in saturated fat (Appel et al.
1997). This dietary approach has been shown in randomized clinical feeding studies of adults to be
associated with lower systolic and diastolic blood pressure that was independent of weight loss. The
DASH eating pattern is not necessarily low in sodium. Subsequent studies have demonstrated that
the DASH diet with the addition of sodium restriction is more effective at lowering blood pressure
than the DASH diet alone in adults (Sacks et al. 2001).
There have been some studies of the DASH diet in children and adolescents. Epidemiologic studies
have shown that individuals with a diet more similar to the DASH diet tend to have lower blood pressure
compared to children and adolescents with a standard Western diet. The Framingham Study showed
that children who had higher dietary intake of fruits and vegetables, plus low-fat dairy in their preschool
years, had a smaller increase in blood pressure with age than those with lower intake of these diet com-
ponents (Moore et al. 2005). Moore (2012) found similar results in a longitudinal study of adolescent
girls. Couch (2008) reported the results of a clinical trial of the DASH diet compared to standard care
over a 3-month period in adolescents with prehypertension or hypertension. They found that those in the
DASH diet group had a significantly greater reduction in systolic blood pressure compared to those in
the standard care group who received general counseling on diet and sodium reduction.
Combining the epidemiologic and clinical trial results suggests that increasing fruit and vegetable
consumption, along with emphasis on low-fat dairy and whole grains with reduction in red meat,
could be beneficial in both prevention of high blood pressure and management of hypertension when
it occurs in children and adolescents. Further research is needed to evaluate whether additional sodium
reduction may be beneficial in children and adolescents on a DASH diet. An advantage of the DASH
diet is that it is a healthful diet pattern for everyone in the family. This means that it can be more eas-
ily implemented in practice and that family members can support each other in adherence over time.
It is clear that diet and nutritional factors are quite important as they relate to blood pressure ele-
vation. Avoidance of excess weight gain and adherence to a DASH diet pattern could be beneficial
in preventing blood pressure elevation in young individuals. For those who have already developed
hypertension, dietary intervention should be a central aspect of any intervention plan.
Cardiovascular Risk
CVD is the leading cause of death worldwide (Mozaffarian et al. 2015). In adults, studies have
demonstrated the association between obesity and the premature development of CVD (Manson
et al. 1990, Ingelsson et al. 2007). Atherosclerosis begins in childhood, notably in those with over-
weight, obesity, and other CVD risk factors.
The Bogalusa Heart study was one of the first studies demonstrating a strong association between
childhood obesity and fatty streaks and fibrous plaques located in the aorta and coronary arteries
(Berenson et al. 1998). Similarly, in the Muscatine Study, Mahoney (2001) found that the greatest
predictor in young adulthood of coronary calcium seen on computed tomography was being over-
weight during childhood. Several studies have also found that obesity in childhood and adolescence,
with and without associated dyslipidemia, predisposes patients to elevated carotid intimal media
thickening and endothelial dysfunction (Freedman et al. 2004, Urbina et al. 2009, Urbina et al.
2010).
In addition, obesity induces structural and hemodynamic changes in the heart, including increased
blood volume and cardiac output. Pulmonary arterial hypertension from sleep apnea and hypoven-
tilation may occur and youth with morbid obesity are at increased risk for developing a cardiomy-
opathy from these abnormalities (Speiser et al. 2005). Along with elevated systolic blood pressure,
obesity is one of the risk factors in the development of increased left ventricular mass in the young
(Yoshinaga et al. 1995, Daniels et al. 1998, Urbina et al. 1999). In adults, left ventricular hypertro-
phy has been found to be an independent risk factor for CVD morbidity and mortality (Flynn and
Alderman 2005).
Dyslipidemia of Obesity
Nearly half of all overweight and obese children have at least one abnormal lipid value (CDC 2010).
The dyslipidemia that is common in this population is called combined dyslipidemia of obesity
(CDO), which consists of elevated TG, low levels of HDL-C, elevated non-HDL-C, and normal or
mildly elevated LDL-C. The LDL particles (LDL-P) tend to be the small, dense subtype.
Indeed, the presence of CDO has been shown to correlate with elevated levels of insulin, over-
weight/obesity, and central fat deposition. In the HEALTHY study, which evaluated lipids in a large
population of sixth-grade children, investigators found that one-third of overweight or obese chil-
dren had a TG/HDL-C ratio > 3.0% and 11.2% had a non-HDL-C >145 mg/dL (Mietus-Snyder et al.
2013). This corroborates with the NHANES data, which found that increased levels of non-HDL-C
in adolescents aged 12–19 years were associated with the MetSyn (Li et al. 2011).
In the long term, the presence of CDO appears to be the most common lipid pattern associated
with clinical CVD in adulthood. In the Framingham Offspring Study, investigators found that CDO
on a standard lipid profile was one predictor of early clinical CVD events, such as myocardial infarc-
tion and/or death from CVD (Robins et al. 2011). It also is a good detector of elevated small LDL-P,
which is a lipid parameter that is also correlated with CVD events (Cromwell et al. 2007). Similarly,
the Princeton Follow-up Study, which evaluated risk factors for early CVD during childhood and
again two decades later, found that individuals with elevated TG levels and TG/HDL-C ratios at
12 years of age, and who maintained these lipid abnormalities into adulthood, were more likely to
experience clinical CVD during adulthood (Morrison et al. 2012).
The treatment of obesity-related dyslipidemia is a focused change in diet and exercise habits.
A referral to a registered dietitian is imperative, not just for the child but for the entire family to
understand how to make appropriate dietary changes (Daniels et al. 2011). In both the pediatric
and adult populations, improvement in CDO has been demonstrated with weight loss, restriction
of simple carbohydrates, and increased physical activity (Nemet et al. 2005, Meyer et al. 2006,
Ebbeling et al. 2007, Ebbeling et al. 2012b, Kirk et al. 2012). Changes of diet and lifestyle act
to decrease TG and non-HDL-C levels, reduce TG/HDL-C ratio, and alter the LDL-P to a larger,
less atherogenic subtype (Becque et al. 1988, Kang et al. 2002, Sondike et al. 2003, Watts et al.
2004, Nemet et al. 2005). In particular, restricting carbohydrates, notably simple sugars and quickly
hydrolyzed starches, has been shown to decrease TG levels significantly (Pieke et al. 2000, Sondike
et al. 2003, Pereira et al. 2004, Ebbeling et al. 2007). When taking a dietary history in the overweight
or obese child, the amount of sweetened beverage consumed should always be assessed. Eliminating
sweetened drinks (e.g., soft drinks, iced tea, lemonade, etc.) almost always results in a reduction in
TGs and a simultaneous weight loss as well (Dornas et al. 2015). Along with reduction in simple
sugars and carbohydrates, there should also be an increase in complex carbohydrates and fiber and
consumption of low-mercury fish twice weekly.
While the majority of obese children with dyslipidemia will demonstrate evidence of CDO, there
are some who may present with isolated elevation of LDL-C or with associated decreased HDL-C.
While weight loss is again necessary for improvement in the cholesterol values, the dietary focus is
somewhat different from those with the CDO lipid profile. In these patients, the focus is on a diet
low in saturated and trans fats, ideally with 25%–30% of calories from fat, <7% of calories from
saturated fats, and approximately 10% of calories from monounsaturated fats. There should be no
dietary intake of trans fat. Food labels are required to indicate the amount of trans fat in all products.
For additional LDL-C lowering, soluble fiber in the form of psyllium should be added at 6 g/day for
children up to age 12 years and 12 g/day for children 12 years and older (Fletcher et al. 2005). The
use of plant stanol or sterol esters, found in certain food products (e.g., special margarines, milk,
oatmeal, nutrition bars, tortilla chips) as well as in pill forms, may reduce LDL-C by an additional
6%–15% at a dose of 2 g/day (Kerckhoffs et al. 2002, Fletcher et al. 2005). These are generally used
in those with more significantly elevated LDL-C levels.
Metabolic Syndrome
In adults, the MetSyn is a constellation of cardiovascular and metabolic risk factors that cluster
together and appear to increase the risk of premature CVD, as well as T2DM (Grundy et al. 2005,
Cook et al. 2008). The risk factors include dyslipidemia (high TG, low HDL-C, increased small,
dense LDL), elevated blood pressure, elevated blood glucose, excess abdominal adiposity, and pro-
inflammatory and/or pro-thrombotic states (Cook et al. 2008). Over the past several years, major
health organizations have proposed somewhat different criteria and cut points for the definition of
MetSyn in adults (World Health Organization 1999, National Institutes of Health 2002, Alberti et al.
2005, Grundy et al. 2005), although, more recently, a joint statement from these health organizations
was released in an attempt to unify the defining criteria (Alberti et al. 2009).
In the pediatric population, definition, assessment, and treatment of the MetSyn have been prob-
lematic. Part of the difficulty is that there are changes in lipid levels, blood pressure, and other
metabolic measures with different ages and stages of puberty. There are also limited long-term data
evaluating the tracking of the MetSyn characteristics from childhood to adulthood and the subse-
quent impact of these risks on premature CVD (Steinberger et al. 2009). However, it is important to
identify at-risk children for MetSyn to help prevent the progression of obesity and the potentially
long-term metabolic, endocrinologic, and cardiovascular ramifications. To this end, in 2007, the
International Diabetes Federation (IDF) published an age-based definition of MetSyn. For children
6–9 years old, it was suggested that the term “MetSyn” not be applied, but that practitioners focus
on weight reduction, especially for those children with abdominal obesity, defined as waist circum-
ference percentile >90th percentile (Zimmet et al. 2007). For children 10–15 years old, MetSyn
can be diagnosed with the presence of abdominal obesity, using the same definition as mentioned
earlier (Zimmet et al. 2007) and at least 2 risk factors, including hypertriglyceridemia, low HDL-
C, elevated blood pressure, and/or elevated fasting glucose. For those 16 years of age or older, the
authors recommended using adult IDF criteria for MetSyn (Zimmet et al. 2007).
Even in children, there are identifiable risk factors for the development of MetSyn. Heredity does
play a role as children of parents who have MetSyn are at increased risk of developing MetSyn, likely
due to a combination of both genetics and environment (Hong et al. 1997, Steinberger et al. 2009).
Ethnicity is another factor in the development of MetSyn, notably with differences in certain com-
ponents of MetSyn between white, black, and Hispanic children. For example, black and Hispanic
children have a higher prevalence of obesity but black youth have lower levels of TC and TG and higher
HDL-C levels when compared with white children (Chen et al. 1999, Ogden et al. 2002).
While there is not a truly agreed upon definition of MetSyn in children, it does appear that
focusing on lifestyle modification, especially directed toward obesity, will help to slow or even halt
the development of risk factors for MetSyn. Studies have shown that modifications to both dietary
composition and physical activity levels can attenuate the risk factors for MetSyn for at least 1 year
(Nemet et al. 2005) and also can improve endothelial dysfunction, notably in children with excess
adiposity (Woo et al. 2004). Indeed, it seems that a concentrated focus on weight control (weight
stabilization or weight loss) through a combination of increased physical activity, along with a diet
rich in whole grains with limited amounts of simple sugars, simple carbohydrates, and saturated fats,
may help to prevent the development of MetSyn and the associated comorbidities as these children
become adults.
Fructose has also been identified as a key player in MetSyn. Since fructose is unable to be regulated
by insulin, fructose intake results in increased insulin resistance. Glucose and fructose are metabolized
differently with the majority of fructose resulting in fat deposition in the liver, promoting fatty liver
disease and increased triglyceride production. Fructose also increases inflammatory markers. Further,
the by-product of metabolized fructose is uric acid, which increases blood pressure (Das 2015). In a
study in obese children with MetSyn, restriction of dietary sugars from 28% to 10% in replacement for
starch improved systolic blood pressure, TG, LDL-C, insulin sensitivity, and glucose tolerance after
9 days (Lustig et al. 2016).
Insulin Resistance and Type 2 Diabetes Mellitus

T2DM is a significant comorbidity of obesity in adolescents. T2DM is characterized by the develop-
ment of hyperglycemia, insulin resistance, and relative impairment in insulin secretion.
Insulin resistance is a state in which a given concentration of insulin is associated with subnormal
glucose response. This can be assessed using a homeostatic model assessment, which is a method used
to quantify insulin resistance and beta cell function. A Homeostatic Model Assessment-Estimated
Insulin Resistance ≥ 3.99 is an indicator for abnormal glucose regulation (Turchiano et al. 2012).
Impaired fasting glucose is defined as a fasting plasma glucose level of 100–125 mg/dL or hemo-
globin A1c of 5.7–6.4%. Insulin resistance may develop in the early stages of childhood obesity and
in very young children and has been associated with higher expression of central obesity and T2DM
(Skoczen et al. 2015).
Diabetes mellitus is diagnosed by the following criteria (American Diabetes Association 2011):
• Fasting plasma glucose ≥126 mg/dL (7 mmol/L)

• Plasma glucose ≥200 mg/dL (11.1 mmol/L) measured 2 hours after an oral glucose toler-
ance test
• Hemoglobin A1C ≥ 6.5% (47 mmol/mL)
Multiple studies have shown that obesity can predict the development of T2DM (Freedman et al.
1999, Copeland et al. 2011, Hannon et al. 2005, Liu et al. 2010). In addition to general lifestyle
management, an increase in dietary fiber, reduction in simple sugars and refined carbohydrates, and
an increase in physical activity are key components to improvement in insulin and glucose regula-
tion (Krauss et al. 2000).
For children above the age of 2, the fiber recommendation is their age + 5 g up to a maximum of
25 g daily (Williams et al. 1995), so that a 10-year-old child should consume a minimum of 15 g of
fiber per day.
Nonalcoholic Fatty Liver Disease

NAFLD is the most common cause of chronic liver disease in childhood and adolescence and is
found in 9.6% of children aged 2–19 (Schwimmer et al. 2006). NAFLD presents a spectrum of
pathology, ranging from simple triglyceride accumulation in hepatocytes to hepatic steatosis with
inflammation (steatohepatitis), fibrosis, and cirrhosis (Neuschwander-Tetri and Caldwell 2003,
Chalasani et al. 2012). NAFLD is defined as macrovesicular fat accumulation in >5% of hepato-
cytes as assessed by liver biopsy, in the absence of excessive alcohol intake or viral, autoimmune,
or drug-induced liver disease. Approximately 25% of children with NAFLD have a progressive sub-
phenotype known as NASH (Molleston et al. 2002). An estimated 7%–10% of children with NASH
will develop cirrhosis and end-stage liver disease (Rubinstein et al. 2008).
Children with NAFLD are usually asymptomatic and are found to have mildly elevated liver
enzymes on screening in the setting of obesity. Elevations in alanine aminotransferase (ALT) are
usually greater than aspartate aminotransferase; however, liver enzymes may be completely normal.
Other forms of liver disease that cause elevations in ALT must also be ruled out (Pardee et al. 2009).
Lifestyle change with weight loss through diet and exercise remains the mainstay of therapy in
NAFLD. This has proven to improve serologic markers and liver histology.
Dietary Intervention and Physical Activity

Weight loss is known to decrease delivery of free fatty acids to the liver and increase extrahepatic
insulin sensitivity by means of improving peripheral glucose utilization and promoting reduction in
reactive oxygen substances, as well as reducing adipose inflammation (Shah et al. 2009). Evidence
to support the utility of weight loss in the treatment of pediatric NAFLD was demonstrated in a
study by Nobili (2006), which evaluated the effect of lifestyle change in 84 children with biopsy-
proven NAFLD. The subjects underwent a 12-month program of lifestyle advice, focusing on physi-
cal activity and dietary modifications, including a balanced, low-calorie diet (25–30 calories/kg/
day; carbohydrate 50%–60%; fat 23%–30%; protein 15%–20%; fatty acid, two-thirds saturated,
one-third unsaturated; ω6/ω3 ratio = 4:1) as recommended by the Italian Recommended Dietary
Allowances. Patients who completed the diet and exercise program had a significant improvement
in BMI, insulin sensitivity, liver enzymes, and liver echogenicity on ultrasound. They also demon-
strated improved liver histology on biopsy (Nobili et al. 2008).
The role of specific dietary macronutrient composition during weight loss has not been exten-
sively studied in children. However, low-glycemic-load diets designed to reduce postprandial rise
in blood glucose and insulin either through carbohydrate restriction or low GI have shown some
promise for use in treatment (Ebbeling et al. 2003). Pozzato et al. (2010) placed 26 obese children
between 6 and 14 years on a normocaloric balanced diet (carbohydrates 55%–60% with <10% high
glycemic index; fat 25%–30% with <10% saturated fat; protein 12%–15%) for 1 year. Mean liver fat
fraction quantified by hepatic proton magnetic resonance spectroscopy declined by 8% (p < 0.0001)
with a decrease in BMI z-score of 0.26 (p < 0.001). The low-GI diet, although effective in reduc-
ing NAFLD, has not been found to be more effective than a low-fat diet in children. A randomized
controlled trial found an estimated 8%–10% reduction in hepatic triglyceride content quantified
on proton magnetic resonance spectroscopy in obese children who underwent either a 6-month
low-glycemic (low-to-moderate glycemic load of carbohydrates 40%, fat 35%–40%) or low-fat
(55%–60% of carbohydrates, 20% fat, <10% saturated fat) dietary intervention with no significant
difference seen between the dietary groups. BMI decreased by −1.3 kg/m2 in the low-glycemic
group (0.0007) and by 1.2 kg/m2 in the low-fat group (p = 0.0004) with no significant difference
between both groups (Ramon-Krauel et al. 2013).
Diets high in fructose are known to increase plasma lipids and oxidative stress. Fructose under-
goes first-pass metabolism in the liver. By-products of fructose metabolism are fat deposition in the
liver, increased triglyceride formation, increased uric acid formation (leading to high blood
pressure), and an increase in the production but not the utilization of insulin (insulin resistance)
(Lim et al. 2010). In addition to overall weight loss, reduction in fructose intake has been proposed
as a potential treatment of NAFLD (Ramon-Krauel et al. 2013). A randomized controlled 6-month
pilot study of 10 children with NAFLD compared the effectiveness of a low-fructose to a low-fat
diet in improving ALT levels. Patients on the low-fructose diet eliminated sugar-containing bever-
ages, fruit juice, and food items in which high-fructose corn syrup was a primary ingredient. At the
end of 6 months, oxidized LDL was significantly lower in the low-fructose group. However, ALT
levels did not change in either group (Vos et al. 2009). The FRAGILE diet is a combination of low
fructose/low glycemic index/low glycemic load. A study evaluated patients on a 6-month FRAGILE
dietary intervention in children with NAFLD (n = 12) compared with healthy lean controls (n = 14).
The investigators found that absolute fructose intake related strongly to plasma aminotransferase,
systolic blood pressure, percent body fat, insulin resistance, and cholesterol level, independent of
weight loss. This suggests that a modest reduction in fructose intake and glycemic index improves
liver dysfunction and cardiometabolic risk (Mager et al. 2015).
Pharmacotherapy and Surgery

As in adults, pediatric pharmacotherapy of fatty liver disease has focused on treating insulin resis-
tance and oxidative stress. Multiple drugs are being evaluated for the treatment of NAFLD, with
only vitamin E demonstrating improvement in biopsy findings (Sanyal et al. 2010). Although vita-
min E may have shown some benefit, it is not superior to placebo in sustained reduction in ALT
(Lavine et al. 2011). The Roux-en-Y GB and the LSG are recommended for severely obese adoles-
cents with NASH (Nobili et al. 2015).
Until the prevalence and severity of obesity improve, the prevalence of NAFLD probably will
continue to rise. Unfortunately, awareness and screening for NAFLD are still lacking. There is cur-
rently no consensus on screening, diagnosis, or management of this patient population. At this time,
lifestyle modification and weight loss through diet and exercise remain the mainstay of treatment.
Research into the pathogenesis, risk factors, natural history, noninvasive modalities, and treatment
of NAFLD is greatly needed.
SUMMARY
In conclusion, obesity in childhood and adolescence has become a major public health issue. As
discussed throughout this chapter, the cardiometabolic consequences are significant and place our
youth at risk of morbidity and mortality not only during childhood but into adulthood as well. We
need better strategies, improved access, and more funding toward prevention and treatment at all
levels of this chronic disease and the health repercussions it causes.
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9 Aging and Cardiovascular
Disease
Lessons from Calorie Restriction
Jasper Most and Leanne M. Redman
CONTENTS
Aging and Cardiovascular Disease.................................................................................................174
Calorie Restriction and Human Aging............................................................................................176
Proposed Mechanisms by Which CR May Affect Human Aging and CVD...................................176
Extension of Life Span due to Slowing of the Rate of Aging....................................................176
Extension of Life Span due to Prevention or Slowing of Age-Related Disease Onset..............177
Evidence for the Antiaging Effects of CR in Humans....................................................................178
An Unintended Study of CR from the Biosphere 2 Experiment................................................178
Self-Administered CR in the Calorie Restriction Optimal Nutrition Society............................178
Short-Duration Interventions with Calorie Restriction..............................................................179
Longer-Duration Interventions with Calorie Restriction...........................................................179
CALERIE 1...........................................................................................................................179
CALERIE 2...........................................................................................................................181
Alternative Strategies for CR..........................................................................................................183
Timed Eating Paradigms............................................................................................................183
Alternate-Day Fasting................................................................................................................183
CR Mimetics..............................................................................................................................184
Conclusion......................................................................................................................................184
References.......................................................................................................................................186
ABSTRACT
The average life expectancy of newborns in the United States is as high as never before. With
advancing age, however, the incidence and prevalence of chronic diseases including cardiovascu-
lar diseases (CVDs) rise. Vice versa, poor cardiovascular health accelerates the aging process and
increases mortality. Calorie restriction may decelerate both the development of CVDs and the pro-
gression of the aging process. In animal models including rodents and nonhuman primates, calorie
restriction has indeed reduced the incidence of CVD and extended life span. In this book chapter,
we review the available evidence for the benefit of calorie restriction on aging and CVD parameters
in humans. Epidemiological data that in fact show reduced CVD mortality during food restriction
are dated a century ago. More recent observational studies and data on long-term voluntary practi-
tioners of calorie restriction offer valuable insight into the potential of calorie restriction to reduce
CVD. The main focus of this chapter will be on the first clinically controlled randomized interven-
tion studies of the CALERIE project. First, three pilot studies have investigated sustained calorie
173
restriction by 25% for 6 or 12 months achieved through different modalities: dietary restriction,
increased exercise, or a combination of both. Thereafter, in the second phase called CALERIE 2,
calorie restriction by 25% was sustained for 2 years to investigate the feasibility and effects of calo-
rie restriction beyond the initial weight loss period. Finally, we discuss alternative strategies that
may achieve comparable effects of calorie restriction but may be easier to implement such as timed
eating paradigms and the calorie restriction mimetic resveratrol.
AGING AND CARDIOVASCULAR DISEASE

Over the past 100 years, food supply and quality, hygiene standards, and health care have improved.
As a result in 2014, the average life expectancy for an individual born in the United States was 78.8
years, 81.2 years for females and 76.4 years for males (Murphy et al. 2015), the highest estimates
ever reported (Murphy et al. 2015; The Board of Trustees of the Federal Old-Age and Survivors
Insurance and Federal Disability Insurance Trust Funds 2015; Xu et al. 2016). In line with this data,
the age-adjusted death rate for the U.S. population in 2014 was at a record low of 725 deaths per
100,000 people (Murphy et al. 2015).
Of all the deaths reported in the United States between 2013 and 2014, 74% were accounted for
by the 10 leading causes of mortality that include heart disease, cancer, chronic lower respiratory
diseases, unintentional injuries, stroke, Alzheimer’s disease, diabetes, influenza and pneumonia, kid-
ney disease, and suicide (Murphy et al. 2015; Xu et al. 2016). Among these, cardiovascular diseases
(CVDs), which include heart disease, hypertension, stroke, pulmonary artery disease, and diseases
of the veins, accounted for approximately 31% or 800,937 deaths. As an individual advances in age,
both cognitive and physical capacities decline and the susceptibility for chronic diseases including
CVD increases (Lakatta and Levy 2003; Mozaffarian et al. 2015). This progressive decline of physi-
ological integrity and body functioning with increasing age has been defined as “primary aging”
(Holloszy 2000). In the United States, CVD-related deaths due to the increasing age of the popula-
tion significantly increased by more than 30% (Roth et al. 2015). Even in the absence of CVD at the
age of 50, the lifetime risk for men and women to develop CVD later in life is still more than 50%
and ~40%, respectively (Lloyd-Jones et al. 2006).
Economically, the aging of the U.S. population has caused the total health care expenditures
to rise up to $632 billion estimated in 2015 and is expected to almost double reaching $1.1 tril-
lion over the next 10 years (Congressional Budget Office’s 2015). Of the health care expenditures
related to CVD, the costs in 2010 are expected to triple by 2030, from $270 billion to $820 billion
(Heidenreich et al. 2011). While these estimations are based on data from 2010, the well-docu-
mented increase in risk factors for CVD such as a lack of physical activity, childhood obesity, and
type II diabetes mellitus will likely contribute to a higher prevalence of CVD than anticipated, hence
leading to a more significant health care burden in the future (Han, Lawlor, and Kimm 2010; Lee
et al. 2012; Menke et al. 2015).
Interestingly, the increasing prevalence of these noncommunicable diseases may even outweigh
the positive effect of improving health care and may cause a stagnation or even decline in life expec-
tancy in the future (Ludwig 2016). This acceleration of aging and the increase in mortality from
external factors such as the influence of lifestyle or the presence of chronic disease such as CVD has
been described as “Secondary aging” (Holloszy 2000).
The progressive and detrimental interaction between aging and the development of CVD is
likely being translated by their common pathologies and clinical manifestations, as summarized
in Figure 9.1. With advancing age, adipose tissue mass increases and muscle mass and strength
decline. The metabolic functions of these tissues including the clearance and storage of postprandial
blood glucose and lipids deteriorate with age, which contributes to a disproportionate accumula-
tion of fat in visceral and ectopic depots such as the liver and skeletal muscle (Atkins et al. 2014;
Onoue et al. 2016). Ectopic fat accumulation is closely associated with disordered handling of lipids
Aging and Cardiovascular Disease 175
• Ectopic fat accumulation

• Visceral adipose tissue
Adiposity
• Hypercholesterolemia
• Hyperlipidemia
Impaired
• Mitochondrial dysfunction
skeletal
• Hyperinsulinemia
muscle
• Insulin resistance
function
Aging
CVD
• Hypertension
Impaired
• Atherosclerosis
vascular
• Arterial stiffness
function
• Systemic inflammation
• Lower heart
Impaired
rate variability
cardiac
• Reduced cardiac output
function
• Reduced exercise capacity
FIGURE 9.1 Overview of symptoms and risk factors of aging and CVDs. CVDs are caused by impaired
functioning of the heart and the vasculature. Fat accumulation in visceral and ectopic depots is closely related
to the development of CVD. They reflect an overload of healthy fat depots and are therefore associated with
increased plasma concentrations of lipids, cholesterol, and inflammatory signals. Ectopic fat accumulation
and inflammation impairs insulin signaling. As a consequence, cells are less responsive to the signals of
insulin (insulin resistance) and take up less glucose. To compensate for this relative lack of insulin, pancreatic
secretion increases and causes hyperinsulinemia. A reduced diameter of the arteries is caused by infiltration of
the arterial walls by cholesterol and inflammatory signals (atherosclerosis) and by vascular resistance to dila-
tation. An impaired blood supply to tissues such as heart and brain can ultimately cause myocardial infarction
or stroke, respectively. To maintain blood supply against a higher resistance (=smaller diameter), heart rate
and blood pressure increase. Chronically elevated blood pressure and vascular resistance force the heart to
pump harder. This compensation however is transient as the heart fatigues and as a consequence its capacity
to pump blood into the circulation (cardiac output) and its reactivity to stimuli that would accelerate or slow
its beating rate (heart rate variability) declines.
(dyslipidemia), insulin resistance, and impaired vascular and cardiac function (Lim and Meigs 2013)
and is predictive of both aging (Liu and Li 2015) and CVD (Britton et al. 2013).
Because of the aging population and the increasing prevalence and costs of managing age-
associated diseases, interventions to attenuate the aging process are desperately needed. The most
promising nutritional intervention with the potential to counteract both primary and secondary
aging in humans is calorie restriction (CR), which is defined as a sustained reduction of habitual
energy intake, typically by 20%–50%, without malnutrition (Holloszy and Fontana 2007). In all
animal models studied to date, including rodents and monkeys, CR has been shown to attenuate
the onset of age-related chronic diseases and prolong life span (Speakman and Mitchell 2011).
Evidence that CR induces a comparable effect on risk factors for CVD in humans is accumu-
lating, giving rise to the potential for CR as an effective intervention for CVD prevention and
treatment.
CALORIE RESTRICTION AND HUMAN AGING

The first study to demonstrate that CR prolonged life span was performed in rats in the 1930s
(McCay, Crowell, and Maynard 1989). Thereafter, similar observations have been reported across
a wide range of species, including yeast, worms, spiders, flies, fish, mice, and rats (reviewed in
Heilbronn and Ravussin (2003)). More recently, the effects of prolonged CR (over 4–20 years)
on longer-lived animals such as nonhuman primates have been studied at different research insti-
tutes across the United States. Collectively, these studies report that CR improves metabolic health,
including reduced weight, improved body composition, and blood lipid profiles, thus reducing the
risk of chronic diseases such as CVD, type 2 diabetes mellitus, and cancer (Bodkin et al. 2003;
Cefalu et al. 2004; Colman et al. 2009, 2014; Mattison et al. 2012). At this stage, however, results on
all-cause mortality and age-related death are conflicting (Colman et al. 2014; Mattison et al. 2012),
which is likely due to differences in diet composition and supplementation, a slight CR in control
animals, and age at time of CR initiation (Mattison et al. 2017). Furthermore, there are notable dif-
ferences in animal husbandry between the primate colonies that interfere with survival rates of the
CR and control animals alike.
Epidemiological data from human studies support a beneficial effect of CR to prevent CVD.
For example, in Denmark and Sweden, CR implemented by government-initiated food restrictions
during World War I (extent of CR not specified) and World War II (20% CR) led to a reduced total
mortality and CVD mortality, respectively (Hindhede 1920; Strom and Jensen 1951). Similarly,
individuals residing on the small Japanese island of Okinawa are estimated to eat a diet that provides
17% fewer calories than their counterparts on mainland Japan, which has been associated with a
reduction in the age-adjusted risks for the development of coronary heart disease (Willcox et al.
2007). Additionally, the CR diet of the Okinawans was associated with an extended life span of
1.5 years as compared to the population of mainland Japan (Willcox et al. 2007). Sadly, an increased
presence of U.S. military on Okinawa starting in 1960 led to the introduction of a Westernized
diet and, consequently, the average level of CR, as well as the quality of the diet, declined. As a
consequence, the life expectancy for children born today is not different between Japanese living
on Okinawa and those residing on the mainland (life expectancy: girls, 87.0 vs. 86.4 years; boys,
79.4 vs. 79.5 years, respectively), whereas elderly Okinawans (>65 years) are still expected to live
longer (women, 89.9 vs. 88.9 years; men, 84.5 vs. 83.8 years) (System of Social and Demographic
Statistics 2016), which likely reflects their exposure to CR early in life.
Within the last decade, the first clinical controlled intervention trials to test the effect of CR on
human aging have been undertaken, and while the results of these studies will not provide conclu-
sions to the impact on life span, these trials allude to the impact of CR on mechanisms of primary
and secondary aging including risk factors for age-related diseases (Das et al. 2007; Heilbronn et al.
2006; Racette et al. 2006; Ravussin et al. 2015).
PROPOSED MECHANISMS BY WHICH CR MAY

AFFECT HUMAN AGING AND CVD
Extension of Life Span due to Slowing of the Rate of Aging
The ability of sustained CR to positively affect health and ameliorate aging is postulated to occur
via two primary and complementary mechanisms; a reduction in energy expenditure and reduced
oxidative stress.
The flux of energy through the body, or “rate of living,” is a strong negative predictor for life span
(Sacher and Duffy 1979). If the metabolic potential, or the energy expendable during a lifetime, is
fixed per species as postulated (Sohal and Allen 1985), a reduction in energy expenditure may extend
life span. During CR, a reduction in energy expenditure can be induced by lowering the metabolic
mass of the individual (e.g., weight loss), by a reduction in physical activity (behavioral adaptation),
or by other metabolic adaptations. CR-induced metabolic adaptations may be due to reduced oxygen
requirements to produce ATP (or increased energy efficiency) or due to slowing of ATP-consuming
processes (metabolic rate) (Liesa and Shirihai 2013). Despite these suggested benefits of reduced
energy expenditure, it needs to be mentioned that a reduction of energy expenditure may increase
the likelihood of a positive energy balance and subsequent weight regain.
Related to the rate of living theory is the oxidative damage theory of aging. Of all oxygen
consumed by the electron transport chain, 1%–3% is reduced to oxygen radicals by leaking elec-
trons. Leaking electrons are electrons that are not properly transferred along the electron transport
chain and therefore diffuse back into the mitochondrial matrix. The generated oxygen radicals can
accumulate in cells as toxic reactive oxygen species (ROS) (Alexeyev, Ledoux, and Wilson 2004),
which in turn leads to damage to the electron transport chain and to the mitochondrial DNA. This
elicits a vicious cycle, because the damaged electron transport chain leads to increased leakage of
more electrons, which ultimately leads to a decline in physiological function and aging (Sohal and
Weindruch 1996). Together these two theories posit that a reduction in the rate of living or meta-
bolic rate due to CR (and hence oxygen trafficked through electron transport chain) causes fewer
ROS to be accumulated. Over time, it is hypothesized that decreased accumulation of ROS yields
less oxidative damage to lipids, proteins, and DNA, thereby leading to an attenuation in the rate of
primary aging.
Extension of Life Span due to Prevention or Slowing of Age-Related Disease Onset

CR prevents overeating and as reviewed earlier, studies in nonhuman primates show a prevention
or an attenuation of the increase in body weight and adiposity including visceral and ectopic fat
accumulation in CR animals compared to the ad libitum fed counterparts that experience age-related
increases in body weight and adiposity (Bodkin et al. 2003; Cefalu et al. 2004; Colman et al. 2009,
2014; Mattison et al. 2012). Consistently, CR animals experience less weight gain than animals on
an ad libitum diet, yet weight trajectories within the CR groups differ between studies dependent on
the study design. While some studies define CR as energy intake clamped to maintain body weight,
whereas the control animals gain weight on an ad libitum diet (Bodkin et al. 2003), in other stud-
ies animals are fed on 30% CR and therefore lose ~10% body weight after 4 years (Cefalu et al.
2004) and 18 months (Colman et al. 1998). Over longer terms, body weight stabilizes and under-
goes age-related changes yet remains lower in CR animals compared to their ad libitum (AL) fed
counterparts (Mattison et al. 2005). Throughout different studies, CR induced significant reductions
of dyslipidemia, hypertension, and incidence of T2DM and CVD (Bodkin et al. 2003; Cefalu et al.
2004; Colman et al. 2009, 2014; Mattison et al. 2012). If CR is similarly effective in humans as in
primates (e.g., there is no age-induced increase in the prevalence of obesity, the age-related decline
in physical activity is no more than 10%, blood pressure is ~25% lower, and there is no evidence
of diabetes mellitus), the American Heart Association and World Heart Federation estimate that
2 million premature deaths due to CVD among men and women could be prevented by CR diets
(Sacco et al. 2016).
Since mortality and longevity are not typically obtainable endpoints in human studies, an under-
standing of the influence of antiaging therapies such as CR on human aging is reliant on the mea-
surement of aging biomarkers. Proposed and well-studied biomarkers indicative of advanced aging
are high body core temperature and high insulin concentrations (Roth et al. 2002). High body tem-
perature likely reflects a high metabolic rate (rate of living) and hence a shortened life span (Keil,
Cummings, and de Magalhaes 2015). Chronically elevated concentrations of insulin may favor CVD
by promoting age-induced increases of adiposity and insulin resistance (Zhang and Liu 2014). The
Baltimore Longitudinal Study of Aging (Shock 1984) investigated the validity of these biomarkers
through their study of mortality in healthy men. A comparison of core body temperature and insulin
levels between those individuals with values in the upper versus lower half of the cohort for these
measures showed that mortality was indeed positively associated with increased core temperature
and insulin concentrations (Roth et al. 2002). The study of these two biomarkers in nonhuman
primate colonies provided additional evidence of the impact of CR diets on the attenuation of aging.
Nonhuman primates consuming a CR diet had reduced rates of mortality and an attenuation of the
age-associated changes in both core body temperature and insulin in comparison to the animals
maintained ad libitum (Roth et al. 2002).
EVIDENCE FOR THE ANTIAGING EFFECTS OF CR IN HUMANS

An Unintended Study of CR from the Biosphere 2 Experiment
Biosphere 2 is a 3.15-acre ecological enclosure that unintentionally provided an opportunity to
study the effects of CR in humans under controlled conditions (Walford, Harris, and Gunion 1992).
A group of eight nonobese individuals between the ages of 25 and 67 entered the Biosphere 2
enclosure for 6 months. Due to unanticipated agricultural problems with the growing and harvesting
of food inside the enclosure, the energy intake of the inhabitants was restricted by ~29%. The diet
composition was diverse, largely vegetarian, and provided adequate protein, high fiber, and low fat.
Over the 6-month observational period, the CR diet resulted in a 15% weight loss.
In line with the hypothesis that CR may reduce the rate of living, 24-h energy expenditure in
the Biosphere inhabitants assessed 1 week and 6 months after exiting the Biosphere enclosure was
less than energy expenditure in 152 non-CR free-living subjects with similar heights and weights,
with appropriate statistical adjustment for age, sex, fat-free mass, and fat mass (Weyer et al. 1999,
2000). The CR diet produced a subtle yet nonsignificant reduction in core temperature; however,
this observation may have been underestimated because the thermostats were not calibrated for tem-
peratures <96°F (Walford et al. 1999). Moreover, a rapid increase in insulin levels within a month
after exiting the enclosure suggested that the CR experienced during the enclosure decreased insulin
concentrations (Walford et al. 2002). The increase in insulin concentrations was accompanied by an
increase in body mass index (BMI) of 1.4 kg/m2. As proposed in previous studies, the CR diet of the
Biosphere inhabitants significantly improved the CVD risk. The 6-month CR diet (~29% reduction
in energy intake from baseline levels) decreased the percentage of body fat, plasma triglycerides,
serum cholesterol, low-density lipoprotein (LDL)-cholesterol, as well as systolic and diastolic blood
pressures during the first 3 months of weight loss, but not further when body weight was main-
tained after 3 months within the enclosure (Walford, Harris, and Gunion 1992; Walford et al. 2002).
Furthermore, a reduction in white blood cell count by 30%–40% may indicate a reduced state of
systemic inflammation.
Self-Administered CR in the Calorie Restriction Optimal Nutrition Society

Members of the Calorie Restriction Optimal Nutrition (CRON) Society report self-prescribing and
administering CR diets for 3–15 years, on a voluntary basis. Observational studies in these individu-
als have allowed for the long-term effects of CR in weight-stable humans to be better understood
(Fontana et al. 2004; Holloszy and Fontana 2007; Yang et al. 2016). As compared to a group of
individuals (matched for age and socioeconomic status) consuming a regular Western diet, members
of the CRON Society consume 30% less energy and their CR diets meet all recommended levels
for essential nutrients (Fontana et al. 2004). They have a low BMI (<20 kg/m2) and a low percent
of body fat (<10%), and none of the 50 men and women (age range 30–82 years) practicing long-
term CR, who have been extensively studied, report taking any medication or show evidence of any
chronic disease.
While data on longevity, or mortality, are not yet available for the CRON Society members, as
compared to body fat–matched athletes consuming Western diets, these self-prescribing CR indi-
viduals have significantly lower core body temperature and slightly lower insulin concentrations,
which could reflect an attenuated rate of primary and secondary aging (Fontana, Klein, and Holloszy
2010; Soare et al. 2011). As to be expected due to the higher volume of training, the group of athletes
consumed more than double the amount of energy, whereas macronutrient composition of the diets
is comparable between groups (Fontana, Klein, and Holloszy 2010). With regard to CVD, clinical
outcome measures for cardiac and vascular function such as carotid artery intima-media thickness,
left ventricular diastolic function, and heart rate variability are significantly improved in the CRON
group compared to age- and socioeconomic status–matched control subjects (Fontana et al. 2007;
Meyer et al. 2006; Stein et al. 2012). Furthermore, systolic and diastolic blood pressures, total cho-
lesterol, LDL-cholesterol, and plasma triglycerides were significantly lower, whereas high-density
lipoprotein (HDL)-cholesterol and free fatty acid concentrations were higher as compared to control
subjects (Fontana et al. 2004, 2007). Reduced concentrations of tumor necrosis factor alpha (TNF
alpha), C-reactive protein (CRP), platelet-derived growth factor AB, resistin, and interleukin 6 in
the CRON group also suggest that CR diets may induce anti-inflammatory mechanisms as well as
ameliorate dyslipidemia and insulin resistance (Fontana, Klein, and Holloszy 2010; Fontana et al.
2004; Meyer et al. 2006).
Short-Duration Interventions with Calorie Restriction

In a randomized, controlled 10-week intervention study, the effects of 20% CR (with adequate
nutritional intake and micronutrient levels “approaching recommended daily allowances”) on
energy metabolism have been investigated. Contrary to the oxidative damage theory of aging, a
reduction in metabolic rate in the CR subjects did not lead to a concomitant reduction of indica-
tors of oxidative stress such as malondialdehyde, LDL-oxidation or 8-hydroxy-2-deoxyguanosine,
catalase, and superoxide dismutase (Loft et al. 1995; Velthuis-te Wierik et al. 1995). However,
CR for 10 weeks significantly reduced body weight (Velthuis-te Wierik et al. 1994) and improved
CVD risk factors including a reduction in systolic and diastolic blood pressure and increased
fibrinolytic activity (Loft et al. 1995; Velthuis-te Wierik et al. 1995). Importantly, after 10 weeks
of intervention, body weight was not stabilized yet, thus no distinction can be made between the
effects of weight loss and CR itself.
Longer-Duration Interventions with Calorie Restriction

The Comprehensive Assessment of Long-Term Effects of Reducing Intake of Energy (CALERIE)
trials were initiated by the U.S. National Institute of Aging to provide the first randomized controlled
clinical trials of longer-duration CR in healthy, nonobese humans. The CALERIE trials were con-
ducted in two phases. The overall purpose of the first three individual studies in Phase 1 (CALERIE 1)
was to inform the design and clinical endpoints in a larger, randomized controlled trial of CR for
longer duration in Phase 2 (CALERIE 2).
CALERIE 1
In CALERIE 1, three pilot studies evaluated the feasibility and effects of different modalities of CR on
biomarkers of aging, and metabolic health after 6 months (Heilbronn et al. 2006) or 12 months (Das
et al. 2007; Racette et al. 2006). Compliance to the CR regimen, or calculation of CR, was determined
by the energy-intake balance method, in which the First Law of Thermodynamics is applied to human
physiology. This states that the amount of energy intake is equal to the amount of energy expendi-
ture plus the changes in body energy stores (de Jonge et al. 2007). In the CALERIE studies, body
composition (vis-à-vis energy stores) and total daily energy expenditure were measured by means of
dual-energy x-ray absorptiometry and doubly labeled water before and after the intervention, which
equate to energy intake throughout the intervention. CR was then the ratio of actual energy intake to
baseline energy requirements. These data and calculations were not available in real time throughout the
trials, and therefore body weight was used as a proxy for CR. The study participants followed a strict
behavioral intervention that required frequent individual meetings with the behavioral counselors and
meetings with the whole CR group at the study center. In addition, foods were provided to participants
at regular intervals and this required a visit to the center twice per day during these periods so that break-
fast and dinner meals were eaten under supervision by the dieticians and the lunch and weekend meals
were packaged to go. Any unconsumed foods were returned and weighed. These strategies helped study
participants comply with the CR diets throughout the trials.
CALERIE 1 at Pennington Biomedical Research Center (PBRC) in Baton Rouge compared a
reduction of energy intake alone (25% CR) for 6 months to a group that had a combined reduction
in energy intake and an increase in energy expenditure through exercise (−12.5% energy intake +
12.5% energy expenditure = 25% CR+EX), to a group that achieved a 15 kg weight loss through a
low-calorie diet, and to a control group eating an ad libitum diet (Heilbronn et al. 2006). Participants
were provided with their food during baseline assessments until week 12 and for 2 weeks prior to
post measurements.
The 25% CR group achieved an actual CR of 18% and lost 10% of body mass, 24% fat mass, and
5% fat-free mass over the 6-month intervention (Heilbronn et al. 2006). Indicated by steady nega-
tive slopes of weekly body weight measurements, weight maintenance was not achieved during the
6-month study. In support of the rate of living hypothesis, all components of energy e xpenditure—
sleep (Heilbronn et al. 2006), rest (Martin et al. 2007), 24-h sedentary (Heilbronn et al. 2006), and
free-living (Redman et al. 2009)—were reduced below baseline levels after 6 months of 25% CR.
The observed reduction in sedentary energy expenditure (sleep and 24 h) was ~6% lower than the
energy expenditure that was expected on the basis of the metabolic mass of the individuals at the
end of the study, indicating metabolic adaptation. Interestingly, thyroid hormone (T3 and T4) and
leptin concentrations were significantly reduced after 6 months and were significantly related to this
metabolic adaptation (Heilbronn et al. 2006; Lecoultre, Ravussin, and Redman 2011). In line
with this suggested antiaging effect, core body temperature (assessed over 24 h) and fasting insulin
concentrations were also reduced by the 25% CR diet (Heilbronn et al. 2006). Importantly, despite
the observed decline in physical activity (total energy expenditure) in the CR group, thigh muscle
mass, lower body skeletal muscle strength (knee flexor and extensor strength), and physical func-
tioning (VO2peak/VO2max on a treadmill, expressed per kilogram of body mass) were preserved or
even increased during CR (Larson-Meyer et al. 2010; Weiss et al. 2007). Since strength and physical
functioning decline with age, it is important that CR does not accelerate these effects that can lead
to increased susceptibility to falls, frailty, and fractures. Interestingly, whether CR was achieved by
diet alone (CR group) or diet in conjunction with exercise (CR+EX group), it had no effect on body
composition outcomes or effects on cardiometabolic risk factors. However, aerobic fitness, which
is also a risk factor for CVD, was notably improved to a greater extent in the CR+EX group. Since
longer-term studies comparing CR and CR+EX have not been undertaken, it is difficult to know if
the outcomes of these two different modalities would remain consistent with each other over time or
if one intervention would prevail with greater CR benefits.
The individuals consuming the 25% CR diet for 6 months had a significant reduction in
subcutaneous adipocyte size, visceral adipose tissue (multislice computed tomography), and
reduced intrahepatic lipid content (magnetic resonance spectroscopy) (Larson-Meyer et al. 2006;
Redman et al. 2007). In line with these improvements in overall adiposity and fat distribution,
plasma triglyceride concentrations and hemostatic Factor VIIc were significantly reduced with
the 25% CR diet (Lefevre et al. 2009). Surprisingly, the 6 months of CR did not affect various
other risk factors that are associated with CVD such as concentrations of lipoproteins (LDL-
cholesterol and HDL-cholesterol), fibrinogen, homocysteine, CRP, or blood pressure (Lefevre
et al. 2009). Also, flow-mediated dilatation as a measure of endothelial function was not changed.
The lack of an effect of CR on these latter measures may have been related to the younger age of
the study participants (<50 years) as well as their impeccable health status even before beginning
the CR intervention. Nevertheless, this 6-month study of 25% CR estimated that based on the
changes in total and HDL cholesterol (expressed as their ratio), systolic blood pressure, as well
as participants age and gender (Anderson et al. 1991), the 10-year risk for CVD was attenuated
by 29% (Lefevre et al. 2009). The reduction in CVD risk by CR is further supported by sig-
nificant improvements in insulin sensitivity and β-cell function (assessed by frequently sampled
intravenous glucose tolerance test [FSIGTT]), which may suggest that CR interventions over the
longer term may also mediate downstream effects on the development of type 2 diabetes mellitus
(Larson-Meyer et al. 2006).
For the CALERIE 1 study at Washington University in St. Louis, 48 individuals who were not
obese and between 50 and 60 years of age were randomly assigned to either 20% CR, or a 20%
increase in energy expenditure through endurance exercise or a control group following a healthy
lifestyle (Racette et al. 2006). Noteworthy, the achieved degree of CR was lower as compared
to the 6-month study at PBRC (13% as compared to 18%), which can be explained by the lower
initiated CR, a lower level of compliance to the dietary intervention throughout the study, and
because the estimated energy intake was based on energy requirements at baseline and was not
adjusted throughout the study as participants lost weight. Interestingly, despite this lower CR,
the observed reductions in weight (10.7%) were similar to those measured at PBRC. While in
the study at PBRC (Heilbronn et al. 2006), reductions in thyroid hormone levels were associated
with the reduction in metabolic rate in both the CR and CR+EX groups, no effect on T3 concen-
trations was observed after weight loss that was exclusively exercise induced (achieved calorie
deficit of 12.8%), suggesting that exercise ameliorates the weight loss–induced decline of T3
(Fontana et al. 2006; Weiss et al. 2008). The 12-month CR intervention reduced concentrations of
LDL-cholesterol and CRP indicative of a reduced metabolic risk profile (Fontana et al. 2007). In
line with these improvements, fat accumulation in visceral adipose tissue was reduced by 37% as
assessed by magnetic resonance imaging and adipose tissue endocrine function was improved as
reflected by increased adiponectin and reduced leptin concentrations after 1 year of CR. In addi-
tion, insulin sensitivity, measured during an oral glucose tolerance test (OGTT), was improved
after 1 year of CR (Villareal et al. 2006; Weiss et al. 2006). Additionally, a CR diet–induced
improvement of left ventricular diastolic function and reductions of oxidative stress to DNA and
RNA in white blood cells suggested a preventive effect of CR on aging and CVD (Hofer et al.
2008; Riordan et al. 2008). Despite the longer duration of the study in St. Louis as compared to
PBRC, the participants of both weight loss groups did not achieve a weight maintenance status
after a year and thus, as we have mentioned before, a distinction between the effects of weight loss
and CR is not possible (Racette et al. 2006).
In the CALERIE 1 study at Tufts University in Boston, 46 young (24–42 years old) men and
women who were overweight (BMI, 25–29.9 kg/m2) were assigned to either a high- or a low-
glycemic-load diet at 30% CR for 1 year (Das et al. 2007). The degree of CR achieved by the 2 CR
groups did not differ significantly (15% CR), and therefore the 8% loss of body weight and 15% loss
of body fat were also similar. CR-induced reductions of the resting metabolic rate and plasma insu-
lin concentrations are in line with previously mentioned studies (Pittas et al. 2006). As in the other
two pilot studies, CR lowered risk factors for CVD including reduced concentrations of plasma
triglycerides, cholesterol, and CRP (Ahmed et al. 2009; Das et al. 2007; Pittas et al. 2006). Insulin
sensitivity and first-phase acute insulin secretion, assessed by an OGTT and a FSIGTT, improved
and may contribute to a reduced risk of type 2 diabetes mellitus and hence to reduced secondary
aging. Contrary to the hypothesis of this pilot, all effects of CR were independent of the glycemic
load of the diet.
CALERIE 2
Most recently, the CALERIE 2 trial was completed and the first results from the trial are start-
ing to emerge. The goal of the CALERIE 2 trial was to investigate the safety and efficacy of a
25% CR diet for 2 years compared to an ad libitum diet. Unlike the individual trials in Phase 1,
CALERIE 2 was conducted as a single protocol across the three CALERIE 1 study sites (PBRC,
Washington University, Tufts University), and 220 healthy individuals of normal weight between
21 and 51 years old were enrolled in the 2-year study (Rochon et al. 2011). Adherence to CR was
supported by a number of intervention enhancements including the supervised delivery of the CR
intervention, and individual and group counseling through the intervention by psychologists and
nutritionists (Rickman et al. 2011). Moreover, the meals for the first 4 weeks were provided as guid-
ance by example. Participants were exposed to 3 different diet patterns (Western, Mediterranean,
low glycemic) to be educated on food selection and portion sizes. In addition, participants received
training on food record-keeping because diets were continually monitored by daily self-monitoring
reports (recorded on PDA devices) and by 6-day food records every 6 months. Provided meals were
fully adequate in all essential nutrients and for self-selected meals, participants were guided to
consume macronutrients as advised by the Dietary Reference Intakes (i.e., 45%–65%, 20%–35%,
and 10%–35% for carbohydrate, fat, and protein, respectively). In addition, all participants received
a daily multivitamin and mineral supplement and additional calcium to ensure that participants in
both treatment arms met the current recommendations for these nutrients (Rochon et al. 2011).
Participants in the AL received no specific intervention or counseling (Rochon et al. 2011). As
planned in the study design, the reduction in body weight (−11.5%) was achieved at 12 months
and maintained thereafter for the rest of the study. Total fat mass was also significantly reduced by
CR (−23%) and maintained over the 2 years (Ravussin et al. 2015; Villareal et al. 2016). Similar to
CALERIE 1, the 25% CR induced a metabolic adaptation in resting metabolic rate at 12 months.
After 24 months, metabolic adaptation was still significant within the CR group, however not sig-
nificantly different from the group adhering to the ad libitum diet any more (Ravussin et al. 2015). In
a similar fashion, reductions in core temperatures after 12 and 24 months were significantly reduced
from baseline with 25% CR, but not significantly different between the CR and control groups
(Ravussin et al. 2015). A diminishing level of CR across the 2 years might explain the lack of signifi-
cance at the 24-month assessment (CALERIE 1, 18% during 6 months; CALERIE 2, 19.5% ± 0.8%
during the first 6 months and 9.1% ± 0.7% on average for the remainder of the study). Importantly,
in an ancillary study of CALERIE 2 at one center and in a subset of participants (ClinicalTrials.
gov Identifier: NCT02695511) that were more compliant (14.8% vs. 11.7% CR during the 2 years),
metabolic adaptation in both 24 h and sleeping energy expenditure (measured in a room calorimeter)
was significant after both 1 and 2 years of intervention in the CR group (Redman et al. 2014). As in
CALERIE 1, metabolic adaptation in sleeping energy expenditure was associated with decreased
leptin and thyroid hormone concentrations (Redman et al. 2014).
In line with previous but mostly shorter-duration studies of CR, 24 months of 25% CR in
CALERIE 2 improved numerous markers of CVD risk. Blood pressure, total cholesterol, LDL-
cholesterol, triglycerides, CRP, and TNF alpha decreased significantly and HDL-cholesterol
increased in the CR group, whereas no changes were observed in the control group (Ravussin et al.
2015). Similarly, the CR group had a significant improvement in insulin sensitivity (HOMA-IR,
1.2–0.9), which was also not observed in the control group. These effects are remarkable consider-
ing that the participants were screened on the basis of their health status and blood pressure, plasma
glucose, insulin, and lipids were all required to be in the normal range at enrollment, prior to the
initiation of the CR intervention.
The safety of CR as well as the quality of life of CR participants was closely monitored in the
Phase 2 study, and measured indicators suggest little concern toward longer-term implementation of
CR, at least in healthy individuals (Ravussin et al. 2015). Monitored safety concerns in the CR group
were low BMI (<18.5 kg/m2, n = 1; resolved and intervention continued), treatment-resistant ane-
mia (n = 4; 2 resolved and intervention continued), and decreased bone mineral density (≥5% from
baseline, n = 3; 2 resolved [returned to <5% from baseline] and intervention continued). A decline in
average bone mineral density (lumbar spine, −1.2%, and femoral neck, −1.7%) was observed in the
CR group but this was found to occur in proportion to the loss of body mass, suggesting that the risk
of bone injury was not increased. The intraindividual variability in bone loss however suggests that
regular, close monitoring of bone health is important for individuals following CR diets (Villareal
et al. 2016). Quality of life evaluated by validated measures of mood, self-reported hunger, sexual
function, and cognitive function remained unchanged or improved (mood) after CR for 2 years
(Martin et al. 2016).
Taken together, the epidemiological studies as well as the controlled clinical trials of CR indicate
that a reduction in usual calorie intake when sustained for a long period of time can induce favor-
able effects on the normal mechanisms of human aging. In particular, the age-induced weight gain
and partitioning of energy in adipose tissues including visceral and ectopic depots are attenuated
with CR diets. Furthermore, circulating levels of insulin, plasma lipids, and inflammatory markers
are improved alongside a slowing of the metabolic rate. Provided that CR interventions preserve
strength and physical function and do not accelerate the age-induced loss of bone, this nutritional
intervention might be impactful to the World Health Organization target to reduce noncommuni-
cable diseases worldwide by 25% in the next 10 years.
ALTERNATIVE STRATEGIES FOR CR

While evidence for the benefit of CR in humans is accumulating, one of the greatest pitfalls of this
nutritional intervention is the difficulty to maintain adherence to CR over a longer period of time,
especially in the current obesogenic environment that is characteristic of energy-dense foods and
sedentary lifestyles. Further research is needed to test whether alternative strategies to CR that are
easier to implement achieve equivalent health benefits.
Timed Eating Paradigms

An emerging approach to dietary interventions is timed eating that follows the idea that meal
times and calorie intake should be more closely linked with circadian rhythms of hormones
rather than just following the traditional 3-meal per day paradigm. It is hypothesized that when
there is a misalignment between eating patterns and circadian rhythms such as secretion of
metabolic hormones (leptin, insulin, and thyroid hormones), there is a greater propensity for
metabolic disorders like insulin resistance, dyslipidemia, and hence development and mortal-
ity of CVD (Gu et al. 2015). This phenomenon also termed as “metabolic jetlag” is commonly
observed, for example, in shift workers, individuals who have been shown to have increased
prevalence of CVD (Copertaro et al. 2008; Gu et al. 2015). Interestingly, in a recent study, a
reduction in erratic eating behavior and eating duration (time between the first and last calorie
intake of the day was 4 h 35 min less than during an observational control period of 3 weeks at
baseline) induced an unintentional CR of 20%, which resulted in a 3 kg reduction in body weight
after 16 weeks (Gill and Panda 2015). To date, altered calorie intake with timed eating paradigms
has not been directly compared to traditional CR diets, and therefore it remains to be investigated
whether timed eating can attenuate biomarkers of human aging or incidences of age-related
diseases (Froy and Miskin 2010).
Alternate-Day Fasting
The first human clinical intervention study on CR (without malnutrition) implemented CR by
alternate-day fasting for 3 years (Vallejo 1957). As the name implies, alternate-day fasting para-
digms involve a day of normal eating followed by a day of either complete fasting or a sig-
nificantly reduced level of dietary intake such as <20%. In the Vallejo study, 60 out of 120 men
received 900 and 2300 kcal on alternate days for 3 years, estimated to be equivalent to 35% CR
overall, whereas the 60 others were fed ad libitum. Strikingly, the death rate (6 vs. 13) and hospi-
tal admissions (123 vs. 219) were ~50% lower in the individuals fed CR through an alternate-day
fasting diet (Stunkard 1976). A more recent study that imposed a complete day of fasting followed
by a day of ad libitum eating for 3 weeks reduced participants’ body weight by 2.5% (Heilbronn
et al. 2005). This suggests that CR can be achieved with these kinds of time-restricted eating
paradigms. In this 3-week study, the alternate-day fasting did not reduce oxidative stress, resting
metabolic rate, or body core temperature. However, a scientific conundrum noted by the authors
is when to collect follow-up measurements, following a day of fasting or a day of feeding. The
ability to sustain these kinds of eating paradigms for longer-term studies is questioned because
lightheadedness, constipation, and irritability (on fast days) were frequently reported (Heilbronn
et al. 2005). Longer-duration studies of alternative-day eating paradigms are lacking, especially
those with a direct comparison to traditional CR diets. The reader is referred to Chapter 27 of
this textbook for a more detailed review of the effects of alternate-day fasting on measures of
cardiometabolic health.
CR Mimetics
Given the long-standing quest for the fountain of youth and development of an antiaging remedy in
a single pill, scientists are screening plants and their bioactive components for potential antiaging
effects. Probably, the most promising compounds with the ability to mimic the effects of CR diets
are activators of the sirtuin-AMPK-PGC1α cascade, which can stimulate glucose uptake, substrate
oxidation, and mitochondrial biogenesis (Bonkowski and Sinclair 2016). To date, the most potent
one is resveratrol, a polyphenolic antioxidant of grapes and red wine. In the past decade, resveratrol
has been added to various diets including high-fat diets administered to numerous rodent models.
Consistently, these studies reported improvements in metabolic health (attenuation or protection of
the negative effects of a high-fat diet on body composition, insulin sensitivity, and dyslipidemia) and
the induction of cellular antiaging pathways (Baur et al. 2006; Lagouge et al. 2006; Pearson et al.
2008). However, despite these improvements, an extension of life span has not yet been achieved
(Marchal, Pifferi, and Aujard 2013). In humans, resveratrol supplementation has induced remark-
ably similar effects to the 25% CR diet in the CALERIE trials. Supplementation with a daily dose
of 80 mg resveratrol reduced sleeping metabolic rate (with no alterations in body mass), improved
mitochondrial capacity (ex vivo respirometry on vastus lateralis), and lowered fasting insulin con-
centrations, blood pressure and intrahepatic lipid content after 30 days in a placebo-controlled cross-
over study with 11 obese men (Timmers et al. 2011). Conversely, a study in healthy, nonobese
women reported no effect of resveratrol supplementation on metabolic health, which questions the
efficacy of resveratrol in healthier populations (Yoshino et al. 2012). Supplementation studies simi-
lar to those of CR are difficult to compare given the differences in dosage and also trial lengths. The
effect of resveratrol and other CR mimetics on human aging remains to be determined. Moreover,
the added benefit of resveratrol to CR diets or the effect of CR diets that provide high levels of poly-
phenols is not yet known.
CONCLUSION
Based on the cumulative data of CR on human aging from epidemiological, observational, and
clinical intervention studies to date, we can conclude that moderate CR (>10% reduction in energy
intake below baseline levels) sustained for at least several months induces significant improve-
ments in the metabolic health profile. The slowing of metabolic processes and a reduction in risk
factors for the development of age-related diseases including CVD are hypothesized to be key
mediators for the antiaging effects of CR on longevity in nonhuman primates. While data on lon-
gevity does not yet exist in human trials of CR, the ability of CR to ameliorate these factors in
shorter-duration studies has been shown consistently.
Calorie restriction
Metabolic rate Insulinemia

Body temperature Lipidemia Adiposity
Oxidative stress Inflammation
Blood pressure Visceral adipose tissue

Insulin sensitivity
Artery thickness Hepatic fat accumulation
Mitochondrial function
Cardiac function
Lifespan
CVD
FIGURE 9.2 A proposed hierarchical model for the effects of CR on parameters of aging and CVDs. CR
reduces metabolic rate through a reduction in body mass, physical activity, and metabolic adaptation. In line
with hypotheses on the progression of aging, a reduction in metabolic rate coincided with less oxidative stress,
assessed in skeletal muscle and plasma cells, and may cause reduced body temperature. In addition, the pro-
longed restriction in energy intake significantly reduces whole-body adiposity and induced a healthier plasma
profile with lower concentrations of insulin, triglycerides, LDL, cholesterol, and inflammatory markers. In line
with these results, visceral and ectopic (hepatic) fat accumulation decreases during CR and skeletal muscle
metabolic function such as insulin sensitivity and mitochondrial function improve. An amelioration of oxida-
tive stress, inflammation, and an improvement in mitochondrial function may contribute to reductions in blood
pressure and artery thickness and thus reduced the risk of atherosclerosis and improved cardiac function.
In concert, these improvements suggest that CR may significantly attenuate the progression of aging and the
development of CVD.
We have proposed a hierarchical model (Figure 9.2) for these well-documented effects of CR on
common parameters of aging and CVD. Future studies need to investigate whether the reductions in
the rate of living and the cardiovascular risk profile indeed translate into prolonged health span and
life span. It has been estimated that if 20% CR is sustained for 50 years, life span could be prolonged
by ~5 years (Figure 9.3). This number may be even higher, because a possible reduction in CVD
mortality is not depicted in this mathematical model because the only data available on the effects
of CR on CVD-specific mortality is from the government-induced food restriction in Norway during
World War II (Strom and Jensen 1951). Because adherence to sustained CR is challenging especially
in the current obesogenic environment, investigation of more plausible eating regimens to induce
CR including alternate-day fasting or the use of nutritional supplements that mimic CR effects may
reveal valuable alternatives to the traditional CR diets.
Albert Einstein on calorie restriction

(March 14, 1879–April 18, 1955)
10 year
CVD risk
28%
Ad libitum
+2 months 19%
30% CR
–29%
15%
Ad libitum
+4.7 year 10%

20% CR
–29%
25 60 76 81 Age (years)
FIGURE 9.3 The impact of CR on CVD and life span in humans. Using the example of Albert Einstein,
we estimated how CR may affect life span by extrapolating data from rodents to humans. Early-onset (at
age 25) CR, sustained throughout life, is estimated to extend life span (gray arrow) by almost 5 years
(white, dotted peak). Initiating CR at middle age (60 years) is estimated to only elongate life span
by 2 months. Adhering to a CR diet over a longer period of time may therefore have a larger impact on
human’s metabolic health over time and consequently on human life span. Prevention of CVD might addi-
tionally contribute to a prolongation of life span. The estimated average 10-year risk for CVD (Anderson
et al. 1991) was reduced by 25%–30% in independent cohorts after 6 months of 25% CR (Lefevre et al.
2009) and after 12 months of 20% CR (Fontana et al. 2007). To date, no data is available on the effects of
CR on CVD-specific mortality, but a 50% lower CVD incidence in nonhuman primates on young-onset CR
shows that CR has a significant impact on CVD and thus likely on CVD mortality. (From Colman, R.J.
et al., Science, 325(5937), 201, 2009.)
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Section II
Dietary Fats and
10 Omega-3 and Omega-6
Fatty Acids*
Roles in Cardiometabolic Disease
William S. Harris
CONTENTS
Introduction.....................................................................................................................................194
Omega-6 Fatty Acids......................................................................................................................195
Effects on Lipids........................................................................................................................195
Effects on Inflammatory Markers..............................................................................................195
Linoleic Acid Metabolites..........................................................................................................196
Associations with CHD Events in Prospective Observational Studies......................................196
Randomized Trials.....................................................................................................................199
Omega-3 Fatty Acids......................................................................................................................200
Effects on Lipids........................................................................................................................200
Effect on Inflammatory Markers................................................................................................201
Associations with CHD Events in Prospective Observational Studies......................................201
Randomized Trials.....................................................................................................................202
Assessing Omega-6 and Omega-3 Fatty Acid Status......................................................................203
Summary.........................................................................................................................................203
References.......................................................................................................................................204
ABSTRACT
Omega-3 and omega-6 FAs both have beneficial roles to play in reducing risk for cardiovascular
disease (CVD). Although this is largely noncontroversial for the former FA family, some researchers
have cautioned that the latter family may actually increase risk for CVD. This review will summarize
the relevant human evidence, which shows that, in fact, both FA families are protective. The pre-
sumed “pro-inflammatory” nature of the omega-6 FAs (which is the basis for their hypothetical
adverse effects) is not supported in a wide variety of human observational and interventional studies.
The individual members of the omega-6 FA family can have completely opposing associations with
risk; therefore there is no rational basis for summing all of the omega-6 FAs into a single metric and
ascribing some health effect to the whole group. The primary omega-6 FA in the diet, linoleic acid,
is associated with lower risk for both coronary heart disease and type 2 diabetes, hence lowering
intakes (and thus blood levels) would be expected to raise, not lower, risk. Recommended intakes of
linoleic acid in the 5%–10% range still apply. Most studies support the view that replacing saturated
and trans fats in the diet with both the omega-6 and omega-3 FAs is the best overall strategy for
reducing cardiometabolic risk.
* Disclosures: WSH is the President of OmegaQuant Analytics, LLC, a laboratory that offers fatty acid testing. He is also a
scientific advisor for the Global Organization for EPA and DHA (fish oil trade association).
193
INTRODUCTION
Long-held dogmas regarding the roles of essentially all of the classes of dietary fatty acids (FAs)
vis-à-vis coronary heart disease (CHD) are being challenged. Saturated FAs, the perennial “bad”
fats, may not be as bad as we’ve thought (see Chapter 11), and the monounsaturated FA oleic acid,
which predominates in olive oil (an important component of the Mediterranean diet), may not be as
cardioprotective as once believed based on the results of recent meta-analyses (Chowdhury et al.,
2014) and animal feeding studies (Brown, Shelness, and Rudel, 2007). Similarly, the marine-derived
omega-3 polyunsaturated fatty acids (PUFAs), which have historically found a place among the
“healthiest” of all dietary fats, have fallen on hard times following the publication of several null
randomized trials (Rizos et al., 2012). A suspicious eye is now being cast on linoleic acid (LA), the
principal vegetable-oil-derived omega-6 PUFA—once taken almost as a medicine using the table-
spoon to lower cholesterol—with some proposing that, instead of preventing, it may be contributing
to CHD (Cunnane and Guesnet, 2011; Ramsden et al., 2013). Even with the trans FAs, which are
almost universally seen as detrimental, there is a controversy regarding the potentially differen-
tial effects of “natural” (i.e., ruminant) derived species versus the industrially produced species
(Bendsen et al., 2011; Gebauer et al., 2015) (see Chapter 12). Understandably, the public is becom-
ing skeptical of official proclamations of what constitutes a “healthy fat.”
The purpose of this chapter is to consider the evidence for and against a cardioprotective role
for the omega-3 and omega-6 FAs. The reader is referred to several prior reviews on omega-3
(De Caterina, 2011; Mozaffarian and Wu, 2011; Khawaja, Gaziano, and Djousse, 2014; Kromhout
and de Goede, 2014) and omega-6 (Harris et al., 2009b; Czernichow, Thomas, and Bruckert, 2010)
that have tread similar ground. These two families comprise all of the PUFAs (those containing at
least 2 double bonds) in the diet and, for the most part, in the body. One member of each is classi-
cally considered to be an “essential” FA because, like vitamins, humans cannot live without them in
the diet. These are LA and its omega-3 counterpart alpha-linolenic acid (ALA), although the rela-
tive essentiality—indeed, even the term “essential”—of each has come under scrutiny (as noted by
Cunnane who enumerates many problems with the current paradigm) (e.g., essentiality may apply
to only some stages of the life cycle, LA needs may be lower in the presence of sufficient ALA,
etc.) (Cunnane, 2003). The structures of these two FA classes and their most important members are
shown in Figure 10.1.
As it constitutes by far the largest amount in the diet, the omega-6 class will be considered first.
Special emphasis will be placed on the pro/anti-inflammatory effects of these FA families.
Omega-6 Non-essential fatty Omega-3

essential fatty acids acids essential fatty acids
COOH COOH
COOH
Palmitic acid (PA) 16:0
Linoleic acid (LA) 18:2n-6 Saturated fatty acid α-Linolenic acid (ALA) 18:3n-3
COOH COOH
COOH Oleic acid (OA) 18:1n-9
γ-Linolenic acid (GLA) 18:3n-6 Monounsaturated fatty acid Eicosapentaenoic acid, EPA 20:5n-3
COOH
COOH
Elaidic acid (EA) COOH
Arachidonic acid (AA) 20:4n-6 18:1n-9 trans; Trans fatty acid Docosahexaenoic acid, DHA 22:6n-3
FIGURE 10.1 The two essential FA families, omega-6 (n-6) and omega-3 (n-3), are shown with important
representative members on the left and right. In the center are three examples of FAs that are not dietary
essentials. The molecular structures assume a carbon atom at each inflection point with 1–3 hydrogen atoms
not shown.
Omega-3 and Omega-6 Fatty Acids 195
OMEGA-6 FATTY ACIDS

Effects on Lipids
The cholesterol-lowering effect of LA is well established from human trials. In a meta-analysis
of 60 feeding studies including 1672 volunteers, the substitution of PUFA (the vast majority of
which was LA, varying from 0.6% to 28.8% energy) for carbohydrates had more favorable effects
on the ratio of total cholesterol to high-density lipoprotein (HDL) cholesterol than any class of
FAs (Siguel, 1996; Mensink et al., 2003). Epidemiologically, the replacement of 10% of calories
from saturated FAs with omega-6 PUFA is associated with an 18 mg/dL decrease in low-density
lipoprotein cholesterol (LDL-C), greater than that observed with similar replacement with carbo-
hydrate (Mensink and Katan, 1992). These findings confirm an LDL-lowering effect of omega-6
PUFA beyond that produced by a lower saturated FA intake. Both baseline and 6-year follow-up
changes in serum omega-6 FA levels, which reflect changes in dietary LA (Bradbury et al., 2010),
were inversely related to the size of very-low-density lipoprotein (VLDL) particles, and directly
related to LDL and HDL particle size (Mantyselka et al., 2014). Others reported that higher
serum concentrations of LA were significantly associated with lower concentrations of total LDL
particles, and higher concentrations of serum LA and arachidonic acid (AA) were significantly
associated with lower levels of large VLDL particles and higher levels of large HDL particles
(Choo et al., 2010). Favorable effects of LA on cholesterol levels are thus well documented and
would predict significant reductions in CHD risk.
Effects on Inflammatory Markers

As noted earlier, some investigators have proposed that LA intakes in America are excessive
(Blasbalg et al., 2011), and far from reducing risk for inflammatory diseases like CHD, they may
actually be increasing risk for them (Ramsden et al., 2013). This perspective builds upon the fol-
lowing logic: (1) LA is the precursor for AA; therefore, higher LA intakes will lead to higher AA
tissue levels; (2) AA is the substrate for the production of certain pro-inflammatory eicosanoids,
so higher AA levels would lead to a greater production of these molecules; (3) AA levels in mem-
branes are rate-limiting for the production of eicosanoids; and (4) CHD is a disease with major
inflammatory components and so the more pro-inflammatory eicosanoids produced, the more
inflammation should be present that should translate into more CHD. Thus, higher intakes of LA
may increase risk for CHD.
Although not intrinsically illogical, this perspective fails to consider several important findings.
First, a systematic review of 36 studies reported that variations in dietary LA, either reduced by up
to 90% or increased by as much as sixfold, did not affect plasma phospholipid AA levels (Rett and
Whelan, 2011). The authors concluded, “Our results do not support the concept that modifying cur-
rent intakes of dietary linoleic acid has an effect on changing levels of arachidonic acid in plasma,
serum or erythrocytes in adults consuming Western-type diets.” On the other hand, a recent feeding
study that aimed to provide a low-LA diet (i.e., 2.4% energy vs. 7.4% energy) for 12 weeks reported
reductions in plasma AA levels (Taha et al., 2014). It is difficult, however, to determine how much
AA levels fell since whole plasma AA was not reported, but only AA in isolated lipid fractions. In
addition, the intake of AA was reduced by about 50% in the low-LA diet. Thus, it is unclear whether
the reduction in circulating LA was the cause of the reduced AA levels.
Second, studies testing the effect of LA on inflammatory status in humans have routinely found
no increase in marker levels. In a recent review (Johnson and Fritsche, 2012), 15 trials were exam-
ined that met inclusion criteria, and the authors concluded that “virtually no evidence is available
from randomized, controlled intervention studies among healthy, non-infant human beings to show
that addition of LA to the diet increases the concentration of inflammatory markers.”
Another potential concern with increasing LA intakes is that it will lower blood levels of
the long-chain omega-3 FAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)
(Cleland et al., 1992) presumably by slowing conversion of ALA to EPA, and/or by competing with

these two FAs for esterification sites in membrane phospholipids. To the extent that EPA/DHA is
cardioprotective, such an effect would be counterproductive. The evidence in this regard is mixed.
For example, one study (Liou et al., 2007) increased the LA intake from 3.8% to 10.5% of total
energy in the diets of 22 men for 4 weeks. The high-LA diet did lower plasma phospholipid EPA
levels slightly (from 1% to 0.6%), but it raised DHA levels by the same amount (from 3% to 3.4%),
leaving the sum of EPA + DHA (4%) unaffected. They also observed no effects on inflammatory
markers or platelet aggregation with the high-LA diet. Another study tested the effects of increasing
amounts of omega-6 FAs in the diet on cardiac tissue omega-3 levels in rats fed various levels of
fish oil (Slee et al., 2010). The authors reported that the omega-6 intake did not affect the uptake of
omega-3 in the heart.
Linoleic Acid Metabolites

As noted, the “LA is harmful” hypothesis depends heavily on the view that an LA metabolite,
AA, is converted to potent pro-inflammatory signaling molecules. While this is partly true, what is
often overlooked is that AA also gives rise to anti-inflammatory metabolites (e.g., lipoxin A4), as
well as anti-aggregatory, vasodilative molecules like prostacyclin. Indeed, the epoxyeicosatrienoic
acids synthesized from AA produce vasodilation, stimulate angiogenesis, have anti-inflammatory
actions, and protect the heart against ischemia–reperfusion injury (Spector, 2009). AA can be
converted into an ever-expanding list of bioactive compounds via cyclooxygenase, lipoxygenases,
and cytochrome P450 monooxygenases (Figure 10.2a). But AA is not the only omega-6 FA with
potential effects on CHD—LA itself can be converted to a wide variety of bioactive molecules by
these enzymes (Figure 10.2a). Not shown in this figure is nitrated LA (LNO2), an LA metabolite
that has been shown to have cardioprotective effects (Baker et al., 2009). In addition, LNO2 is a
powerful ligand for peroxisome proliferator activator receptor-gamma (PPAR-γ) (Schopfer et al.,
2005), a nuclear transcription factor that controls cell differentiation as well as production of met-
abolic and anti-inflammatory signaling molecules. At physiologically relevant levels, LNO2 rivals
the effects of the thiazolidinediones on PPAR-γ (Schopfer et al., 2005). LA can also be converted
to a growing number of oxygenated metabolites (i.e., oxylipins) by cyclooxygenase, lipoxygen-
ases, and/or cytochrome P-450 epoxygenases (Figure 10.2a), the individual and aggregate effects
of which have not been systematically examined.
LA can also be metabolized to dihomo-gamma-linolenic acid from which other bioactive lipids
can be produced including prostaglandins of the 1-series (Wang, Lin, and Gu, 2012). Within the
LA “metabolome,” one finds a constellation of products including a variety of prostaglandins, leu-
kotrienes, ligands for endocannabinoid receptors, lipoxins, isoprostanes, nitrated AA, and epox-
ides, among others. Considering only the cyclooxygenase, lipoxygenase, and cytochrome P450
pathways, Tam lists 8 metabolites of LA and 41 from AA (Tam, 2013) (Figure 10.2a). Some are
“pro-inflammatory” but some are “anti-inflammatory” or promote the resolution of inflammatory
insults. Often these effects have only been observed in certain cell/tissue types and under potentially
non-physiological conditions, and their effects or those of other metabolites in normal physiology
remain to be discovered. The net impact on human metabolism (and CHD risk) of this multitude
of products will ultimately be determined by their interaction among themselves (and with their
omega-3 FA analogs), and is virtually impossible to predict. Hence, to label the entire class of
omega-6 FA metabolites as “proinflammatory” is far too simplistic.
Associations with CHD Events in Prospective Observational Studies

The classic tool of nutritional epidemiology has been the prospective cohort study in which large
numbers of healthy subjects are recruited, their diets analyzed by a variety of techniques [24-h recall,
3-day food record, food frequency questionnaire, etc. (Shim, Oh, and Kim, 2014)], and a wide range
13-Oxo 9,10
9-Oxo
ODE DiHOME
ODE
12,13
PGB2 DiHOME
15k- 9- 13- sEH
PGF2α HODE HODE
sEH
9,10
PGC2 EpOME 12,13
alox15
Plasma membrane EpOME
PGF2α
PGA2 pla2g4a cyp2j fam 5,6-
Linoleic acid
cyp2c fam diHETrE
PGE2
16- sEH
HETE
TXB2 8,9-
ptges1/2/3 5,6- diHETrE
Omega-3 and Omega-6 Fatty Acids
EET
TXA2 PGG2 ptgs1/2 Arachidonic acid cyp2j fam sEH

ptgs1/2
cyp2c fam 8,9-
tbxas1 PGH2
cyp2b fam EET
ptgds1/2 ptgis alox5 alox15 alox8 alox12 11,12-

EET
14,15-
EET sEH
PGD2 PGI2 5- 15- 8- 12- 11-
HPETE HPETE HPETE HPETE HETE
sEH 11,12-
diHETrE
6k- 5- 8- 12-
PGJ2 15-
PGF1a HETE HETE HETE 14,15-
HETE diHETrE
LTA4 alox5
15d-
PGJ2 alox12 LXA4 HXA3 HXA3
Ita4h Itc4s
LXB4
LTB4 LTC4
LTD4
LTE4
(a)
197
FIGURE 10.2 (See color insert.) An overview of metabolites of omega-6 FAs (a). (Continued)
198
13- 9-
HOTrE HOTrE
22- 21-
HDoHE HDoHE
PD1
alox15 alox5
20- 10S, 17S-

5- HDoHE DiHDoHE
Epoxide
HEPE hydrolase
Linolenic acid
22- RvD1
21-
8- HpDHA
HpDHA
HEPE 20- 17-
HpDHA HDoHE
alox5 RvD2
9- 17-
HEPE EPA DHA alox15 HpDHA RvD5
16-
alox15 HpDHA
16-
alox5 alox12 HDoHE
11-
14-
HEPE
13- HpDHA
18- HpDHA 14-
12- HEPE 4- 7- HDoHE
HEPE HpDHA HpDHA
8- 11-
10- Maresin
HpDHA HpDHA
15- HpDHA
HEPE
11-
RvE1 4-
7- HDoHE
HDoHE
HDoHE 13-
8- 10- HDoHE
HDoHE HDoHE
(b)
FIGURE 10.2 (Continued) (See color insert.) An overview of metabolites of omega-3 FAs (b). Boxes show enzymes responsible for production of metabolites (brown
boxes designate processes that can be accomplished nonenzymatically). (Taken from Tam, V.C., Semin. Immunol., 25(3), 240, 2013. With permission; Abbreviations given
in the original paper.)
of biometric and health-related measures are collected. In some studies, biological samples are taken
for biomarker measurement. The cohort is then followed without any prescribed interventions for
a number of years, and the incidence of different diseases is tracked. With these data, sophisticated
statistical analysis is applied to explore the question of how food/nutrient/pattern is associated with
an outcome of interest. The strengths of such studies are their “real world” setting and the ability
to include many thousands of subjects. Their weaknesses include not being able to control every
aspect of a person’s life, and the very real possibility that, even though nutrient X (whether from
dietary data or biomarker level) is strongly associated with incident disease, it may be other factors
that track with nutrient X that are the real reason for the observed relationships. This “unmeasured
confounding” makes it impossible to conclude that a cause and effect relationship exists between
the nutrient and the disease outcome. Nevertheless, such associations (if they are seen in multiple
studies conducted under a variety of conditions) build a strong circumstantial case for a link between
nutrient and disease.
Many studies have been performed in the area of FAs and cardiovascular disease (CVD). They
are best summarized in meta-analyses. The most comprehensive meta-analysis in recent years
examined the relations between dietary and circulating levels of all major FAs and CHD out-
comes (Chowdhury et al., 2014). Here, contra the “omega-6-are-harmful” hypothesis, there was
no association between LA levels when replacing saturated fat and CHD in the full analysis, and
a favorable association when the controversial Sydney Heart Study (Ramsden et al., 2013) was
excluded. Even more contra was the finding, based on 10 studies including some 23,000 indi-
viduals with over 3,700 CHD events, that higher levels of circulating AA—the presumed toxic
omega-6 mediator—were associated with lower risk for CHD events (HR, 0.83, 95% confidence
interval [CI], 0.74–0.92).
Farvid et al. have published the largest and most recent (as of December 2015) meta-analysis
that specifically addressed the relations between omega-6 FAs (primarily LA and AA) when
replacing either carbohydrates or saturated fat and CHD morbidity and mortality (Farvid et al.,
2014). Utilizing data from both published and unpublished studies (via direct investigator contact),
13 cohort studies involving about 310,000 individuals with over 12,000 CHD events and about
5,900 CHD deaths were examined. The primary outcome was myocardial infarction, ischemic
heart disease, sudden cardiac arrest, acute coronary syndrome, and CHD deaths. Intakes of LA
were estimated by a variety of types of dietary intake instruments, and follow-up ranged from
5 to 30 years. Comparing the highest to the lowest intake groups (10th to 90th percentiles for
LA intake ranged from 1.1% to 9.5% energy) and risk for CHD events was lower by 15% (0.85
(0.78, 0.92)), and for CHD death by 21% (0.79 (0.71, 0.89)), both statistically significant. Viewed
another way, risk for events was increased by 18% and death by 27% in the lowest intake group
compared to the highest.
The fact that these relations were observed using such blunt instruments as dietary question-
naires could suggest that the findings are robust. However, as noted earlier, unmeasured confound-
ing factors could be influencing the outcomes. The observation that replacing either saturated fats
or carbohydrates with vegetable oils produced essentially the same CHD benefit suggests that it is
not the reduced intake of the nutrient being replaced by LA that affords the benefit, but LA itself.
Finally, since the LA effect was independent of the intake of ALA (the omega-3 FA found primarily
in soybean oil where it constitutes about 6% of total FAs compared with 54% as LA), the benefit
observed cannot easily be attributed to co-consumption of the ALA as some have hypothesized
(Ramsden et al., 2013).
Randomized Trials
Of course, the most direct way to test the hypothesis that higher LA intakes reduce risk for CHD
is to perform a randomized controlled trial (RCT). This has been attempted many times, and
nearly as many meta-analyses have been employed to summarize their findings. Depending on
which trials one includes, there is either a significant reduction in risk (Mozaffarian, Micha, and
Wallace, 2010) or no effect (Chowdhury et al., 2014; Ramsden et al., 2013; Schwingshackl and
Hoffmann, 2014) of higher omega-6 intakes. In a meta-analysis including only the four trials
utilizing soybean oil (which contains about 50% LA and 7% ALA), there was a significant 22%
reduction in CHD events (Ramsden et al., 2010). In these trials, omega-6 PUFA consumption
was often raised to very high levels (far exceeding the currently recommended 5%–10% energy
from PUFA and producing, in three trials, omega-6:omega-3 PUFA ratios ranging from 7 to 21)
and demonstrated CHD benefit, not detriment. Thus, these results directly contradict the view
that high omega-6 PUFA intakes or “high” omega-6:omega-3 PUFA ratios increase the risk of
CHD (Hibbeln et al., 2006). Ramsden examined the effects on CHD events in two other trials
utilizing corn oil (no ALA) and found no significant effects (Ramsden et al., 2010). The findings
from this study stand in contrast to the authors’ statement that “advice to specifically increase
omega-6 PUFA intake is unlikely to provide the intended benefits, and may actually increase the
risk of CHD and death.”
All this being said, this is a difficult hypothesis to properly test in large-scale, multiyear inter-
vention trials because one major dietary component like LA is (must be) substituted for another
potentially active (vis-à-vis CHD) component. Hence, interpretation is challenging. Because of
these concerns, it has been argued that prospective cohort data should be given the same eviden-
tiary weight as RCTs in nutrition because each has relevant strengths and weaknesses (Harris et al.,
2009a). When these two types of data (and others) are viewed in the aggregate, a strong case for a
protective effect of LA on CHD can be made (Harris et al., 2009b).
OMEGA-3 FATTY ACIDS

Effects on Lipids
The first and most well-characterized effect of the marine omega-3 FAs was triglyceride lowering.
This effect was first summarized in 1989 (Harris, 1989) and has been confirmed in more recent
meta-analyses (Leslie et al., 2015). Reductions ranging from 10% to 50% can be achieved with
“pharmacological” doses of EPA+DHA (e.g., 3–4 g/day) with the variability largely associated
with the degree of hypertriglyceridemia (Harris et al., 1997; Davidson et al., 2007). In patients
with very elevated triglyceride levels, LDL-C levels have been observed to rise (Jacobson, 2008),
an effect attributed to the DHA component of marine oils (Wei and Jacobson, 2011). Whether the
small increase in LDL-C impacts risk for CHD in the patient taking 3–4 g of EPA+DHA per day
is unknown, but in the view of the author, it seems unlikely given the fact that the mechanisms
responsible for the reduction in risk for CVD may have little to do with reducing serum triglyceride
levels as discussed in the following text.
Beneficial effects on CVD endpoints have been observed with long-term dietary intakes of <1 g/day
of EPA+DHA, and the doses used in major clinical CVD trials (typically 1–2 g) have little or no
impact on serum triglyceride levels (Radack, Deck, and Huster, 1990; Roche and Gibney, 1996;
Investigators, 1999; Schwellenbach et al., 2006). It is beyond the scope of this review to cover
in detail the proposed nonlipoprotein-related mechanisms of omega-3 FAs (besides inflammation;
see as follows). Briefly, however, these include increased myocardial resistance to arrhythmias
(Reiffel and McDonald, 2006), enhanced plaque stability (Thies et al., 2003), reduction in heart rate
(Mozaffarian et al., 2005b), improved endothelial function (Nestel et al., 2002; Xin, Wei, and Li,
2012), increased heart rate variability (Xin, Wei, and Li, 2013), and a variety of other antiatheroscle-
rotic and antithrombotic processes (Robinson and Stone, 2006). The cellular and molecular bases
for these effects of omega-3 FAs are multiple. The interested reader is referred to recent reviews
(De Caterina, 2011; Poudyal et al., 2011; Serhan and Petasis, 2011; Rangel-Huerta et al., 2012;
Shearer, Savinova, and Harris, 2012; Calder, 2013).
Effect on Inflammatory Markers

The anti-inflammatory and inflammation-resolving properties of the omega-3 FAs are relatively
well documented (Calder, 2013). Such documentation comes from observational studies and
randomized trials, and mechanistic insights have been obtained from cell culture work. With
respect to the former, a recent report from the Framingham Offspring Study documented the
significant inverse correlations between erythrocyte EPA+DHA levels [the Omega-3 Index
(Harris and von Schacky, 2004)] and eight different biomarkers of inflammatory processes
across a wide spectrum of systems (Fontes et al., 2015). As summarized in Fontes et al., 6 of 8
prior studies using dietary omega-3 PUFA data found a significant inverse association between
at least one inflammatory biomarker and intakes. In addition, in 10 of 12 biomarker-based stud-
ies, significant inverse relations for at least one inflammatory marker with EPA and/or DHA
levels were observed. Similarly, a recent meta-analysis of 68 trials in patients with chronic,
non-autoimmune diseases found overall significant reductions in C-reactive protein and inter-
leukin 6 and marginally significant reductions in tumor necrosis factor alpha after omega-3
PUFA supplementation (Li et al., 2014). Another marker of inflammation more directly tied to
vascular inflammation is lipoprotein-associated phospholipase A2 (LpPLA-2), which has been
linked closely with CVD events and is localized in atherosclerotic plaques (Mallat, Lambeau,
and Tedgui, 2010). Levels of LpPLA-2 were reduced by treatment with 4 g/day of EPA (Bays
et al., 2013) and with 3.4 g/day of EPA+DHA (Davidson et al., 2009). Hence, there is sup-
port both from observational and from interventional studies for an anti-inflammatory effect
of omega-3 FA. The extent to which this is the basis for the cardioprotective effects of these
long-chain marine FAs is unclear.
The mechanisms by which EPA and/or DHA exert their anti-inflammatory effects appear to
ultimately derive from their effects on membrane biophysics. By altering lipid raft composition
(Williams et al., 2012; Turk and Chapkin, 2013), the long-chain omega-3 FAs can alter endocytic
activity (Pinot et al., 2014), L-type calcium channels, the Na+–Ca2+ exchanger, and other signal-
ing pathways involving activation of phospholipases, synthesis of eicosanoids, and regulation of
receptor-associated enzymes and protein kinases (Siddiqui, Harvey, and Zaloga, 2008). Like AA,
EPA and DHA can be metabolized into resolvins, protectins, and a number of other oxylipins
(Figure 10.2b), which can impede inflammatory processes. By serving as a ligand for transcription
factors such as PPAR-γ, they can interfere with the production of nuclear factor kappa B (NFκB)
that is the transcription factor that ultimately controls the synthesis of a variety of cytokines and
adhesion molecules as well as cyclooxygenase 2, inducible nitric oxide synthase, and matrix metal-
loproteinases (Figure 10.3).
NFκB synthesis is also inhibited by activation of transcription factor I kappa B, the production of
which is stimulated by activation of GPR120 by these same FAs [as summarized in (Calder, 2013)].
Higher levels of EPA and DHA also reduce membrane phospholipid levels of AA, thereby reduc-
ing the pro-inflammatory cytokines and oxylipins derived from this important omega-6 FA. It is by
these interacting and concerted mechanisms that fish oil exerts its anti-inflammatory effects across
many cell types.
Associations with CHD Events in Prospective Observational Studies

While prospective cohort studies also have significant limitations, the relations between nutrient
intakes (or, better, nutrient biomarker levels) and disease outcomes should be considered comple-
mentary to, and equally important as, RCT data. There have been at least 16 cohort studies that
have used dietary estimates of EPA+DHA intake as the exposure marker for CHD endpoints, and
13 studies that have used circulating EPA+DHA levels. These have been included in a major meta-
analysis that examined all FAs, not just the omega-3 FAs (Chowdhury et al., 2014). In this analysis,
Inflammatory Extracellular Extracellular

stimulus EPA and DHA AA
Cell membrane
Raft EPA and DHA in AA in

TLR4 GPR120 assembly phospholipids phospholipids
Free EPA and DHA Free AA
Resolvins,
NFκB PPAR-γ protectins Eicosanoids
and maresins
Cytokines
Adhesion molecules
COX-2
iNOS
MMPs
FIGURE 10.3 Summary of the anti-inflammatory actions of marine omega-3 PUFAs. COX, cyclooxygenase;
GPR, G-protein coupled receptor; iNOS, inducible nitric oxide synthase; MMP, matrix metalloproteinase.
Dotted lines indicate inhibition. (Taken from Calder, P.C., Br. J. Clin. Pharmacol., 75(3), 645, 2013. With
permission.)
upper tertile (vs. lower) intakes of long-chain omega-3 FA were associated with a 13% (95% CI,
3%–22%) reduction in CHD events, and this class of FAs was the only one linked to a lower risk of
events. Consistent with this, higher circulating levels of these two omega-3 FAs (and levels of doc-
osapentaenoic acid) were significantly associated with reduced risk for CHD events. This indicates
that, in the long run and in more “natural” settings outside of clinical trials, long-chain omega-3
FAs are consistently and directly associated with better cardiac health. Of course as alluded to in
the omega-6 FA section, unmeasured confounding should always be considered as a possible con-
tributing explanation for the “benefits” of omega-3 FA. That is, there are many lifestyle, medical,
and social factors associated with higher omega-3 levels (Harris et al., 2016). It is always possible
that benefits seen in “association” studies may not be attributable to EPA+DHA per se but to other
behaviors or factors found in people who have higher blood levels of these FAs.
Randomized Trials
As summarized earlier, there are a wealth of data from population (He et al., 2004), case-control
(Siscovick et al., 1995), prospective cohort (Albert et al., 2002; Iso et al., 2006), and RCTs (Burr
et al., 1989; Marchioli et al., 2002; Yokoyama et al., 2007; Investigators, 2008) supporting a car-
dioprotective effect of omega-3 FAs. With respect to the latter study designs, there have been eight
major RCTs (each including at least 2000 patients) examining the effects of omega-3 FAs and
risk for cardiovascular endpoints and/or death. These have been widely reviewed and summarized
(Saravanan et al., 2010; De Caterina, 2011; Mozaffarian and Wu, 2011; Kromhout et al., 2012). This
extensive literature has generated multiple meta-analyses over the years, with four being published
in 2012 alone (Kotwal et al., 2012; Kwak et al., 2012; Rizos et al., 2012; Trikalinos et al., 2012). The
report by Rizos et al. garnered the greatest attention as it was published in JAMA (Rizos et al., 2012).
This group concluded that there was insufficient evidence to conclude that omega-3 FA supplements
reduced risk for CVD. This was a surprising conclusion given that they reported a significant 9%
reduction in risk for cardiac death (p = 0.01). The authors took the position that the alpha (normally
0.05) should be adjusted for multiple testing (to <0.006). This highly unusual (and controversial)
statistical maneuver in a meta-analysis converted a positive finding to a null finding and injected
considerable confusion into the omega-3 and CVD issue (Harris, 2013). Other recent meta-analyses
have reached different conclusions (Musa-Veloso et al., 2011; Delgado-Lista et al., 2012; Trikalinos
et al., 2012; Casula et al., 2013) finding overall benefit for omega-3 FAs (as also was found in the
data, if not the interpretation, in Rizos et al. (2012)).
ASSESSING OMEGA-6 AND OMEGA-3 FATTY ACID STATUS

Whether in reference to dietary FA intakes or blood-based biomarker FA levels, there are many
ways to express what is sometimes called “omega status.” Our bias over the last 12 years has been
to use the Omega-3 Index (erythrocyte EPA+DHA, expressed as a percent of total FAs) (Harris
and von Schacky, 2004; von Schacky, 2014). This marker has gained widespread use in the FA
research community owing to its ease of analysis, intuitive meaning, low within-person vari-
ability (Harris and Thomas, 2010), insensitivity to acute omega-3 FA loads (Harris et al., 2013),
strong correlation with cardiac EPA+DHA levels (Harris et al., 2004), responsiveness to omega-3
supplementation (Flock et al., 2013), and utility as both a biomarker and risk factor for CHD
(Harris, 2009). An alternate expression of “omega status” is the omega-6/omega-3 ratio. This
pools all omega-6 FAs and all omega-3 FAs, regardless of chain length or double bond number,
and then divides the former by the latter. In the author’s opinion, this metric is of little to no use
for reasons previously outlined (Harris, 2006). Among the theoretical weaknesses of this ratio are
(1) the failure to distinguish among the specific FA species within each class, effectively allow-
ing ALA to “count” as equivalent metabolically to EPA, DPA, and DHA, and LA as equivalent
to AA (or gamma-linolenic or adrenic acid); (2) the imprecision that arises from the fact that a
virtually endless array of FA levels can all produce the same ratio; (3) the implicit presumption
that the omega-6 FAs are “bad” and the omega-3 FAs are “good”; and (4) that lowering a “high”
ratio (which is presumably bad) can be accomplished in five ways, at least one of which involves
actually lowering omega-3 levels. In a workshop sponsored by the UK Food Standards Agency
that addressed the utility of the omega-6/omega-3 ratio, the panel concluded, “On the basis of this
review of the experimental evidence and on theoretical grounds, it was concluded that the n-6:n-3
FA ratio is not a useful concept and that it distracts attention away from increasing absolute
intakes of long-chain n-3 FAs which have been shown to have beneficial effects on cardiovascular
health” (Stanley et al., 2007).
SUMMARY
Omega-3 and omega-6 FAs may be viewed as “partners in prevention” as they relate to cardiometa-
bolic diseases (Harris, 2010). Synthesizing evidence from a wide variety of investigations supports
the view that higher versus lower intakes of both LA and EPA+DHA have favorable cardiometabolic
effects (Mozaffarian et al., 2005a). While opposing views remain regarding LA (Ramsden et al.,
2012, 2013; Bazinet and Chu, 2014), the consensus in this author’s view continues to support the
recommendation of many health authorities for 3%–11% of energy as LA (Harris et al., 2009b; FAO,
2010; Vannice and Rasmussen, 2014). Given the safety record of EPA+DHA (Villani et al., 2013),
recommendations for the marine omega-3 FAs of between 250 and 1000 mg/day (Kris-Etherton,
Harris, and Appel, 2002; Harris, Kris-Etherton, and Harris, 2008; Vannice and Rasmussen, 2014) or
even higher as suggested from Japanese studies (Sekikawa, Doyle, and Kuller, 2015) are reasonable
for reducing the risk for cardiometabolic diseases.
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11 Evolving Role of
Saturated Fatty Acids
Patty W. Siri-Tarino and Ronald M. Krauss
CONTENTS
Primer on SFA Biochemistry and Physiology................................................................................209
Evolving History of Studies of SFA Effects on Lipids, Lipoproteins, and CVD Risk...................210
Metabolic Studies Evaluating Diet and Lipid Profiles...............................................................211
Diet and CVD Studies................................................................................................................211
Refined CVD Risk Assessment..................................................................................................212
SFAs in Context.........................................................................................................................213
Replacement Nutrient............................................................................................................213
Atherogenic Dyslipidemia and the Obesity and Diabetes Epidemics...................................214
Food Sources of SFAs and Dietary Patterns..........................................................................214
SFA Effects on Other Cardiometabolic Health Risk Factors..........................................................215
Variability in Response of LDL-C to Saturated Fat........................................................................216
Applying New Knowledge to Dietary Guidelines..........................................................................216
Conclusions.....................................................................................................................................217
References.......................................................................................................................................217
ABSTRACT
Given their cholesterol-raising ability and (like other fats) higher caloric content per gram compared
to proteins or carbohydrates, saturated fatty acids (SFAs) have been a focus of dietary guidelines for
cardiovascular health since their inception. Restriction of this class of fatty acids was advised, how-
ever, without reference to the replacement nutrient, in part contributing to food formulations high
in sugars and refined carbohydrates that can adversely affect cardiometabolic health. Moreover, in
light of more recent research highlighting factors that modulate the effects of SFAs on cardiometa-
bolic risk, including the heterogeneity of SFAs themselves, food sources of SFAs, and variation in
individual responsiveness to diet, a foods-based approach that moves away from targeting restric-
tion of SFAs as a whole is warranted. A reevaluation of how nutritional science is communicated
to the public is further informed by recent research demonstrating diets high in SFAs and low in
carbohydrate as an effective therapeutic regimen for some individuals with obesity and/or diabetes.
Future dietary recommendations should emphasize individualized approaches that consider both the
context of the SFAs consumed and the individuals consuming them.
PRIMER ON SFA BIOCHEMISTRY AND PHYSIOLOGY

Saturated fatty acids (SFAs) contain no double bonds as a result of having all of their carbons
“saturated” with hydrogen molecules. The most abundant SFAs contain between 12 and 22 carbons
(Chow 2000). SFAs between 12 and 18 carbons in chain length can be desaturated to their respective
monounsaturated products via stearoyl-CoA-desaturase, with increasingly more efficient conversion
of SFAs of longer chain length according to cellular studies conducted in rats (Legrand and Rioux
2010). Tracer studies in humans have also documented greater rates of desaturation of C18:0 versus
209
TABLE 11.1
Common SFAs and Their Typical Food Sources
Common Name Carbon Chain Length Typical Food Sources
Butyric acid C4:0 Butter and dairy fat
Lauric acid C12:0 Coconut oil
Myristic acid C14:0 Coconut oil and dairy fat
Palmitic acid C16:0 Palm oil, meat, and dairy fats
Stearic acid C18:0 Meat fat and cocoa butter
C16:0 (Rhee et al. 1997), although on an absolute level, the rates of conversion are relatively small,
that is, 14% and 2%, respectively. It is unclear whether the biological desaturation of SFAs affects
their hypercholesterolemic properties (Rhee et al. 1997).
SFAs are structural components of sphingolipids and ceramides, which are contained in cell
membranes, skin, and myelin. SFAs have specific physiological functions, including protein fatty
acid acylation, specifically N-terminal myristoylation and side chain palmitoylation (Nettleton,
Legrand, and Mensink 2015). The myristoyl moiety mediates protein subcellular localization
and protein–protein or protein–membrane interactions that enable the biological functions of the
myristoylated proteins. Further, various SFAs have different cellular and biological functions.
Myristic acid may enhance the bioavailability of docosahexaenoic acid and eicosapentaenoic acid
at an optimal level of 1.2% of total energy (at 1.8%, it reduced bioavailability); butyric acid may
protect against early tumorigenic events; and medium-chain fatty acids appear to be differen-
tially metabolized, resulting in less adipose tissue deposition compared to long-chain fatty acids
(Legrand and Rioux 2010). More recent evidence suggests that very-long-chain saturated fatty
acids (C20:0–C24:0) may also have metabolic benefits (Mozaffarian 2016).
Because humans can synthesize them de novo, there is no dietary requirement for SFAs. Further,
it is important to note that plasma concentrations of even-chained SFAs do not accurately reflect
their dietary consumption given endogenous metabolic pathways. In fact, SFAs present in blood are
not strongly associated with dietary SFA consumption (Ma et al. 2015, Morio et al. 2016). Rather,
dietary carbohydrates appear to be an important determinant of plasma SFA concentrations by driv-
ing de novo lipogenesis (Forsythe et al. 2010).
The major dietary sources of SFAs in the U.S. diet are red meat and dairy foods (Table 11.1).
Palmitic acid (C16:0) is the most abundant SFA in both categories, along with stearic acid in meat,
and myristic acid in dairy. Tropical oils, including palm and coconut oils, are also sources of SFAs,
specifically lauric and myristic acids as well as palmitic acid.
EVOLVING HISTORY OF STUDIES OF SFA EFFECTS

ON LIPIDS, LIPOPROTEINS, AND CVD RISK
In 1909, as reviewed in Steinberg (2004), the St. Petersburg-based pathologist Alexander Ignatowski
fed rabbits high-protein diets containing meat, eggs, and milk and showed that such diets induced
arterial lesions that resembled human atherosclerosis (Ignatowski 1909). Several years later, Nikolai
Anichkov at the same institute showed that the same phenomenon could be induced by feeding rab-
bits purified cholesterol (Anitschkow 1913). Anichkov would subsequently conduct an elaborate
series of experiments using various animal models over the next decades to establish a role for
blood cholesterol in the pathogenesis of atherosclerosis as reviewed in Konstantinov, Mejevoi, and
Anichkov (2006), a finding that would set the stage for the many experimental studies to follow
that were aimed at understanding atherosclerotic progression and the dietary and pharmacological
therapies that could effectively modulate it.
Evolving Role of Saturated Fatty Acids 211
Metabolic Studies Evaluating Diet and Lipid Profiles

Experimental studies in humans during the mid-twentieth century showed that blood cholesterol
level was increased by dietary SFAs relative to other macronutrients as reviewed in Konstantinov,
Mejevoi, and Anichkov (2006). Substituting plant foods for animal foods led to a significant decrease
in blood cholesterol levels (Kinsell et al. 1952), an effect found to be due to the unsaturation of veg-
etable fats (Ahrens, Blankenhorn, and Tsaltas 1954). Metabolic studies in men conducted by Ancel
Keys evaluated effects on blood cholesterol concentrations of varying quantities of dietary saturated
and polyunsaturated fats (Keys, Anderson, and Grande 1957). Consistent with the earlier findings,
increases in serum cholesterol were a function of higher dietary saturated fat and lower polyunsatu-
rated fat content of the diets (Keys, Anderson, and Grande 1957). The effects of monounsaturated
fats were estimated to be neutral. Further, the investigators noted that these relationships were based
on group averages, whereas the reliability of individual prediction of change in cholesterol level
was low due to significant variations in dietary response (Keys, Anderson, and Grande 1957).
Hegsted et al. subsequently reported that SFAs with chain lengths of 14 and 16 carbons, but not
those with 10, 12, and 18 carbons, raised total serum cholesterol and also considered a role for
dietary cholesterol (Hegsted et al. 1965).
A more recent meta-analysis of data from 27 controlled diet trials published between 1970 and
1991 that evaluated effects of replacing SFAs with other macronutrients on total cholesterol (TC),
low density lipoprotein-cholesterol (LDL-C), and high density lipoprotein-cholesterol (HDL-C)
was in good agreement with the Keys and Hegsted equations, with changes in total serum choles-
terol increased by C14:0 and C10:0 SFAs and decreased by polyunsaturated fats (Mensink and
Katan 1992). Notably, Mensink and Katan showed that the replacement of fat with carbohydrate
led to increases in plasma triglycerides independent of the type of fat replaced (Mensink and
Katan 1992). Further, replacement of saturated fats with unsaturated fats decreased the LDL
to HDL ratio, whereas replacement with carbohydrates had no effect. Thus, it was concluded
that under isocaloric, metabolic ward conditions, the most favorable lipoprotein profiles were
achieved when saturated fats were replaced with unsaturated fat, without decreasing the total fat
intake. A similar evaluation by the same investigators showed that replacement of SFAs and trans
fatty acids with unsaturated fatty acids was associated with decreases in the TC:HDL-C ratio,
whereas replacement with carbohydrate was associated with no change (Mensink et al. 2003). Of
note, LDL-C may only be increased with SFAs when polyunsaturated fatty acid (PUFA) intake
is below ~5% (Hayes et al. 1997). As for specific SFAs, when replacing carbohydrate, lauric
acid (C12:0) raised LDL-C, the most compared to myristic (C14:0) and palmitic (C16:0) acids.
However, lauric acid also raised HDL-C significantly, thus resulting in the largest reduction in
the TC:HDL-C ratio (Mensink et al. 2003).
Diet and CVD Studies

Clinical trials evaluating the effect of replacing saturated fats with polyunsaturated fats on cardio-
vascular disease (CVD) events, for example, myocardial infarction and CHD death, in the 1960s
and 1970s, generally showed reduced CVD risk (Dayton and Pearce 1969, Leren 1970, Turpeinen
et al. 1979), although there were also trials, including some completed nearly 20 years later,
that showed no effects on CVD of this replacement scenario (Burr et al. 1989, Frantz et al. 1989,
Watts et al. 1992).
Positive associations of dietary SFAs with CVD were observed in an epidemiological study
across seven countries conducted by Keys around the same time (Keys et al. 1966). More recently,
the Seven Countries Study has been criticized for including only those data that supported a posi-
tive linear relationship between SFAs and CVD (Lustig 2012, Taubes 2007). As with all epide-
miological studies, associations are not evidence of causality. It is also important to note that
consideration of a single macronutrient in isolation, that is, not in relation to what is being replaced,
is necessarily limiting. Nonetheless, the findings from the Seven Countries Study among other
epidemiological studies along with the aforementioned metabolic trials formed the basis for the
“diet-heart” hypothesis, which pointed to SFAs as a major dietary determinant of the increasing
CVD rates that were occurring in the United States in the decades after the Second World War.
Based on epidemiological, metabolic, and clinical trial evidence available at the time,
restriction of both SFAs and total fats was advised in the first U.S. dietary guidelines (United
States Department of Agriculture and Department of Health and Human Services 1980). The
advice to reduce total fats in the diet was thought to be a pragmatic approach that would enable
the reduction of SFAs. Since these guidelines were issued, an increasing body of research, as
described in the following, speaks to the newly appreciated complexity of the SFA–CVD rela-
tionship, including a refinement in lipoprotein measures as biomarkers of CVD, the importance
of specifying the replacement nutrient, the heterogeneity of SFAs, variable effects of different
SFA food sources, and interindividual variability in response to diets (Siri-Tarino et al. 2015).
Further, the epidemics of obesity and diabetes have pushed to the forefront the need for appro-
priate lifestyle therapies for their management, and diets lower in carbohydrates, and higher in
SFAs, provide an option. These developments support a shift in dietary guidance toward foods
and dietary patterns rather than macronutrients.
Refined CVD Risk Assessment

The concept that LDL particles provide a better assessment of risk than cholesterol alone dates
to the 1950s and has been carried through to current investigations (Krauss 2014). LDL particles
exist along a spectrum of sizes and densities with differing cholesterol content and associations
with CVD risk. The distribution of the LDL particle subclasses varies between individuals, with
medium particles generally the most abundant in healthy individuals and smaller and more dense
LDL (sdLDL) particles associated with increased CVD risk compared to larger LDL. Recent large
prospective cohort studies have shown that levels of sdLDL predict CVD risk in various populations
independently of LDL-C concentrations (Hoogeveen et al. 2014, Tsai et al. 2014). Smaller LDL
have reduced LDL receptor affinity, and hence slower plasma clearance, as well as greater binding
to arterial proteoglycans and greater oxidative susceptibility (Berneis and Krauss 2002). sdLDL par-
ticles may also contain more apoCIII and be subject to increased nonenzymatic glycation, features
that have been associated with greater atherogenicity (Siri-Tarino et al. 2015).
LDL-C concentrations can misrepresent the number of LDL particles in persons with a pre-
dominance of sdLDL. Thus, in two individuals with a similar LDL cholesterol concentration, there
may be differential CVD risk based on the number and quality of LDL particles, with persons with
increased particle numbers and/or increased smaller and more dense LDL particles at increased
CVD risk. Half of the general population demonstrates discordance between LDL-C and LDL par-
ticle number, where discordance is defined as a differential in the population percentile of 12% or
more, and in such cases, LDL particle number more accurately predicts CVD risk (Davidson et al.
2011). Of note, discordance occurs in as many as 75% of persons with type 2 diabetes mellitus
(T2DM) or metabolic syndrome or in people taking statins (Davidson et al. 2011).
Importantly, diet can shift LDL particle distribution and composition as reviewed in Siri-Tarino
et al. (2015), such that dietary carbohydrate, which increases triglycerides, is associated with a shift
in distribution toward sdLDL particles. In contrast, higher saturated fat content of diets has been
associated with larger and more buoyant particles (Dreon et al. 1998, Krauss et al. 2006). Self-
reported changes in intake of total SFAs from 6%E to 18%E in the context of high-fat, moderate-
carbohydrate diets and lower-fat, higher-carbohydrate diets (46% fat:39% carbohydrate vs. 24%
fat:59% carbohydrate, respectively) were positively associated with increases in the mass of large
LDL particles, but not with changes in LDL-C concentrations (Dreon et al. 1998). In a carefully
executed feeding intervention, high versus low levels (15% and 8%, respectively) of SFAs in the
context of a lower carbohydrate (26%) diet showed that the increase in LDL-C on the higher SFA diet
was attributable to an increase in larger and medium LDL particles without effects on small, dense
particles (Krauss et al. 2006). In contrast, in the context of a diet whose primary protein source was
beef (Mangravite et al. 2011) versus mixed protein sources, for example, chicken, fish, and tofu, as
provided in the aforementioned dietary intervention trial (Krauss et al. 2006), effects of higher satu-
rated fat (15% vs. 8%) led to increases in sdLDL particles as well as increases in apoB, total, LDL,
and non-HDL cholesterol, thus suggesting that very high red meat intake may modify the effects of
SFAs on lipid and lipoprotein profiles (Mangravite et al. 2011). Finally, a recent study in persons
with pattern B—defined as lipoprotein profiles with a preponderance of sdLDL—showed that very
high concentrations of SFAs (18%E vs. 8% E), provided mostly from dairy fat in the context of mod-
erate carbohydrate intake (40% E), resulted in significant increases in the concentrations of small
and medium LDL (Chiu et al. 2016b). Thus, the preferential effect of SFAs on larger, more buoyant
LDL particles likely depends on context, including but not limited to the food sources of SFAs, the
total carbohydrate content of the diet, the metabolic characteristics of the individuals consuming the
diet, and/or possible threshold effects for SFAs above which LDL particles of all sizes are affected.
SFAs in Context
Replacement Nutrient
The effects of SFAs on CVD risk in weight-stable individuals are intrinsically a function of the
nutrient that replaces it. Although this premise dates back to early metabolic studies conducted in
the 1950s, dietary recommendations and public health messaging have generally focused on reduc-
ing SFAs without specifying appropriate replacement nutrients (DGAC 2015, Eckel et al. 2014). In
meta-analyses summarizing the effects of prospective cohort studies evaluating diet–CVD relation-
ships, SFAs per se have not been associated with CVD (Mente et al. 2009, Siri-Tarino et al. 2010,
Skeaff and Miller 2009). In part, the lack of association may have been due to the inability to discern
from the component studies the effects of the replacement nutrient. In particular, it would be relevant
to assess the adverse effects of trans fatty acids and refined carbohydrates versus the potential for
beneficial effects of PUFAs on CVD risk (Mente et al. 2009, Skeaff and Miller 2009). Importantly,
the replacement of SFAs with refined carbohydrates—as commonly occurs in practice—has been
associated with no improvement, or a worsening, of CVD risk (Hu 2010).
A meta-analysis of RCTs that specifically considered PUFA replacement of SFAs showed CVD
benefit of this replacement scenario at a level comparable to what would have been predicted based
on changes in lipid profiles (Micha and Mozaffarian 2010). More recent meta-analyses that included
different sets of component studies have presented inconsistent results, with neutral (Chowdhury
et al. 2014, Ramsden et al. 2013) or even adverse effects (Ramsden et al. 2010) of PUFAs reported.
However, these analyses were compromised by the inclusion of component studies that used trans fats
in either the control or intervention arms (Willett, Stampfer, and Sacks 2014). Trans fats were often
used to replace SFAs in food products, but have since been shown to increase LDL-C and triglyceride
and decrease HDL-C, and thus increase CVD risk, relative to SFAs (see Chapter 12). A reanalysis
of one of the aforementioned meta-analyses with the confounded study excluded showed significant
benefit when SFAs were replaced with PUFAs, that is, relative risk for CVD = 0.81 (0.68–0.98)
(Chowdhury et al. 2014), in line with a Cochrane review that showed modest but significant benefit
on CVD events of this replacement scenario (Hooper et al. 2015).
The type of PUFAs, that is, omega-3 fatty acids and omega-6 fatty acids, used to replace SFAs
can differentially affect CVD risk (Ramsden et al. 2013). Omega-3 fatty acids have been associated
with CVD benefit in observational studies, although these results have not been supported by recent
clinical trials in higher-risk populations (Siri-Tarino et al. 2015). The role of omega-3 fatty acids
in primary prevention is currently being investigated. Epidemiological data also support beneficial
effects of omega-6 fatty acids (Farvid et al. 2014). Further, plasma biomarkers for omega-6 fatty
acids, which can be used to assess their consumption since they cannot be synthesized in vivo, have
been associated with improved cardiometabolic risk (Chapter 10).
Given the adverse effects of dietary carbohydrates on components of atherogenic dyslipidemia as

described in the following, monounsaturated fatty acids (MUFAs) were shown to be a better replace-
ment for SFAs than carbohydrates in persons at high cardiometabolic risk (Berglund et al. 2007).
However, there are observational data in a Finnish population that suggest that MUFA replacement
of SFAs, trans fats, or carbohydrates is associated with increased CHD risk (Virtanen et al. 2014).
There are also intervention studies in African green monkeys on high-cholesterol diets that indicate
comparable effects of dietary MUFAs and SFAs on aortic atherosclerosis when compared to PUFAs
(Rudel, Parks, and Sawyer 1995). These results, as well as similar findings in hypercholesterolemic
mouse models (Degirolamo, Shelness, and Rudel 2009), have been attributed to a more rigid choles-
teryl ester structure with MUFAs and SFAs than with PUFAs.
Atherogenic Dyslipidemia and the Obesity and Diabetes Epidemics

As reviewed elsewhere (Siri-Tarino et al. 2015), both excess calories and dietary carbohydrates,
particularly refined and processed carbohydrates, can induce or amplify atherogenic dyslipidemia, a
trait characterized by a cluster of interrelated lipid and lipoprotein changes, namely elevated triglyc-
eride, reduced HDL-C, and increased sdLDL. Importantly, each of these components has been asso-
ciated with increased CVD risk (Ballantyne et al. 2001, Manninen et al. 1992, Sarwar et al. 2007).
Of note, nonfasting triglyceride, representing in part remnant lipoproteins derived from chylomicron
and VLDL triglyceride hydrolysis, can be more strongly predictive of CVD than fasting triglyceride
(Bansal et al. 2007, Nordestgaard et al. 2007).
Food Sources of SFAs and Dietary Patterns

The food source of SFAs can modulate their association with CVD risk. In the Multi-Ethnic Study
of Atherosclerosis, SFAs from meat were associated with increased CVD risk, whereas consump-
tion of comparable amounts of SFAs from dairy sources was associated with decreased CVD risk
(de Oliveira Otto et al. 2012). Epidemiological studies have suggested that red meat intake is more
strongly associated with CVD risk than other sources of protein (Bernstein et al. 2012, Sinha et al.
2009) and that the major determinant of this association may be processed red meats (Micha,
Wallace, and Mozaffarian 2010). More recently, a carefully controlled feeding trial that compared
cheese and meats as food sources matched for SFA content in the context of a diet with 36% of
energy from fat relative to a lower-fat (23%E), higher-carbohydrate diet showed a 5% increase in
HDL-C and an 8% increase in apoAI in both the cheese and meat groups relative to a lower-fat
(23%E), higher-carbohydrate diet (Thorning et al. 2015). However, effects of the high SFAs in the
context of either meat or cheese were not observed for LDL or total cholesterol as would have been
expected, a finding that may have been due to the high MUFA content of both the cheese and meat
diets. Thus, SFA effects on lipoprotein profiles in the context of meat were no different than those
for cheese, and these two diets led to less atherogenic profiles compared to a lower-fat, higher-
carbohydrate diet (26% energy).
Food sources of dairy have been shown to be relevant, with several studies showing that the con-
sumption of full-fat cheese relative to butter and other nondairy sources of SFAs resulted in lower
total and LDL cholesterol (Siri-Tarino et al. 2015). Further, specific evaluation of the SFA content
of dairy has not been shown to have adverse effects on lipid and lipoprotein profiles in several recent
metabolic studies (Raziani et al. 2016, Thorning et al. 2015). Full-fat versus regular-fat cheese did
not adversely alter LDL-C, triglyceride, insulin, glucose, blood pressure, or waist circumference in
a real-world study in which these foods were substituted for components of habitual diets (Raziani
et al. 2016), suggesting that SFAs in the context of full-fat cheese can be consumed as part of a
healthy diet. However, possible attenuation of a cholesterol-raising effect could have been related to
weaknesses in study compliance, statistical power, and/or unspecified food replacement scenarios
(Siri-Tarino and Krauss 2016). Nonetheless, the neutral or beneficial effects of dairy foods on cardio-
metabolic profiles irrespective of SFA content may be due to other components of the food source,
including vitamin D, calcium, magnesium, potassium, and whey protein. Fermented dairy products,
such as yogurt, may be particularly effective at improving cardiometabolic profiles (Astrup 2014,
St-Onge, Farnworth, and Jones 2000), possibly through effects on the gut microbiome.
Although tropical oils such as coconut and palm do not represent a major source of SFA con-
sumption in the United States, there is some evidence that lauric acid, the main SFA contained in
these foods, does not adversely affect the TC:HDL ratio or other CVD biomarkers (Mensink et al.
2003, Voon et al. 2011).
Variations in SFA content have also been examined in the context of established heart-healthy
dietary patterns such as the Dietary Approaches to Stop Hypertension (DASH) diet, which emphasizes
fruits, vegetables, low-fat dairy products, whole grains, poultry, fish, and nuts (Appel et al. 1997).
SFA EFFECTS ON OTHER CARDIOMETABOLIC HEALTH RISK FACTORS

Studies of dietary SFAs relative to other macronutrients in the context of weight stability have
generally not shown an association with insulin sensitivity and T2DM (Forouhi et al. 2014, Ma
et al. 2015, Morio et al. 2016, Siri-Tarino et al. 2015). In particular, replacing SFAs with MUFAs
(Chiu et al. 2014, Jebb et al. 2010, Tierney et al. 2011), PUFAs (Tierney et al. 2011), or carbo-
hydrates (Jebb et al. 2010) had no or only modest (Vessby et al. 2001) effects on insulin sensitiv-
ity. Dietary SFA intake was not associated with incident diabetes in a meta-analysis (Micha and
Mozaffarian 2010).
Plasma concentrations of SFAs have also been evaluated in relation to diabetes risk. Several
studies have documented associations of levels of even-chain-length SFAs, that is, C14:0 (myris-
tic), C16:0 (palmitic), and C18:0 (stearic) with increased T2DM risk (Forouhi et al. 2014, Ma et al.
2015). However, it is not clear to what extent these reflect dietary SFA intake as opposed to de novo
lipogenesis, which is driven by dietary carbohydrates.
In contrast, blood concentrations of odd-chained SFAs have been inversely associated with T2DM.
These SFAs (C15:0 and C17:0) reflect the consumption of dairy fats as does the plasma biomarker
C16:1n-7, trans-palmitoleic acid, which has also been shown to be inversely associated with insulin
resistance (Mozaffarian et al. 2010). As covered in Chapter 25, an emerging literature suggests that dairy
foods are neutrally or inversely associated with T2DM (Sluijs et al. 2012). Although some studies point
to low-fat, fermented dairy products as being most beneficial (O’Connor et al. 2014), recent studies have
suggested that the effects on insulin sensitivity of full-fat dairy are just as beneficial as low-fat dairy
products (Benatar, Sidhu, and Stewart 2013). In addition to other components of dairy foods such as
calcium, magnesium, and other nutrients that have been associated with improved cardiometabolic risk,
dairy foods also contain short- and medium-chain SFAs that have different biological properties and
affect health differently (inverse) compared to long-chain SFAs. Thus, the diversity of foods containing
SFAs and the heterogeneity of SFAs themselves may modulate insulin sensitivity and provide further
substantiation for the concept that consideration for total SFA content of the diet does not provide a
meaningful measure of the overall quality of the diet.
Cellular and animal data suggest that diets high in SFAs adversely affect inflammation, but data
supporting effects of SFAs on inflammatory markers in humans are more limited (Siri-Tarino et al.
2015). Although one study showed a decrease in two inflammatory markers, interleukin-6 (IL-6)
and E-selectin, when oleic acid was replaced with stearic acid or a combination of lauric, myristic,
and palmitic acids (Baer et al. 2004), other studies have not demonstrated such effects. Diets high
in SFAs, that is, lauric, myristic, and palmitic, did not alter the inflammatory biomarkers TNF-α,
IL-1β, IL-6, IL-8, high-sensitivity C-reactive protein (hs-CRP), or interferon-γ (Voon et al. 2011).
There was also no effect of high SFAs in the context of a very-low-carbohydrate diet on hs-CRP,
IL-6, IL-8, TNFα, or MCP-1 (Forsythe et al. 2010).
Keogh et al. reported impaired flow-mediated dilation (FMD) with administration of diets high
in SFAs versus PUFAs, MUFAs, or carbohydrates (Keogh et al. 2005) as did de Roos and Fuentes
(de Roos, Bots, and Katan 2001, Fuentes et al. 2001). Sanders et al. showed no changes in FMD
with diets high in SFAs (~15% total energy) compared to diets high in MUFAs or carbohydrates
with SFAs at ~9% of total energy in 121 insulin-resistant men and women (Sanders et al. 2013).
Notably, in the latter study, SFAs were provided by palm oil and milk fat rather than meat fats, and
refined high oleic sunflower oil rather than extra-virgin olive oil was used as a source of MUFAs.
In the context of weight loss, very-low-carbohydrate diets high in SFAs were associated with
lower concentrations of inflammatory markers compared to a low-fat diet (Forsythe et al. 2008);
although weight loss was consistently greater with the very-low-carbohydrate diet, changes in
inflammatory markers were not associated with the magnitude of weight loss.
Some evidence exists for modest but significant adverse effects of meals high in SFAs on post-
prandial inflammatory responses compared to meals rich in PUFAs, MUFAs, or carbohydrates
(Masson and Mensink 2011, Nappo et al. 2002, Raz et al. 2013). In contrast, several studies have
shown no differences in postprandial inflammatory markers in response to meals of varying food
and macronutrient composition (Jimenez-Gomez et al. 2009, Manning et al. 2008, Teng et al. 2015).
High versus low PUFA:SFA ratios also did not affect postprandial inflammatory responses (Poppitt
et al. 2008). Differences in study population or meal composition may explain the inconsistent
results, but overall, no strong evidence in humans is available for SFA effects on inflammation.
As regards blood pressure, while one trial showed a decrease with MUFA, but not SFA
(Rasmussen et al. 2006), and another showed a decrease with PUFA or MUFA replacement of
SFAs (Lahoz et al. 1997), seven other trials showed no effects of replacing SFAs with PUFAs,
MUFAs, or carbohydrates on blood pressure (Micha and Mozaffarian 2010). Importantly, high
saturated fat (14% of energy) in the context of the DASH diet was shown to lead to comparable
reductions in blood pressure compared to a DASH diet with low saturated fat (8% of energy) (Chiu
et al. 2016a), emphasizing the role of dietary pattern over macronutrients in the determination of
cardiometabolic health.
VARIABILITY IN RESPONSE OF LDL-C TO SATURATED FAT

Persons with a higher baseline LDL-C have been shown to respond to reductions in SFAs with
greater reductions in LDL-C (Denke 1995). In addition, overweight (Chiu et al. 2014) and insulin-
resistant (Lefevre et al. 2005) individuals have been reported to be less responsive to the effects of
SFAs on LDL-C, as have women versus men (Weggemans et al. 1999).
A number of genetic variants have been reported to be associated with LDL-C response to SFA
intake. Among the most consistent of these is the relation of the apoE4 isoform with a greater
LDL-C response (Dreon et al. 1995). Similarly, the presence of serine to threonine at position 347
in the apoAIV gene was associated with a greater LDL response to a change from a higher SFA
diet to a National Cholesterol Education Program Step 1 diet (Jansen et al. 1997). Replacement
of dietary SFAs with PUFAs was associated with no changes in LDL-C but greater decreases in
dense LDL-C in persons with a glutamine to histidine substitution at position 360 (Wallace et al.
2000). Polymorphisms in apoB have also been shown to modulate effects of changes in dietary fat
on LDL-C, although none of these were specifically related to changes in dietary SFAs (Masson,
McNeill, and Avenell 2003). Cumulatively, however, the effect sizes for these genetic influences
on dietary LDL-C are small and account for only a minimal portion of interindividual variation
in response.
APPLYING NEW KNOWLEDGE TO DIETARY GUIDELINES

The message to reduce SFAs has been an oversimplified one, which may have unintentionally led to
the increased consumption of refined and processed carbohydrates, a replacement scenario that has
been associated with no benefit or even a worsening of CVD risk, at least in part through its induc-
tion of atherogenic dyslipidemia. In light of the current epidemics of obesity and insulin resistance,
reduction of sugars and refined carbohydrates as sources of high calories but also for their specific
adverse metabolic effects may be more effective in improving cardiometabolic health. Of note,
processed food formulations that combine high sugars and high SFAs and are stripped of nutritional
content may represent a different category of “foods” and may be particularly detrimental.
The 2015 Dietary Guidelines for Americans have moved toward recommending foods and
dietary patterns, but the specification of a limitation on SFAs is retained (DGAC 2015). In contrast,
the Dutch have recently moved away from nutrient recommendations as first issued in 1986 toward
guidelines that are exclusively food based (Kromhout et al. 2016). In France, dietary guidelines
advise the limitation of total SFAs to less than 12% of energy, but also consider SFA subtypes, with a
specific limitation of lauric, myristic, and palmitic to less than 8% (Nettleton, Legrand, and Mensink
2015). Thus, an appreciation for the complexity and subtleties of the SFA–CVD relationship is now
beginning to shape recommendations for public health.
CONCLUSIONS
Investigation of the effects of dietary SFAs on cardiometabolic risk over the last half century has
revealed the complexity and nuances of this relationship. Although it is clear that SFAs in relation
to other macronutrients can affect lipid and lipoprotein profiles, the evidence for effects of dietary
SFAs on other CVD risk factors such as blood pressure, insulin sensitivity, inflammation, and endo-
thelial function is equivocal. The diversity of SFAs and the variable food sources and dietary pat-
terns in which they are consumed modulate their effects.
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12 Effects of Dietary Trans Fatty
Acids on Cardiovascular Risk
Ronald P. Mensink
CONTENTS
Abbreviations..................................................................................................................................223
Introduction.....................................................................................................................................224
TFA and Risk Markers for Cardiovascular Disease........................................................................225
Serum Lipids and Lipoproteins..................................................................................................225
Mechanistic Studies...............................................................................................................226
LDL Particle Size.......................................................................................................................227
LDL Oxidation...........................................................................................................................227
Lipoprotein(a)............................................................................................................................227
Glucose Homeostasis.................................................................................................................228
Blood Pressure...........................................................................................................................228
Hemostatic Function..................................................................................................................228
Vascular Function.......................................................................................................................229
Inflammation..............................................................................................................................229
TFA and Cardiovascular Disease....................................................................................................229
Intakes and Recommendations.......................................................................................................230
Conclusion......................................................................................................................................231
References.......................................................................................................................................231
ABSTRACT
Most double bonds of the unsaturated fatty acids in the diet have the so-called cis configuration, but
fatty acids with double bonds in the trans configuration also exist. These so-called trans fatty acids
(TFA) are mainly produced by the industry during the partial hydrogenation of vegetable oils rich
in cis-polyunsaturated fatty acids (linoleic acid and α-linolenic acid) like sunflower oil and soybean
oil. However, TFA are also formed by the bacterial transformation of polyunsaturated fatty acids in
the first stomach of ruminant animals. This chapter first summarizes the effects of TFA intake on the
serum lipoprotein profile and other markers related to cardiometabolic health. Then, relationships
with cardiovascular risk are discussed.
ABBREVIATIONS
CHD Coronary heart disease
HDL High-density lipoproteins
iTFA Industrially produced trans fatty acids
LDL Low-density lipoproteins
rTFA Ruminant trans fatty acids
TFA Trans fatty acids
223
INTRODUCTION
Most double bonds of the unsaturated fatty acids in the diet have the so-called cis configuration,
but fatty acids with double bonds in the trans configuration also exist. These so-called trans fatty
acids (TFA) are mainly produced during the partial hydrogenation of vegetable oils rich in cis-
polyunsaturated fatty acids (linoleic acid and α-linolenic acid) like sunflower oil and soybean oil.
Through partial hydrogenation, the liquid oils are converted into fats with increased functionality
and stability that can be used for frying and baking and for the manufacturing of foods such as bis-
cuits, shortening, and margarines with longer shelf life. Fats rich in these industrially produced TFA
(iTFA) were mainly used as substitutes for natural fats rich in saturated fatty acids such as butter,
lard, and tropical oils. Typically, these iTFA have 18 carbon atoms and one double bond, mainly
located between the (n-5)-carbon and the (n-12)-carbon atom. When the double bond is located at
the (n-9)-position, this specific TFA isomer is called elaidic acid (trans-C18:1n-9). In contrast to
partial hydrogenation, the full hydrogenation of vegetables oils does not result in the production of
TFA but of stearic acid (Figure 12.1). However, TFA are also formed by the bacterial transforma-
tion of polyunsaturated fatty acids in the first stomach of ruminant animals. In ruminant fats, trans
isomers with 18 carbon atoms and one double bond also dominate, but trans isomers with 14 and 16
carbon atoms are present as well. As bacterial transformation is a more selective process, the double
bond in ruminant TFA (rTFA) is mainly, but not exclusively, located at the (n-7)-carbon atom. This
TFA is called trans-vaccenic acid or briefly vaccenic acid (trans-C18:1n-7). Most rTFA in the diet
are from dairy origin. Though most TFA in the diet have one double bond, TFA isomers of linoleic
acid and α-linolenic acid also exist. In this respect, conjugated linoleic acid (CLA) is well known.
CLA refers to a mixture of positional and geometric isomers of linoleic acid, whose double bounds
can be in either trans or cis configuration. CLA differs from most natural polyunsaturated fatty acids
in that the double bounds are not separated by a methylene carbon but are conjugated. One common
CLA isomer has a cis double bond at the (n-9) carbon atom and a trans double bond at the (n-7)
position and is present in ruminant fat. However, it can also be formed in the human body by the
desaturation of vaccenic acid.
In this chapter the effects of TFA on cardiovascular risk in humans will be reviewed. The focus
will be on dietary intervention studies and meta-analyses as related to the intakes of TFA with
18 carbon atoms and one double bond.
Trans-MUFA:
Elaidic acid and its
positional isomers
O
OH
Trans-C18:1n-9
Linoleic acid Stearic acid

O O
OH OH
C18:2n-6 C18:0
Cis-MUFA:
Oleic acid and its
positional isomers
O
OH
Cis-C18:1n-9
FIGURE 12.1 Simplified scheme of partial and full hydrogenation. Partial hydrogenation of linoleic acid
leads to the formation of trans- and cis-monounsaturated fatty acids (MUFA), while full hydrogenation leads
to the formation of stearic acid.
Effects of Dietary Trans Fatty Acids on Cardiovascular Risk 225
TFA AND RISK MARKERS FOR CARDIOVASCULAR DISEASE

Numerous human intervention studies have addressed the effects of iTFA on risk markers for
cardiovascular disease (CVD). Typical intakes of iTFA in these studies were >4% of energy. In
most of these studies, the distribution of the double bond over the TFA molecule was not reported.
Therefore, it is not known if the different isomers of the iTFA molecule exert different metabolic
effects. Further, TFA from dairy origin have been studied less extensively and generally at intakes
<3% of energy. Only more recent studies have specifically focused on rTFA. As already described,
TFA from dairy origin have predominantly the double bond at the (n-7)-position. It should be kept
in mind, however, that iTFA also include isomers with the double bond at this position. Thus, iTFA
and rTFA are composed of a mixture of different positional isomers that partly overlap.
Serum Lipids and Lipoproteins

More than 40 years ago, a few studies addressed the effects of iTFA on serum total cholesterol
concentrations. Results, however, were conflicting. In one study, it was reported that the cho-
lesterolemic effects of iTFA were comparable to those of oleic acid (Mattson, Hollenback, and
Kligman 1975), the most common cis-monounsaturated fatty acid in the diet. Two other studies,
however, suggested that these trans-isomers increased serum total cholesterol concentrations, but
less than saturated fatty acids did (Vergroesen 1972, Vergroesen and Gottenbos 1975). In these
studies, effects on lipoproteins were not reported. About 25 years ago, a paper was published
reporting the results of a well-controlled dietary intervention study with 59 healthy subjects who
received three mixed natural diets in random order for three weeks each (Mensink and Katan
1990). The nutrient composition of the diets was similar, except for 10% of the daily energy
intake, which was provided as oleic acid, as trans isomers of oleic acid (iTFA), or as a mixture
of saturated fatty acids. It was reported that iTFA increased the concentration of cholesterol in
the atherogenic low-density lipoproteins (LDL) as compared with oleic acid. Effects of iTFA
on LDL-cholesterol, however, were slightly less than those of a mixture of saturated fatty acids.
More importantly, however, was the finding that iTFA lowered high-density lipoprotein (HDL)
cholesterol concentrations as compared with the other two diets. Together, these changes resulted
in the most unfavorable lipoprotein profile following a diet high in iTFA. This study was criti-
cized, also because the intake of iTFA exceeded habitual intakes at that time, which were typically
less than 4% of daily energy intake (De Vries et al. 1997). Later studies, however, using lower
intakes of iTFA largely confirmed these effects.
In 2010, Brouwer, Wanders, and Katan (2010) published a meta-analysis describing the effects of
TFA on plasma lipoproteins. Effects were expressed relative to those of an isocaloric amount of cis-
monounsaturated fatty acids. In the end, 23 trials were identified, which provided 28 data points that
were used to estimate the effects of iTFA. It was calculated that LDL-cholesterol increased by 0.048
mmol/L and HDL-cholesterol decreased by 0.010 mmol/L for each 1% of energy from iTFA replacing
cis-monounsaturated fatty acids (Figure 12.2). For the plasma LDL-cholesterol to HDL-cholesterol
ratio, this estimate was 0.055. A high plasma LDL-cholesterol to HDL-cholesterol is strongly associ-
ated with coronary heart disease (CHD). Effects of iTFA on other plasma lipids and lipoproteins were
not estimated. However, an earlier meta-analysis including eight studies reported that iTFA increased
concentrations of serum triacylglycerol and apoB100 and decreased those of apoA-I as compared with
cis-monounsaturated fatty acids (Mensink et al. 2003). Thus, it is evident that the effects of iTFA have
adverse effects on the serum lipoprotein profile.
The question then arises if TFA from ruminants have comparable effects. Five trials were iden-
tified that provided six data points that could be used to calculate the effects of rTFA on plasma
lipids (Brouwer, Wanders, and Katan 2010). It was estimated that increasing the intake of 1% of
energy from rTFA at the expense of cis-monounsaturated fatty acids increased LDL-cholesterol by
0.045 mmol/L, decreased HDL-cholesterol by 0.009 mmol/L, and increased the LDL-cholesterol to
0.12
iTFA
Change when 1 En% of TFA

0.09
rTFA
replaces cis-MUFA 0.06
0.03
0.00
–0.03
LDL-cholesterol HDL-cholesterol Ratio of LDL- to
(mmol/L) (mmol/L) HDL-cholesterol
FIGURE 12.2 Effects on serum lipids when 1% of energy (En%) of industrially produced trans fatty acids
(iTFA) or of trans fatty acids from ruminant fats (rTFA) in the diets replaces cis-monounsaturated fatty
acids (cis-MUFA). Data are means (±95% confidence intervals) and are derived from Brouwer, Wanders,
and Katan (2010).
HDL-cholesterol ratio by 0.038. These effects were slightly less than those of iTFA, although differ-
ences did not reach statistical significance. It should be noted, however, that the confidence intervals
for the estimates of rTFA were very wide and estimates for LDL-cholesterol and HDL-cholesterol
were not significantly different from zero (Figure 12.2). The wide confidence intervals for rTFA
are, at least partly, due to the fact that intakes in the various intervention studies were in general
quite low, which makes it difficult to obtain precise estimates for their effects. Based on their meta-
analysis, however, Gayet-Boyer et al. (2014) reported that rTFA does not affect the LDL-cholesterol
to HDL-cholesterol ratio. Thus, earlier reports on the effects of iTFA and rTFA on the serum lipo-
protein profile were controversial. Recently, however, Gebauer et al. (2015) published the results of
a double-blind randomized crossover trial involving 106 healthy subjects. In that well-designed and
controlled study, diets differed by about 3.0%–3.5% of daily energy intakes from iTGA, rTFA, or
stearic acid. Compared with the diet rich in iTFA, the diet rich in rTFA increased concentrations of
total cholesterol, LDL-cholesterol, HDL-cholesterol, triacylglycerol, apoA-I, and apoB. Effects on
the total cholesterol to HDL-cholesterol ratio were comparable between the diet rich in iTFA and
that rich in rTFA. The diet high in stearic acid had a more favorable effect on total cholesterol, LDL-
cholesterol, the total cholesterol to HDL cholesterol ratio, and apoB than either the diet rich in rTFA
or rich in iTFA. Stearic acid and rTFA had comparable effects on HDL-cholesterol, triacylglycerol,
and apoA-I concentrations. Finally, the effects of stearic acid and rTFA on triacylglycerol concen-
trations were comparable. Thus, this adequately powered study shows that effects of rTFA on the
serum lipoprotein profile are not favorable as compared with those of iTFA.
Mechanistic Studies
To explain the effects of iTFA on the lipoprotein profile, several mechanistic studies have been car-
ried out. Effects of rTFA are less well documented. Several studies have focused on cholesteryl ester
transfer protein (CETP). CETP is a plasma protein that is involved in the exchange of cholesteryl
esters from HDL for triacylglycerols from very-low-density lipoproteins (VLDL) and LDL. Thus,
a high-CETP activity could lead to decreases in HDL-cholesterol and increases in LDL-cholesterol
and VLDL-cholesterol. Indeed, iTFA increased CETP activity as compared with saturated fatty
acids, cis-monounsaturated fatty acids and linoleic acid (Abbey and Nestel 1994, Lichtenstein
et al. 2001, van Tol et al. 1995). Chardigny et al. (2008), however, did not observe any changes
in CETP activity after consumption of diets rich in iTFA or rTFA. In other studies, milk fat rich
in rTFA had comparable effects on CETP activity as compared with milk fat with lower levels of
rTFA (Malpuech-Brugère et al. 2010, Lacroix et al. 2012). Thus, these findings suggest that iTFA
increases CETP activity, especially at high intakes. Further, Sundram, French, and Clandinin (2003)
have reported that iTFA increases endogenous cholesterol synthesis as compared with a mixture of
palmitic acid and oleic acid. In contrast, no specific effects of iTFA on cholesterol synthesis were
observed in another study (Matthan et al. 2000). Finally, Matthan et al. (2004) concluded that the
catabolism of apoA-I from HDL was increased and that of LDL apoB-100 was decreased after
intake of iTFA. Thus, the underlying mechanism by which iTFA and rTFA affect lipoprotein metab-
olism remains to be elucidated, but it is likely that multiple, interrelated pathways are involved.
LDL Particle Size

LDL particles vary in size, density, and composition. It is thought that small and dense LDLs are
more atherogenic than larger, less dense LDLs. Studies relating TFA intake to LDL particle size are
equivocal. Mauger et al. (2003) have reported that increasing the intake of iTFA at the expense of a
mixture of other fatty acids dose-dependently decreased LDL particle size. Chardigny et al. (2008)
showed that iTFA and rTFA do not have different effects on the concentrations of small, dense LDL
particles. Finally, Tricon et al. (2006) concluded that dairy products naturally enriched in rTFA have
comparable effects on small, dense LDL concentrations as regular dairy products.
LDL Oxidation
Oxidation of polyunsaturated fatty acids in the LDL particle may lead to the formation of oxidized
LDL (oxLDL) or minimally modified LDL (mmLDL). These are pro-inflammatory and proatherogenic
lipoprotein particles that can activate pathways involved in the development of atherosclerotic lesions.
However, there is no generally accepted method to examine in vivo susceptibility of LDL to oxidation.
Resistance of LDL toward an oxidative challenge can be determined in vitro by measuring the lag-
phase, which is the time before oxidized products of fatty acids within the LDL particle start to arise.
Also, the rate of oxidation and the amount of oxidative products formed can be measured. Copper ions
are frequently used to initiate the oxidation process. Using this approach, effects of diets rich in satu-
rated fatty acids, cis-monounsaturated fatty acids, and cis-polyunsaturated fatty acids had comparable
effects on in vitro LDL oxidizability to iTFA (Cuchel et al. 1996, Halvorsen et al. 1996, Nestel et al.
1992). In addition, the uptake of LDL by macrophages or the relative electrophoretic mobility of LDL
was not changed (Halvorsen et al. 1996). These latter two parameters may be affected by the degree
of modification of the LDL particle. Smit et al. (2011), however, showed that consumption of iTFA
increased urinary 8-iso-PGF2α levels, which is an isoprostane produced by the nonenzymatic peroxida-
tion of arachidonic acid and serves as an in vivo marker for free radical–induced lipid peroxidation and
may be increased in patients with CHD. It was, however, discussed that increased urinary 8-iso-PGF2α
levels may also be the result of a shift from a decreased endogenous breakdown of 8-iso-PGF2α to an
increased urinary excretion of this isoprostane. At much lower intakes, Tholstrup et al. (2006) did not,
however, observe any effects of butter rich in rTFA on urinary 8-iso-PGF2α levels as compared with
regular butter. Also, in vitro LDL oxidizability did not change when subjects were fed diets with dairy
products naturally enriched in rTFA (Tricon et al. 2006). Taken together, these studies suggest that iTFA
and rTFA do exert adverse effects on in vitro LDL oxidation or in vivo lipid peroxidation.
Lipoprotein(a)
Lipoprotein(a) [Lp(a)] is an LDL particle with an apolipoprotein (a) molecule covalently bound to
apoB100. Serum concentrations of Lp(a), which have a very strong genetic component, are positively
related with cardiovascular risk. The effects of iTFA have been examined in many intervention studies.
In most of these studies, Lp(a) concentrations were increased after consumption of diets rich in iTFA, as
compared with diets rich in saturated or cis-unsaturated fatty acids (Almendingen et al. 1995, Aro et al.
1997, Clevidence et al. 1997, Judd et al. 1998, Mensink et al. 1992, Nestel et al. 1992). Larger increases
were observed in subjects with higher baseline Lp(a) (Clevidence et al. 1997, Mensink et al. 1992). In
three other studies, Lp(a) concentrations were not significantly increased after iTFA intake (Lichtenstein
et al. 1999, 2006, Müller et al. 1998). Effects of rTFA have been studied less extensively. Chardigny
et al. (2008) have reported that iTFA and rTFA had comparable effects on Lp(a) concentrations after the
consumption of iTFA or rTFA. In contrast, Gebauer et al. (2015) found that rTFA increased Lp(a) as
compared with iTFA and stearic acid.
Glucose Homeostasis
Aronis, Khan, and Mantzoros (2012) carried out a systematic review on the effects of iTFA on fasting
glucose and insulin concentrations. In total, seven randomized controlled trials were identified. Diets
were provided for 4–16 weeks, and a total of 208 subjects were included. Intake of TFA varied between
2.6% and 9.0% of energy. In all studies, iTFA were replaced by other fatty acids and total fat intake
within a study was therefore constant. Even at these relatively high intakes, no effects were observed
on fasting plasma glucose or insulin concentrations. Studies that specifically focused on dairy products
also do not suggest that this source of TFA affects glucose metabolism (Tholstrup et al. 2006, Tricon
et al. 2006, Werner et al. 2013). Thus, there is no evidence that the effects of iTFA or rTFA on glucose
homeostasis are different from those of saturated fatty acids, oleic acid, or linoleic acid.
Blood Pressure
No intervention studies have been carried out that were specifically designed to examine the effects
of TFA on blood pressure. However, blood pressure has been measured in several randomized con-
trolled trials as a secondary outcome. In these trials, iTFA were exchanged for an isocaloric amount
of saturated fatty acids, oleic acid, or linoleic acid. No effects on systolic or diastolic blood pres-
sure were reported (Lichtenstein et al. 2003, Mensink, de Louw, and Katan 1991, Zock et al. 1993).
Likewise, studies focusing on dairy products naturally enriched in TFA did not demonstrate effects
on blood pressure (Chardigny et al. 2008, Lacroix et al. 2012, Malpuech-Brugère et al. 2010). In all
these studies, subjects were normotensive. It is therefore still possible that TFA may increase blood
pressure in hypertensive subjects, but effects—if any—are likely to be small.
Hemostatic Function
A proper hemostatic function is the result of a complex interplay between molecules derived and
secreted from the endothelium, platelets, and leukocytes and from factors that are part of the coagu-
lation and fibrinolytic pathways. If disturbed, a thrombus may be formed within the blood stream,
which may lead to a myocardial infarction or stroke. In contrast, impaired hemostasis can lead to
excessive bleeding.
Only a few intervention studies have examined the effects of TFA on markers for hemostatic
function. Almendingen et al. (1996) reported that a diet rich in partially hydrogenated soybean oil
unfavorably affected plasminogen activator inhibitor type 1 (PAI-1) antigen and PAI-1 activity as com-
pared with a diet high in butter fat. PAI-1 is part of the fibrinolytic system, which breaks down clots.
However, other markers related to platelet activation (β-thromboglobulin), the coagulation cascade
(factor VIIc, fibrinopeptide A), and the fibrinolytic system (D-dimers, tissue plasminogen activator)
were not affected. Mutanen and Aro (1997) concluded that stearic acid and iTFA had similar effects on
markers of coagulation and fibrinolysis. Collagen-induced platelet aggregation, however, was reduced
in the diet rich in TFA, as compared with the diet rich in stearic acid. Other parameters related to
platelet function or to endothelial function did not change. Finally, Louheranta et al. (1999) reported
that oleic acid and iTFA have comparable effects on factor VII, a coagulation factor. These three stud-
ies were carried out in the fasted state. Effects of a single meal rich in elaidic acid on activated FVII
concentrations (FVIIa) during the postprandial phase were studied by Sanders et al. (2000). However,
no evidence was found that the meal rich in elaidic acid had a different impact on postprandial changes
in FVIIa compared with meals enriched with oleic acid, stearic acid, or medium-chain triglycerides. In
a later study, coagulant and fibrinolytic markers were also not different during the fasting and postpran-
dial phase, when subjects had consumed a diet rich in iTFA or oleic acid for 2 weeks (Sanders et al.
2003). For rTFA, no difference in effects on FVIIc and PAI-1 was found between butter rich in rTFA
and regular butter (Tholstrup et al. 2006). Gebauer et al. (2015), however, reported that rTFA lowered
fibrinogen concentrations as compared with iTFA and stearic acid, while no effects on factor VII were
observed. Overall, these intervention studies do not provide clear evidence that iTFA or rTFA have an
adverse effect on hemostatic function as compared with other fatty acids.
Vascular Function
Many markers exist to investigate the different aspects of the vasculature. One frequently used
method is flow-mediated vasodilation (FMD) of the brachial artery, which is considered as the
noninvasive gold standard technique to measure vascular endothelial function. Brachial FMD is
inversely associated with future cardiovascular events (Ras et al. 2012). Plasma biomarkers are also
used to assess vascular function such as the soluble forms of vascular cell adhesion molecule 1,
intercellular adhesion molecule 1, and endothelial selectin (sE-selectin). Increased concentrations of
these molecules, which are involved in the binding of white blood cells to the vascular endothelium,
are positively related to future cardiovascular events (Blankenberg, Barbaux, and Tiret 2003).
In one of the few studies on the effects of iTFA on FMD, de Roos, Bots, and Katan (2001) reported
impaired fasting FMD as compared with an isocaloric amount of saturated fatty acids. In contrast, no
postprandial impairment in FMD was found after the intake of a meal rich in iTFA (de Roos et al. 2002).
Five weeks of iTFA consumption increased plasma sE-selectin levels as compared with saturated and
cis-monounsaturated fatty acids (Baer et al. 2004). These detrimental effects of iTFA on sE-selectin,
however, were not confirmed by Smit et al. (2011). In the study of Gebauer et al. (2015), sE-selectin
concentrations were comparable after the consumption of diets rich in iTFA, rTFA, or stearic acid.
Inflammation
Inflammatory signals play a major role in all stages of atherosclerosis and evoke many other responses.
Vascular inflammation, for example, stimulates the generation of vascular cell adhesion molecules. In
addition, the inflammatory network is complex. Frequently measured markers used to measure low-
grade systemic inflammation include TNFα, interleukin-6 (IL-6), and C-reactive protein (CRP). Baer
et al. (2004) have reported that a diet high in iTFA significantly increased IL-6 and CRP concentrations
as compared with oleic acid. These effects were not specific for iTFA as diets rich in stearic acid and
a mixture of saturated fatty acids had similar effects on IL-6. Surprisingly, when part of the iTFA was
replaced by stearic acid (C18:0), effects were no longer evident. Smit et al. (2011), however, found no
effects of a diet rich in iTFA (7.3% of energy) on various inflammatory markers—including IL-6 and
CRP—as compared with a control diet rich in oleic acid. Other studies also reported no effect of either
iTFA or rTFA on plasma CRP or IL-6 concentrations (Gebauer et al. 2015, Lichtenstein et al. 2006,
Motard-Bélanger et al. 2008, Tardy et al. 2009, Tholstrup et al. 2006, Werner et al. 2013).
TFA AND CARDIOVASCULAR DISEASE

In addition to effects on cardiovascular risk markers, the relationship between TFA intake and CVD
itself has also been studied. It is clear that, for evident reasons, no randomized controlled trials have
been carried out in this field and one has to rely on prospective cohort studies. In these studies, TFA
intake has been assessed in two ways. First, TFA consumption was estimated using recorded food
intake using, for example, food frequency questionnaires or food records. One disadvantage of this
approach is that it is difficult to obtain precise estimates of nutrient intakes, in particular, at the indi-
vidual level. For example, the composition of food products between different batches and different
brands can be highly variable, fatty acid composition in nutrient databases are not complete, and
people do not remember accurately what—and how much—was actually consumed. Such sources
of errors may attenuate possible relationships between TFA intake and CVD risk. For the second
approach, TFA levels can be assessed in plasma or fat tissue. As the majority of TFA in the human
body are derived from dietary sources, this biomarker approach may provide a good estimate for
TFA intakes over the past weeks or even years.
Chowdhury et al. (2014) identified five prospective cohort studies that examined the relationship
between TFA intake and coronary risk. These studies were from the United States and Europe, and
among the 155,270 participants, 4,662 coronary events were recorded. It was estimated that subjects
in the top tertile of TFA intake had a 16% higher risk for coronary events compared to those in the
bottom tertile of TFA intake (RR: 1.16, 95% CI: 1.06–1.27). In an earlier meta-analysis using data
from four prospective cohort studies, including 4,965 coronary events among 139,836 participants, it
was estimated that a 2% higher energy intake from TFA as an isocaloric replacement for carbohydrate
was associated with a RR of 1.23 (95% CI: 1.11–1.37) (Mozaffarian and Clarke 2009). In another
meta-analysis, Bendsen et al. (2011) estimated the effects of total TFA, iTFA, and rTFA on total CHD.
For total TFA (6 cohorts; 186,531 participants; 6,135 cases for total CHD), the RR was 1.22 (95% CI:
1.08–1.38). Similar calculations were done for iTFA (3 cohorts; 91,778 participants; 1,089 cases). The
RR for CHD events was 1.21 (95% CI: 0.97–1.50). For rTFA (4 cohorts; 95,464 participants; 1,463
cases), the RR was 0.92 (95% CI: 0.76–1.11). These findings, however, do not prove that the source of
TFA is important for CHD risk. First of all, the RR estimates for iTFA and rTFA were not significantly
different from one and overlapped. Secondly, at the time the study was performed, the intake of iTFA
and its range was much larger than that of rTFA, which may have affected estimates. A comparison of
the RR at similar ranges of intakes would therefore be very informative. In fact, Weggemans, Rudrum,
and Trautwein (2004) concluded that no differences in the risk of CHD were evident between total,
ruminant, and industrial TFA when the intake is below 2.5 g per day.
In the meta-analyses discussed earlier, the focus was on the relationship between the intakes of
TFA with 18 carbon atoms and one single double bond. However, as discussed earlier, diets also
provide minor amounts of other TFA. These intakes are even more difficult to estimate. Therefore,
the most reliable estimate of intakes due to the low levels in the diet is to estimate levels in tissue
lipid fractions. Still, it has to be acknowledged that TFA are metabolized in the human body and
one TFA molecule can be converted into another TFA molecule. Vaccenic acid, for example, can be
desaturated by a Δ9-desaturase (stearoyl-CoA desaturase-1), giving rise to the formation of a spe-
cific CLA isomer. Relationships between TFA in plasma lipids on CVD risk have been examined in
several prospective cohort studies. In the Multi-Ethnic Study of Atherosclerosis cohort of 2837 U.S.
adults, the proportion of trans-palmitoleic acid (trans-C16:1n-7) in the plasma phospholipid frac-
tion was not related to incident CVD or CHD (de Oliveira Otto et al. 2013). In the Cardiovascular
Health Study, a prospective cohort of older U.S. citizens, trans-isomers of linoleic acid in plasma
phospholipids with two double bonds in the trans configuration were adversely related with total
mortality, especially with CVD (Wang et al. 2014). The study cohort consisted of 2742 elderly indi-
viduals. No associations were observed for trans-monounsaturated fatty acids with 16 or 18 carbon
atoms. Thus, these studies suggested that differences between the various TFA might exist, although
further investigation and confirmation of these findings is needed.
INTAKES AND RECOMMENDATIONS

A very detailed report on worldwide fatty acid intake was published a few years ago (Micha et al.
2014). It was estimated that in 2010, the mean global TFA intake was 1.4% of energy with the highest
intakes in Egypt (6.5% of energy) and the lowest intakes in the Caribbean region (0.2% of energy).
Only 12 of the 187 countries included had mean intakes below 0.5% of energy, while 12 countries had
intakes above 2% of energy. For the U.S. population, mean daily iTFA intake in 2006 was estimated to
be 1.3 g per person with a 90th percentile of 2.6 g. Assuming a daily energy intake of about 2000 kcal,
this would equal 0.6% and 1.2% of energy, respectively (Doell et al. 2012). Trends in TFA intakes
between the 1980–1982 and 2007–2009 periods have been reported for the Minnesota Heart Survey
(Honors et al. 2014). It was estimated that for women, intakes decreased gradually during this period
from 2.9% to 1.9% of energy and for men, from 2.7% to 1.7% of energy. Also, results among the
Framingham Heart Study participants showed that between the 1991–1995 and 2005–2008 periods,
TFA intake had decreased from 1.5% to 1.2% of energy (Vadiveloo et al. 2014).
Various governmental agencies have issued recommendations for the intake of TFA. Although
different wordings are used, the general consensus is that intakes should be as low as possible. If an
upper limit is formulated, it is frequently mentioned that intakes should not exceed 1% of energy,
as a zero-TFA diet is virtually impossible, due to the presence of naturally occurring TFA. Despite
decreases in iTFA intake, also due to the reformulation of fats used for food formulations, it is
evident that a large part of the global population still consumes levels above recommended intakes.
CONCLUSION
Prospective epidemiological cohort studies consistently support the finding that iTFA intake is asso-
ciated with an increased risk of CHD. Evidence from many controlled human intervention studies
suggests that these effects are mediated, at least in part, through adverse effects on the serum lipo-
protein profile. Intakes show a linear-dose response with serum LDL-cholesterol, demonstrating
that effects are proportional to the amounts of iTFA consumed. Elevated LDL-cholesterol has been
causally linked to CHD. Although a protective causal role for HDL in CHD has not been estab-
lished, iTFA also dose-dependently decrease HDL-cholesterol concentrations as compared with
other fatty acids. Diets rich in iTFA also increase fasting triacylglycerol concentrations, which are
also positively related to CHD risk. Therefore, there is a solid, evidence-based basis to minimize the
use of iTFA and to keep intakes as low as possible. Finally, there is evidence indicating that iTFA
increase Lp(a), especially in people with elevated concentrations, but the significance of this finding
for CVD risk is unclear. At least for LDL-cholesterol, effects of iTFA and rTFA are comparable.
Relationships between the intakes TFA with CVD risk are less clear, also due to lower intakes of
rTFA than of iTFA at the time the studies were performed. However, while differences in effects
of iTFA and rTFA are of interest scientifically, this may not be a critical public health issue as long as
intakes of rTFA remain at current low levels.
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Section III
Dietary Carbohydrates and
13 Epidemiologic and
Mechanistic Studies of
Sucrose and Fructose
in Beverages and Their
Relation to Obesity and
Cardiovascular Risk
George A. Bray
CONTENTS
Introduction.....................................................................................................................................238
Effects Attributable to the Energy in Beverages.............................................................................239
Epidemiology.........................................................................................................................239
Clinical Studies and Randomized Clinical Trials..................................................................240
Mechanisms for Weight Gain Associated with Soft Drinks..................................................241
Cardiometabolic Effects Attributable to the Sucrose (Sugar) and Fructose in Beverages..............242
Epidemiology.........................................................................................................................242
Mechanisms for the Effects of Sucrose and Fructose............................................................244
Schematic Model of the Relation of Sugar, Caffeine, and Fructose to Cardiometabolic Disorders.....245
Conclusions and Recommendations...............................................................................................246
References.......................................................................................................................................246
ABSTRACT
Consumption of calorie-sweetened beverages has continued to increase and plays a role in the epi-
demic of obesity, the metabolic syndrome, and fatty liver disease. Data from the Global Burden of
Disease Study, NHANES, and USDA dietary surveys are used to understand changes in sugar and
fructose consumption in beverages. Meta-analyses, randomized clinical trials, and clinical studies
were used to evaluate outcomes of beverage and fructose intake. About 75% of all foods and bever-
ages contain added sugar in a large array of forms. Consumption of soft drinks has increased five-
fold since 1950. Meta-analyses suggest that consumption of sugar-sweetened beverages is related
to the risk of obesity, diabetes, cardiovascular risk factors, and the metabolic syndrome. Drinking
two 16 ounce sugar-sweetened beverages per day for 6 months induced features of the metabolic
syndrome and fatty liver in healthy human beings. Fructose increases blood pressure and serum
lipids. Randomized, controlled trials in children and adults lasting 6 months to 2 years have shown
that replacing the intake of sugar-sweetened soft drinks with low-energy drinks reduces weight gain
or induces weight loss. Recent studies suggest a gene-SSB relationship in modulating weight gain.
237
INTRODUCTION
The rapid and steady increase of sugar-sweetened beverage intake of the past 50 years of the
twentieth century appears to have declined recently, but overall the level of intake is still very high
(Ng et al. 2012; Ford and Dietz 2013; Kit et al. 2013; Ludwig 2013). This chapter will argue that
the amount of sugar (sucrose), and the fructose which it contains, poses a significant health risk for
many Americans and that something needs to be done about it (Nestle 2015). A major source of
sucrose in our diet is in the beverages we drink, and this will be a principal focus of this chapter.
These sugar-sweetened beverages have several components. The first is the “energy” that is pro-
vided when the sucrose (or high fructose corn syrup, HFCS) is metabolized to glucose and fructose
and then to water and carbon dioxide in the body. The second is the fructose moiety of the sucrose
molecule (or HFCS), which seems to have detrimental effects independent of the energy that it pro-
vides. The third component of many beverages is caffeine, which heightens alertness and may be
mildly addictive, leading to increased consumption of sugar-sweetened beverages. Finally, there are
the other components of SSB including flavorings, sodium, potassium, and other additives that may
have metabolic and/or clinical effects.
Sugar is sweet, and the fructose it contains even sweeter. There is an innate human desire
for sweetness. If sugar, HFCS, and fructose were not sweet there would be little or no debate
about their risk because their consumption would undoubtedly be less. A preference for sweet
taste is present in newborn infants and increases in intensity throughout childhood. It may even
be that the “craving” for sweets can be enhanced further by early exposure to intense sweeten-
ers. Human beings have consumed almost all of the sugar that has been produced, which drives
increasing production. At the time of the American Revolution in 1776, Americans consumed
about 4 pounds of sugar per person each year. By 1850, this had risen to 20 pounds per capita
and by 1994 to 120 pounds each year (Johnson et al. 2007). The food industry has used sugar as
a major sweetener for delivery of increasing amounts of beverages and food over the past half
century (Moss 2013). The result has been that the consumption of sugar-sweetened beverages
rose by a startling 38.5 gallons per person between 1950 and 2000 (10.8 gallons per person
in 1950 to 49.3 gallons per person in 2000) (Putnam and Allshouse 2003a,b). We have seen
small declines since then. Between 2001–2002 and 2009–2010, total daily beverage consump-
tion (excluding water) decreased from 24.4% to 21.1% of energy intake. Significant decreases
occurred in sugar-sweetened sodas (13.5%–10.2% energy), whole milk (2.7%–1.6% energy),
fruit juices with sugar added (2.3%–2.1% energy), and fruit-flavored drinks (1.6%–0.8%
energy). Significant increases occurred for sweetened coffees/teas, energy drinks, sport drinks,
and unsweetened juices though the contribution of each to total energy intake remained <1%
(Nielsen et al. 2002). Low-/no-calorie drink consumption also increased, rising from 0.2 to
1.3 oz/d (Mesirow and Welsh 2015).
Food manufacturers continue to find new ways to increase sugar consumption by constantly
manufacturing new food and beverage products, be they in fruit juice, energy drinks, vitamin waters,
protein waters, or sports drinks (Moss 2013). This chapter concludes that sugar and related caloric
sweeteners provide nutritionally invisible calories that in the amounts now consumed pose a health
risk to many Americans (Bray 2013).
My research interest in the role of sugar and fructose in human health was stimulated by a
quotation from John Yudkin, professor of nutrition at Queen’s college, London: “If then there is
reason to be concerned about a dietary cause of a widespread disease [obesity], one should look
for some constituent of man’s diet that has been introduced recently or has increased considerably,
recently” (Yudkin 1972). There is clear evidence that dietary factors are driving weight up, but
genetic variation also plays a key role. Some genes such as the OB gene and the MC4R gene have
a major effect on obesity (Loos 2012), while others contribute only a small amount individually
but collectively provide the background for individual responses to diet (Speliotes et al. 2010;
Locke et al. 2015). A gene–environment interaction has been demonstrated for soft drinks and
Epidemiologic and Mechanistic Studies of Sucrose and Fructose in Beverages 239
weight gain (Qi et al. 2012). We might state the relation of diet and genes this way: “Genetic
variability loads the gun; the diet and environment pull the trigger” (Bray 2015).
A key paper about fructose and health was published in 2004 showing a correlation between
rising consumption of HFCS and obesity just as Yudkin had predicted (Bray et al. 2004). Although
correlation does not prove causation, the data presaged an increasing body of scholarly work that
has highlighted the important relation between sucrose and HFCS (and the fructose they contain)
and the risk of obesity and several diseases. The decade of the 1980s began quietly enough for
obesity research and for the sugar industry. The prevalence of obesity, though rising slowly through
the first half of the twentieth century, was still only 14% in 1960–1962 (Ogden et al. 2007, 2014).
Sugar had received a relatively clean bill of health from the National Academy of Science in the
Diet and Health Report—its only health problem according to this authoritative book was its role
in dental caries (National Research Council 1989). The metabolic syndrome was yet to be clearly
defined (Reaven 1988); we did not know that calories in sweetened beverages were nutritionally
invisible and were not adequately compensated by a reduction in food intake. Finally, the concept
of non-alcoholic fatty liver disease was only beginning to emerge (Abdelmalek et al. 2010; Vernon
et al. 2011); up to that time it was “alcohol” that was the major nutritional factor producing liver
disease. This was the calm before the storm. What a difference 25 years can make! As I look back
it is clear that the increasing consumption of sugar of HFCS and the fructose that they both contain
has dramatically increased the health risks for many people (Bray et al. 2004; Bray 2013; Bray and
Popkin 2013). This chapter focuses on this issue.
In the 1960s and 1970s, sugar from beverages represented only a third of our total added sugar
intake, but this rose to two-thirds of our sugar intake by the year 2000 and has subsequently been
declining to about 40% of total added sugar intake (Duffey and Popkin 2008; Slining and Popkin
2013). Finally after a long period of refusing to put an upper limit on sugar intake, the 2015 Dietary
Guidelines Advisory Committee has recommended a 10% upper level. This is the level recommended
by the American Heart Association (AHA) and the Food and Agriculture Organization (FAO).
EFFECTS ATTRIBUTABLE TO THE ENERGY IN BEVERAGES

Epidemiology
Several meta-analyses have shown a strong relationship between sugar-sweetened beverages and obe-
sity. One of the earliest of these was by Vartanian et al. (2007). This meta-analysis showed a clear
relationship between beverage intake and obesity and weight gain, which was largely eliminated with
adjustment for energy intake. That is, the effect of the beverages could be largely attributed to the extra
sugar energy that they provided. The results of their meta-analysis are summarized in Table 13.1.
Other meta-analyses followed, almost all of which showed a relationship between sugar-sweet-
ened beverage consumption and obesity. Another meta-analysis by Olesen and Heitman (2009)
included 14 prospective and 5 experimental studies, which found a positive association between
intake of calorically sweetened beverages and obesity. Three experimental studies also found posi-
tive effects of calorically sweetened beverages and changes in body fat, but two did not find these
effects; none were negative. Eight prospective studies adjusted for energy intake and seven of these
found essentially the same associations. Olesen and Heitman concluded that a high intake of calori-
cally sweetened beverages can be regarded as a determinant for obesity. Other reviews and meta-
analyses have reached generally similar conclusions (Malik et al. 2006, 2010a,b, 2013; TeMorenga
et al. 2012, 2014). In addition to increasing the risk of obesity, sugar and sugar-sweetened beverages
enhance the risk of diabetes (Malik et al. 2010a) and cardiovascular disease (Yang et al. 2014).
In contrast to this consistent body of data, a recent meta-analysis concluded that the relationship
between sweetened beverage intake and obesity was incorrect since adjustment for energy intake
and physical activity removed the effect (Trumbo and Rivers 2014). If it is the “energy” in the bev-
erage that is the culprit, their conclusion could not have been otherwise because they eliminated
TABLE 13.1
Effect Size for Energy Intake and Body Weight in the Meta-Analysis
by Vartanian et al. (2007)
Research Design Energy Intake Body Weight
Cross-sectional studies 0.13 0.06
(0.12, 0.14) (0.03, 0.08)
Longitudinal studies 0.24 0.03
(0.23, 0.26 (0.00, 0.06)
Short experimental studies 0.24 0.24
(0.16, 0.31) (0.18, 0.28)
Overall effects 0.16 0.06
(0.15, 0.16) (0.05, 0.08)
Data are effect size and 95% CI for the effect size.
“calories” by their adjustment for this central factor in the relationship (Trumbo and Rivers 2014).
This study was supported by industry, and as Bes-Rastrollo et al. (2013) point out nearly 90% of
papers supported by industry that studied the relation of beverages to obesity found no relationship
whereas nearly 90% of those with independent funding did find this relationship. The importance
of calorie intake, but not physical activity, on the effect of sugar-sweetened beverages on body was
shown by Tucker et al. (2015). In this 4-year study of 170 non-smoking women, 4-year weight
gain was significantly greater (2.7 kg) in the women drinking sugar-sweetened beverages than in
the women who consumed artificially sweetened beverages (−0.1 kg) or no soft drinks (0.5 kg).
Adjusting for energy intake weakened the effect as one would expect if these invisible “calories”
were the driving force. Adjusting for physical activity had no effect on risk (Tucker et al. 2015).
Clinical Studies and Randomized Clinical Trials

Clinical studies have also shown that over a period of 10 weeks, subjects drinking a set amount
of sugar-sweetened beverages gained weight compared to subjects drinking a comparable amount
of artificially sweetened beverages who lost weight. In this trial, 41 overweight men and women
entered a 10-week parallel arm study. One group (n = 21) received 3.4 MJ (813 kcal) of sugar-
containing beverages and the other group (n = 20) received artificially sweetened beverages contain-
ing about 1 MJ (240 kcal) and no sugar. After 10 weeks, energy intake had increased by 1.6 MJ/d
(382 kcal/d) and sucrose to 28% of energy intake. Protein and fat intake declined in the sugar
group. Body weight and fat mass increased by 1.6 and 1.3 kg, respectively, in the sugar group and
decreased by 1.0 and 0.3 kg in the group drinking aspartame-sweetened beverages. Blood pressure
increased by 3.8/4.1 mmHg in the sugar-consuming group (Raben et al. 2002). Concentrations of
several inflammatory markers were also changed. Haptoglobin rose by 13%, transferrin by 5%, and
C-reactive protein by 6% in the sucrose group, but fell by 16%, 2%, and 26%, respectively, in the
aspartame-sweetened beverage group (Sørensen et al. 2005).
In a study by Maersk et al. (2012), 47 overweight men and women completed a 6-month trial.
Participants were randomized to receive 1 of 4 treatments: 1 liter/d of sugar-sweetened cola (≈ two
16 oz beverages), 1 liter/d of diet cola, 1 liter/d of milk, or 1 liter/d of water. Carbohydrate was
100 g/d from cola (1/2 fructose) and 47 g/d from milk (no fructose). During this 6-month period,
visceral, liver, and muscle fat increased in those drinking the sugar-sweetened cola beverage even
though total fat and subcutaneous fat did not. Systolic blood pressure, plasma triglycerides, and
total cholesterol were also higher in those drinking the sugar-sweetened cola beverage than in those
receiving one of the other beverages.
A series of randomized, controlled trials in children and adults lasting 6 months to 2 years have
shown that weight gain is slowed by replacing sugar-sweetened beverages with alternative bever-
ages. The two most noteworthy were done by a group from Boston (Ebbeling et al. 2012) and by a
group from Amsterdam (de Ruyter et al. 2012). After 1 year, the group drinking artificially sweet-
ened beverages in the Boston study gained significantly less weight than the group receiving the
sugar-sweetened beverages. The Amsterdam study went further and provided either 250 mL of an
artificially sweetened beverage or a sugar-containing beverage providing 104 kcal to 641 youth over
an 18-month period. The BMI, weight, and skinfold-thickness and fat mass increased significantly
less in the low-caloric beverage group.
In a post hoc analysis of the Amsterdam study, the authors examined the predicted weight change
from computer models with the actual changes to calculate the energy deficit. They found that the
heavier children (the upper half of the group) in terms of weight category failed to compensate
adequately. That is, they ate more calories than predicted for their age, sex, and size. These “invis-
ible” calories are a major concern for the problem of weight gain with sugar-sweetened beverages.
Mechanisms for Weight Gain Associated with Soft Drinks

One of the detrimental consequences of ingesting the nutritionally invisible calories in beverages
is that the mechanisms that regulate caloric intake do not recognize them, and they thus become
add-on calories (Rolls et al. 1990; Mattes 1996, 2006). Beverages do not suppress the intake of
other food calories to an appropriate degree to prevent weight gain. Thus, beverage calories can be
viewed as “add-on” calories enhancing the risk of obesity. The ground-breaking works of Mattes
and his associates and Rolls and her colleagues have led to dozens of replications highlighting
this relationship (Rolls et al. 1990; DiMeglio and Mattes 2000; DellaValle et al. 2005; Flood
et al. 2006; Mattes 2006; Mourao et al. 2007). A recent meta-analysis confirmed the relationship
between intake of low-energy beverages versus sugar-containing beverages on energy intake and
body weight (Rogers et al. 2016). Rogers et al. (2016) examined both animal and human studies
where a low-energy substitute was compared to a control that included sugar-containing beverages
or water. In the 129 human studies of short-term interventions, energy intake from a preload +
ad-libitum meal was significantly lower with the low-energy drink than a sugar-sweetened drink.
Among 10 sustained intervention studies, the absolute value for total or change in energy intake
was lower with the low-energy beverage than a sugar-sweetened beverage. Finally, in studies last-
ing more than 4 weeks (12 comparisons), difference in weight loss or weight gain favored the
low-energy drink by 1.41 kg in adults and 1.04 kg in children, comparing the low-energy drink to
a sugar-sweetened one. They conclude that both single-meal and longer-term studies consistently
showed a reduction in energy intake and/or body weight in the low energy intake group compared
to the group with sugar-sweetened beverages (Rogers et al. 2016).
Studies of the brain response to fructose and glucose have added to our understanding of the
problem (Page et al. 2013). A study of adolescents who were either lean or obese showed important
differences in the brain’s response to oral fructose or glucose. In the adolescents who were lean, both
glucose and fructose increased perfusion of brain areas involved in “executive function and control”
(prefrontal cortex) but did not activate the “homeostatic” appetite control areas (hypothalamus)
(Jastreboff et al. 2016). A very different picture was seen in the adolescents with obesity where
ingestion of either fructose or glucose reduced perfusion of the executive region of the brain (pre-
frontal cortex) and increased activity in the “reward” or “pleasure” centers. This suggests that obese
adolescents may lack the ability to downregulate the hedonic and homeostatic regions of the brain
after oral ingestion of fructose or glucose. In addition, the ingestion of fructose produced a greater
increase in perfusion of the pleasure or reward centers in the adolescents with obesity—something
not seen in the lean adolescents. The authors speculate that the reduced response of the executive
centers to fructose/glucose may reduce their ability to control intake of sugar-sweetened beverages
(Jastreboff et al. 2016; Bray 2016).
CARDIOMETABOLIC EFFECTS ATTRIBUTABLE TO THE

SUCROSE (SUGAR) AND FRUCTOSE IN BEVERAGES
Epidemiology
In addition to the data consistently showing that the energy intake of sugar-sweetened beverages can
increase body fatness, there is a body of studies showing that these same beverages have detrimental
effects on cardiometabolic risk (Malik et al. 2010). A recent meta-analysis showed that adding fruc-
tose to the diet in controlled studies produced weight gain unless calories were reduced somewhere
else in the diet, that is, a hypercaloric diet produced by adding fructose produced weight gain as one
would expect. However, when the fructose was substituted for starch isocalorically, there was no
weight gain as one would anticipate (Ng et al. 2012; Sievenpiper et al. 2012). In addition, this group
showed that substituting fructose for starch in the diet did not appear to have the detrimental effects
noted above for sugar-sweetened beverages. Addition of fructose to the diet or in foods independent
of its presence in sugar or HFCS represents only a small amount of fructose. By focusing only on
studies with fructose replacing other carbohydrate, the authors appear to me to have overlooked the
importance of the invisible calories from sugar that are provided by sugar-sweetened beverages.
In an analysis of worldwide burden of diseases related to consumption of sugar-sweetened bever-
ages, Singh et al. (2015) estimated that there were 184,000 deaths/y attributable to the consumption
of sugar-sweetened beverages, another 133,000 attributable to diabetes mellitus, 45,000 to cardio-
vascular disease, and 6,450 to cancers.
Malik et al. evaluated sugar-sweetened beverage intake and body weight and cardiometabolic
disease in a meta-analysis of 8 prospective cohort studies of weight gain and 10 prospective cohort
studies of cardiometabolic disease risk. They showed a clear and consistent positive association
between sugar-sweetened beverage intake and weight gain. Among the 294,617 participants in
the meta-analysis, the highest level of intake had a 24% greater risk of cardiometabolic diseases
than those in the lowest group. They concluded that higher consumption of calorically sweetened
beverages is associated with both the risk of weight gain and the risk of cardiometabolic diseases
(Malik et al. 2010a).
Data from the Health Professionals Follow-up study have added to evidence for the detrimen-
tal effects of beverages on risk for CVD. This cohort provides a prospective group for analyzing
the 3683 CHD cases over 22 years of follow-up in 42,883 men. Participants in the top quartile of
sugar-sweetened beverage intake had a 20% higher relative risk of CHD than those in the bottom
quartile after adjustment for age, smoking, physical activity, alcohol, multivitamins, family history,
diet quality, energy intake, body mass index, preenrollment weight change, and dieting. Artificially
sweetened beverage consumption was not significantly associated with CHD. Adjustment for self-
reported high cholesterol, high triglycerides, high blood pressure, and diagnosed type 2 diabetes
mellitus slightly attenuated these associations. Intake of sugar-sweetened but not artificially sweet-
ened beverages was significantly associated with increased plasma triglycerides, C-reactive protein,
interleukin-6, and tumor necrosis factor receptors 1 and 2 and decreased high-density lipoprotein
cholesterol, lipoprotein(a), and leptin (P < 0.02) (de Koning et al. 2012).
Data from the National Center for Health Statistics has also been used to examine the trends
in sugar consumption and its impact on cardiovascular mortality. Among US adults, the adjusted
mean percentage of daily calories from added sugar increased from 15.7% in 1988–1994 to 16.8%
(P = 0.02) in 1999–2004 and decreased to 14.9% in 2005–2010. Most adults consumed 10% or
more of calories from added sugar (71.4%), and approximately 10% of adults consumed 25% or
more in 2005–2010. This rise and then stabilization or decrease is consistent with the observations
of Mesirow and Welsh noted elsewhere (2015). During 14.6 years of follow-up, 831 CVD deaths
were identified during 163,039 person-years. The adjusted hazard ratios (HRs) for CVD mortality
across quintiles of the percentage of daily calories consumed from added sugar were 1.00, 1.09,
1.23, 1.49, and 2.43 (P < 0.001), respectively (Yang et al. 2014). Adjusted HRs were 1.30 and
2.75 (P = 0.004), respectively, comparing participants who consumed 10.0%–24.9% or 25.0% or

more calories from added sugar with those who consumed less than 10.0% of calories from added
sugar. These findings were largely consistent across age groups, sex, race/ethnicity (except among
non-Hispanic blacks), educational attainment, physical activity, health eating index, and body mass
index (Yang et al. 2014).
Sweetened beverage consumption was significantly positively associated with risk of total stroke
and cerebral infarction but not with hemorrhagic stroke (Larsson et al. 2014). The multivariable RRs
comparing ≥2 (median: 2.1) servings/d (200 mL/serving) with 0.1 to <0.5 (median: 0.3) servings/d
were 1.19 for total stroke and 1.22 for cerebral infarction. These findings suggest that sweetened
beverage consumption is positively associated with the risk of stroke (Larsson et al. 2014).
In addition to cardiovascular disease, consumption of sugar and sugar-sweetened beverages
increases the incidence of diabetes mellitus (Bazzano et al.2008). In a meta-analysis of 17 cohorts
(38,253 cases/10,126,754 person-years), higher consumption of sugar-sweetened beverages was
associated with a greater incidence of type 2 diabetes (Imamura et al. 2015). An increase of one
serving per day increased the risk of diabetes by 18% (95% confidence interval 9%–28%, and 13%
after adjustment for adiposity) and for artificially sweetened beverages, 25% and 8% (respectively)
and for fruit juice, 5% and 7% (respectively). Potential sources of heterogeneity or bias were not evi-
dent for sugar-sweetened beverages. However, for artificially sweetened beverages, publication bias
and residual confounding were indicated. For fruit juice, the finding was nonsignificant in studies
ascertaining type 2 diabetes objectively (P for heterogeneity = 0.008). Under specified assumptions
for population attributable fraction, 20.9 million events of type 2 diabetes were predicted to occur
over 10 years in the USA (absolute event rate 11.0%), 1.8 million would be attributable to consump-
tion of sugar-sweetened beverages (population attributable fraction 8.7%), and of 2.6 million events
in the UK (absolute event rate 5.8%), 79,000 would be attributable to consumption of sugar-sweet-
ened beverages (population attributable fraction 3.6%). Imamura et al. (2015) conclude that habitual
consumption of sugar-sweetened beverages was associated with a greater incidence of type 2 dia-
betes, independently of adiposity measured as BMI. Although artificially sweetened beverages and
fruit juice also showed positive associations with incidence of type 2 diabetes, the findings were
likely to involve publication bias and residual confounding. Nonetheless, both artificially sweetened
beverages and fruit juice are unlikely to be healthy alternatives to sugar-sweetened beverages for the
prevention of type 2 diabetes (Imamura et al. 2015).
Although the intake of sugar-sweetened soft drinks has been reported to be associated with an
increased risk of type 2 diabetes, it is unclear whether this is because of the sugar content or related
lifestyle factors, whether similar associations hold for artificially sweetened soft drinks, and how
these associations are related to BMI. Greenwood et al. (2014) conducted a systematic literature
review and dose-response meta-analysis of evidence from prospective cohorts to explore these
issues. They found 11 publications on 9 cohorts. Consumption values were converted to mL/d, per-
mitting the exploration of linear and nonlinear dose-response trends. The summary relative risks
for sugar-sweetened beverages were 1.20/330 mL per d (P = X), and 1.13/330 mL per d (P = 0.02)
for artificially sweetened soft drinks. The association with sugar-sweetened soft drinks was slightly
lower in studies adjusting for BMI, consistent with BMI being involved in the causal pathway.
There was no evidence of effect modification, though both these comparisons lacked statistical
power. Heterogeneity between studies was high. The included studies were observational, so their
results should be interpreted cautiously, but findings indicate a positive association between sugar-
sweetened soft drink intake and type 2 diabetes risk, attenuated by adjustment for BMI. The trend
was less consistent for artificially sweetened soft drinks.
Finally, fructose intake clearly influences the prevalence of gout. The prevalence of gout
was studied in the 49,166 men from the Health Professionals Follow-up Study using the Food
Frequency Measures of beverage intake and the incidence of gout. The men in the top quintile
of fructose intake had an approximately 80% increased risk compared to men is the lowest quin-
tile of fructose intake (Choi and Curran 2008).
Mechanisms for the Effects of Sucrose and Fructose

Sucrose and fructose increase blood pressure in acute and longer-term studies. Acute ingestion of
fructose orally increased blood pressure in healthy volunteers compared to the same quantity of
glucose or saline. Fifteen healthy men drank 500 mL volumes of water (placebo) alone or with 60 g
of fructose or glucose on three occasions and blood pressure, metabolic rate, and autonomic nervous
system activity were measured for 2 hours (Brown et al. 2008). Fructose, but not water or glucose,
increased diastolic blood pressure by 4 mmHg within 20 minutes.
In longer-term studies, Raben et al. (2002) showed that weight gain over 10 weeks in individu-
als drinking sugar-sweetened beverages was associated with increased blood pressure, but not in
the group drinking the artificially sweetened beverages who actually lost weight. An increase in
blood pressure was also evident in the study by Maersk et al. (2012) where those drinking the
sugar-sweetened cola beverage for 6 months had an increase in systolic blood pressure without any
significant change in body fat or body weight.
Sucrose and fructose also affect lipid metabolism (Teff et al. 2009). In an early clinical study compar-
ing the effect of 50 g of glucose, 50 g of fructose, and 100 g of sucrose on plasma triglycerides, Cohen
and Schall (1988) found that both fructose in the amount found in sucrose and sucrose increased plasma
triglycerides following a meal, but that glucose did not, leading them to conclude that the rise in triglyc-
erides was due to the fructose either alone or as part of sucrose (table sugar), and not glucose.
A 10-week study comparing beverages providing 25% of calories from fructose with a bever-
age providing 25% of calories from glucose showed that fructose increased postprandial triglyc-
erides, particularly at night. This study also showed that de novo fat synthesis was increased in
those consuming fructose-containing drinks. Most importantly, visceral fat, a depot which has
the strongest association with cardiovascular risks, increased with only 10 weeks of drinking a
fructose beverage compared to the glucose beverage (Stanhope and Havel 2009; Stanhope et al.
2011; Stanhope 2012). In this study, 32 men and women were randomly assigned to either 25%
of calories as fructose in beverages or glucose in beverages for 10 weeks along with ad-lib intake
of a weight maintenance diet. Subjects were studied before and after ingesting their respective
beverages (Stanhope and Havel 2009). Triglycerides at night were significantly higher in those
receiving the beverages with fructose than glucose.
In a somewhat-longer clinical study, the daily intake of 1 liter per day (approximately two 16 oz
servings) of cola, diet cola, milk, or water were compared in parallel study groups. The sugar-
sweetened cola consumed for 6 months increased liver fat, visceral fat, muscle fat, and plasma tri-
glycerides compared to the other beverages (Maersk et al. 2012). From a public health perspective
it is concerning that drinking as little as two sugar-sweetened beverages per day for 6 months can
increase risk of fatty liver and induce features of the metabolic syndrome. These studies certainly
need to be repeated, but if replicated, the public should be warned about the hazards of drinking
sugar-sweetened beverages in much the same way as the Food and Drug Administration warns
people about risk of taking medications.
Fructose also has interesting effects at the cellular level by inducing inflammatory markers
in human kidney cells (Cirillo et al. 2009). Incubation of HK-2 (human kidney cells) with fruc-
tose increases monocyte chemotactic protein-1. Knock-down of ketohexokinase, the enzyme that
phosphorylates fructose, blocks this effect as do antioxidants. Fructose also increased intracel-
lular uric acid (Johnson et al. 2010).
Fructose is a sweet-tasting sugar that is found naturally in fruits and some vegetables. In modest
amounts, it has been part of the human diet for eons (Wolf et al. 2008). It has the highest sweet-
ness taste of all natural components of sugar. It is the dramatic increase in its consumption over
the past 30 years that has led to concerns of its detrimental health effect (Bray 2013). It is not the
only caloric sweetener found in our food supply. As noted above, about 75% of all U.S. foods
and beverages contain added sugars (Ng et al. 2012). The large increase in added sugar intake
has led to a major increase in total fructose intake, an increase that has occurred since about 1980
(Bray et al. 2004; Duffey and Popkin 2008). While many health problems are linked with this increase
in fructose intake, fatty liver disease is one whose increase is noted in both the United States and
Europe and may be linked with the rising fructose intake (Dekker et al. 2010; Ouyang et al. 2008).
SCHEMATIC MODEL OF THE RELATION OF SUGAR, CAFFEINE,

AND FRUCTOSE TO CARDIOMETABOLIC DISORDERS
A schematic representation of the health consequences of the sucrose and fructose in soft drinks
is shown in Figure 13.1 (Bray and Popkin 2014). This figure pulls together the findings from the
studies described above into a single model. The high levels of consumption of sugar-sweetened
beverages and other sugary beverages (Duffey and Popkin 2008; Marriott et al. 2009; Popkin 2010;
Welsh et al. 2010; Bray 2013) is viewed as the driver for the increase in energy and fructose
intake, which play a role in the development of obesity and the metabolic consequences depicted
in Figure 13.1 (Bray 2013). The caffeine present in these beverages may serve as a positive feed-
back signal for continuing ingestion due to its ability to stimulate the central nervous system.
Interestingly, the FDA is currently reviewing use of caffeine, a drug which some consider to be
mildly addictive. Even 3 weeks of sugar-sweetened beverage ingestion was sufficient to alter lipid
metabolism by increasing low-density and decreasing high-density lipoprotein cholesterol, which
is a marker of increased CVD risk (Aeberli et al. 2011; Bray 2013). This and other research by
60 Beverage intake
50
Positive
Soft drink intake
(gallons/person)
40
feedback
30
20
10
Caffeine 0
1955 1975 1995
Years
Obesity Energy Sucrose/fructose
Liver
Hepatic Fat in Triglyceride

insulin liver cholesterol
resistance muscle/VAT
Metabolic Nonalcoholic
syndrome fatty liver Cardiometabolic
disease disease
Gout in men Uric acid
FIGURE 13.1 Model showing some potential consequences of increasing fructose and energy intake from
sugar or high fructose corn syrup in beverages.
Aeberli et al. (2013) provide insights into the unique role of fructose in initiating liver dysfunction
and possibly leading to non-alcoholic fatty liver disease and the metabolic syndrome, which have
become increasingly prevalent (Dekker et al. 2010; Bray 2013).
One key question that Aeberli et al. (2013) began to address is whether the detrimental effects of
fructose are simply the result of a linear dose-response to our increasing dietary intake of fructose, or
whether there is a threshold below which fructose is without harm. Stanhope et al. (2015) have addressed
this question and found a linear response to increasing fructose intake. Consuming beverages containing
10%, 17.5%, or 25% of daily energy requirement from HFCS produced significant linear dose-response
increases of lipid/lipoprotein risk factors for CVD and uric acid. Compared with beverages containing 0%
HFCS, all 3 doses of HFCS-containing beverages increased concentrations of postprandial triglyceride,
and the 2 higher doses increased fasting and/or postprandial concentrations of non-HDL cholesterol, LDL
cholesterol, apolipoprotein B, apolipoprotein CIII, and uric acid (Stanhope et al. 2015). This increase in
CVD risk factors with increasing amounts of HFCS is particularly important as many studies have shown
that there is a group of adolescents and young adults that consume large amounts of sugar-sweetened bev-
erages both in the US as well as other countries (Barquera et al. 2008; Duffey and Popkin 2008; Ng et al.
2011). In fact, it appears that a major push toward marketing sugar-sweetened beverages exists in low- and
middle-income countries (Kleiman et al. 2011).
CONCLUSIONS AND RECOMMENDATIONS

This chapter has reviewed data on the effects of sugar-sweetened beverages on obesity and cardio-
metabolic disease. The various original studies, reviews, and meta-analyses that have been included
provide a damning picture of both the invisible calories that sugar-sweetened beverages provide
to people susceptible to obesity and of fructose with its many adverse metabolic effects on blood
pressure, circulating lipids, and lipid storage. In the author’s opinion, the intake of sugar-sweetened
beverages is too high, particularly in younger individuals. Replacement of sugar-sweetened bever-
ages with water from safe drinking sources would be a valuable change in drinking patterns.
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14 Effects and Mechanisms of
Fructose-Containing Sugars
in the Pathophysiology of
Metabolic Syndrome
Kimber L. Stanhope and Peter J. Havel
CONTENTS
Introduction.....................................................................................................................................252
Description of Metabolic Syndrome...............................................................................................252
Affirmed Concepts.....................................................................................................................252
MetS Risk Factors......................................................................................................................253
Formally Defined Diagnostic Criteria...................................................................................253
Other Risk Factors That Are Not Formally Recognized.......................................................253
Residual Risk Factors............................................................................................................254
Lifestyle Risk Factors............................................................................................................254
Added Sugars................................................................................................................................. 254
Consumption Levels of Added Sugars.......................................................................................254
Sources of Added Sugars...........................................................................................................254
Plausible Mechanisms by Which Consumption of Fructose-Containing Sugar May
Promote the Development of MetS............................................................................................255
Unregulated Hepatic Uptake and Metabolism of Fructose...................................................255
DNL, Hepatic Lipids, and Hepatic Insulin Resistance..........................................................255
Very Low–Density Lipoprotein Production and Secretion...................................................257
Dyslipidemia.........................................................................................................................257
Intramyocellular Lipid Accumulation and Whole-Body Insulin Resistance.........................257
Hyperuricemia.......................................................................................................................258
Visceral Adipose Accumulation............................................................................................258
Inflammation.........................................................................................................................258
Fructose Overload versus the Established Paradigm.................................................................259
Dietary Sugar Consumption as a Modifiable Risk Factor for MetS: Scientific Evidence..............259
Observational Studies.................................................................................................................259
Dietary Intervention Studies......................................................................................................260
Effects of Sugar Consumption on the Formally Defined Components of MetS...................260
Effects of Sugar Consumption on Components of MetS That Are Not Formally
Defined: Liver Lipid Accumulation, Insulin Resistance, and other Biomarkers of MetS.....263
Conclusion......................................................................................................................................265
Acknowledgments...........................................................................................................................265
Conflicts of Interest.........................................................................................................................265
References.......................................................................................................................................265
251
ABSTRACT
The objective of this chapter is to present the mechanisms and evidence that support the h ypothesis
that increased consumption of added sugars contributes to the development of metabolic s yndrome
(MetS). MetS is described along with its formally defined diagnostic criteria and less well-recognized
risk factors. The amount and types of sugar consumed in the United States are discussed. The mech-
anistic scenario by which consumption of fructose or fructose-containing sugar may promote the
development of components of MetS and how this scenario differs from the established paradigm
are described. We cite the epidemiological studies that demonstrate associations between sugar con-
sumption and all the formally defined, and some of the less well-recognized, risk factors for MetS.
Results from dietary intervention studies showing that many of these risk factors are adversely
affected when sugar intake is increased and beneficially affected when sugar intake is decreased are
presented. We conclude that specific recommendations to reduce consumption of added sugars to
the levels recommended by the 2015–2020 Dietary Guidelines for American, or to the even lower
levels recommended by the American Heart Association, may be beneficial in the prevention and
management of MetS.
INTRODUCTION
The objective of this chapter is to present the mechanisms and evidence that support the hypothesis
that increased consumption of added sugars contributes to the development of metabolic syndrome
(MetS). MetS is described along with its formally defined diagnostic criteria and less well-recognized
risk factors. The amount and types of sugar consumed in the United States are discussed. The term
added sugars in this chapter refers to sugars not naturally occurring in foods, and these consist mainly
of the fructose-containing sugars (sucrose) and high-fructose corn syrup (HFCS). It also refers to the
sugars added to both beverages and solid foods, even though as previously detailed (Stanhope 2016), it
cannot be assumed that sugar in solid food and sugar in beverage have equivalent effects. We describe
the mechanistic scenario by which consumption of fructose or fructose-containing sugar may promote
the development of components of MetS and how this scenario differs from the established paradigm.
We present the epidemiological and the direct experimental evidence concerning the effects of fructose
or added sugar consumption on each of the formally defined and some of the less well-recognized risk
factors for MetS. We conclude with consideration of whether prevention and treatment of MetS should
include specific nutrition advice to reduce the consumption of added sugars.
DESCRIPTION OF METABOLIC SYNDROME

MetS is well described by the MetS concepts that were affirmed and recently reported by the
Cardiometabolic Think Tank (Sperling et al. 2015). This group of individual experts representing
more than 20 professional organizations convened in June, 2014, to focus on a new care model for
the MetS (Sperling et al. 2015).
Affirmed Concepts
The six affirmed concepts (AC.) presented below represent the evidence-based consensus of the
group (Sperling et al. 2015):
AC.1. MetS is a progressive pathophysiological state associated with substantially increased

risk for development of type 2 diabetes (T2D) and atherosclerotic cardiovascular disease.
AC.2. MetS is clinically manifested by a cluster of risk factors that are causally interrelated
(not aggregating by chance alone).
AC.3. Risk for adverse health outcomes increases substantially with the accumulation of com-
ponent MetS risk factors, in addition to unmeasured (“residual risk”) factors. The timely
Fructose-Containing Sugars in the Pathophysiology of Metabolic Syndrome 253
recognition of MetS risk factors helps to identify individuals at high risk for ASCVD and
T2D and to initiate preventive strategies before end-organ damage occurs.
AC.4. Obesity is a MetS risk factor that is imperfectly gauged by body mass index and/or
waist circumference and is modulated by adipocyte distribution, size, and function, as well
as race, behavior, and lifestyle. Excess ectopic and/or visceral adiposity is fundamental to
the pathophysiology of MetS.
AC.5. Treatment of MetS should prioritize therapeutic lifestyle changes, including a healthy
diet and regular physical activity, to address all risk factors. Treatment should also continue
to be focused on specific interventions for component MetS risk factors.
AC.6. The term “MetS” will be used to designate a portfolio of descriptors that have previ-
ously included the terms cardioMetS, insulin resistance syndrome, syndrome X, and others.
MetS Risk Factors

A key finding of the Cardiometabolic Think Tank is that MetS is a cluster of risk factors that include
those that are formally defined and those that are less well-recognized (Sperling et al. 2015).
Formally Defined Diagnostic Criteria

The formally defined risk factors, shown in Table 14.1, are included in the MetS diagnostic crite-
ria. The current criteria were proposed in a joint interim statement of the International Diabetes
Federation Task Force on Epidemiology and Prevention; National Heart, Lung, and Blood Institute;
American Heart Association; World Heart Federation; International Atherosclerosis Society; and
International Association for the Study of Obesity (Alberti et al. 2009). A person with 3 out of the
5 formally defined risk factors included in Table 14.1 qualifies for a diagnosis of MetS. The formally
defined risk factors are easily measured in clinical practice.
Other Risk Factors That Are Not Formally Recognized

Insulin resistance is a critical feature of MetS, which associates with all five of the formally defined
criteria (Sperling et al. 2015). It is not included in Table 14.1 because it is not formally defined or
easy to measure in clinical practice. Also not included in Table 14.1 are other less well-recognized
risk factors that include elevated levels of apolipoprotein B, small dense LDL, C-reactive protein,
fibrinogen, and microalbuminuria (Grundy et al. 2005, Sperling et al. 2015). The Cardiometabolic
Think Tank did not include fatty liver (Fan and Peng 2007, Fabbrini et al. 2009, Bremer, Mietus-
Snyder, and Lustig 2012, Gaharwar et al. 2015, Marino and Jornayvaz 2015) or uric acid (Chaudhary
et al. 2013, Yadav et al. 2013, Billiet et al. 2014, Liu et al. 2015, Nejatinamini et al. 2015) on this list;
however, recent evidence and reviews of the literature support their inclusion.
TABLE 14.1
Criteria for Clinical Diagnosisa of the Metabolic Syndrome
Risk Factor Categorical Cut Points
Elevated triglycerides (or drug treatment for elevated triglycerides) ≥150 mg/dL (1.7 mmol/L)
Reduced HDL-C (or drug treatment for reduced HDL-C) <40 mg/dL (1.0 mmol/L) in males,
<50 mg/dL (1.3 mmol/L) in females
Elevated blood pressure (antihypertensive drug treatment) Systolic ≥130 and/or diastolic ≥85 mm Hg
Increased waist circumference Population- and country-specific definitions
Previous cutoffs: ≥102 cm for males, ≥88 cm for females
Elevated fasting glucose (or drug treatment of elevated glucose) ≥100 mg/dL (5.6 mmol/L)
a Metabolic syndrome is diagnosed by a co-occurrence of three of the above risk factors.

Residual Risk Factors

The Cardiometabolic Think Tank also recognized the existence of residual risk indicators. These can
include low birth weight, birth from a mother with gestational diabetes, parental history of MetS,
and low socioeconomic status (Sperling et al. 2015). Not only do these add to the risk burden, they
can impose an increased risk very early in life. Both of these, total risk burden (Berry et al. 2012)
and early life risk exposure (DeBoer et al. 2015a,b), markedly increase the risk of adverse health
outcomes (e.g., T2D and CVD) later in life.
Lifestyle Risk Factors

The Cardiometabolic Think Tank recognizes that lifestyle, specifically physical activity and nutri-
tion, are modifiable factors crucial to prevent and treat MetS and its consequences (Sperling et al.
2015). For nutrition, it is recommended that emphasis should be placed on dietary patterns such as
the Dietary Approaches to Stop Hypertension (DASH) and Mediterranean-style diets rather than
specific macronutrients (Sperling et al. 2015). This lack of focus on specific macronutrients con-
trasts with the key recommendation of the 2015–2020 Dietary Guidelines for Americans (United
States Department of Health and Human Services and U.S. Department of Agriculture 2015) and the
World Health Organization guideline (World Health Organization 2015) that added sugar consump-
tion be limited to less than 10% of daily calories. However, for both the US Dietary Guidelines and
the World Health Organization, the main rationale for the recommendation to limit added sugars
relates to meeting food group and nutrient needs within calorie limits rather than the possibility that
consumption of added sugars may specifically contribute to the development of MetS or chronic
disease compared with other macronutrients.
ADDED SUGARS
The remainder of this chapter will address the questions of whether consumption of added sugars
should be considered a modifiable risk factor for MetS and whether the nutrition emphasis proposed
by the Cardiometabolic Think Tank (Sperling et al. 2015) should be expanded to include a specific
recommendation to reduce consumption of added sugars.
Consumption Levels of Added Sugars

Americans consume more than three times more energy as processed sugar, 368 kcal/day, than
naturally occurring sugars. Sugar-sweetened beverages (SSBs) contribute 33% of these calories,
solid sugar and candy contribute 16%, cakes and other baked goods contribute 13%, and ice cream
and other dairy desserts contribute 9% (Johnson et al. 2009). Self-reported food intake data suggests
that 13% of the U.S. population consume 25% or more of their daily energy as added, processed
sugar (Marriott et al. 2010). The average level of added sugars intake in the U.S. is approximately
13%–15% of daily calories for adults 20–60 years of age (Ervin and Ogden 2013, Yang et al. 2014)
and 16% for children and adolescents (Ervin et al. 2012). These figures may underestimate con-
sumption of added sugars, as self-reported food intake is often underreported (Livingstone and
Black 2003), and sugar is one of the foods most likely to be underreported (Rangan et al. 2014). The
United States leads the world in sugar consumption with levels more than double the world average.
Sugar consumption in Brazil, Australia, Argentina, and Mexico is close to the U.S. level, while it is
75% lower in China and much of Africa (Suisse Research Institute Credit 2013).
Sources of Added Sugars

The predominant source of dietary added sugars throughout the world is sucrose, which
is extracted and purified from sugarcane and sugar beets. However, in the United States, a
significant amount of the added sugars energy is provided by HFCS. HFCS is derived from the
hydrolysis of cornstarch, which produces glucose syrup, and then isomerization of the glucose
syrup to produce syrup containing 42% fructose. The fructose in this syrup can be extracted
to produce syrup that has 90% fructose. The proportion of fructose to glucose in the final
HFCS product is variable depending on how much of the 90% fructose syrup is added to the
42% fructose syrup. Food nutrition labels do not provide information regarding the propor-
tion or amount of fructose in the HFCS that is contained in the product. Information from the
Corn Refiners Association (The Corn Refiners Association n.d.) states that HFCS comes in two
formulations—HFCS-42, which has 42% fructose, and HFCS-55, which has 55% fructose.
However, analyses of popular SSB showed the mean fructose content in the HFCS used was
59% (range 47%–65%), and several major brands appear to be produced with HFCS that is
65% fructose (Ventura, Davis, and Goran 2011). Therefore, while consumers can be certain that
sucrose always contains 50% fructose and 50% glucose, they cannot determine from the food
label the fructose content of the HFCS used.
Plausible Mechanisms by Which Consumption of Fructose-Containing

Sugar May Promote the Development of MetS
The proportion of fructose in added sugars is an important issue because a large body of scientific
evidence from studies in animals and humans indicates that consumption of fructose results in
more adverse changes in components of MetS than consumption of glucose. This includes a study
from our group in which subjects consuming 25% of their energy requirement (Ereq) as fructose-
sweetened beverages for 10 weeks exhibited increases of de novo lipogenesis (DNL), visceral
adipose tissue deposition, dyslipidemia, and circulating uric acid and markers of inflammation
and reductions of fatty acid oxidation and insulin sensitivity, while subjects consuming glucose-
sweetened beverages did not, despite comparable body weight gain (Stanhope et al. 2009, Cox
et al. 2011, 2012a,b). These results, our more recent reports (Stanhope et al. 2011a, 2015), and the
findings from a number of other investigators support the plausibility of the mechanisms, described
below and illustrated in Figure 14.1, by which consumption of added, fructose-containing sugars
may mediate or contribute to MetS.
Unregulated Hepatic Uptake and Metabolism of Fructose

Hepatic glucose metabolism is regulated by insulin and hepatic energy needs, and this allows most
of the ingested glucose, from starch or a glucose-sweetened beverage, arriving via the portal vein
to bypass the liver and reach the systemic circulation. In contrast, the initial phosphorylation of
dietary fructose is largely catalyzed by fructokinase, which is not regulated by hepatic energy status
(Mayes 1993, Havel 2005). This results in unregulated fructose uptake by the liver, with most of
the ingested fructose being metabolized in the liver and very little reaching the systemic circulation
(Teff et al. 2009). The excess substrate generated from the unregulated metabolism of fructose in
the liver leads to increased DNL (Stanhope et al. 2009). Recent evidence from the research group
of Schwarz and colleagues demonstrates that fructose consumption can increase DNL more than
isocaloric complex carbohydrates even in subjects consuming energy-balanced, weight-maintaining
diets (Schwarz et al. 2015).
DNL, Hepatic Lipids, and Hepatic Insulin Resistance

DNL increases the hepatic lipid supply directly (Maersk et al. 2012, Sevastianova et al. 2012,
Schwarz et al. 2015), via synthesis of fatty acids, and indirectly, by inhibiting fatty acid oxidation
(Cox et al. 2012a, Schwarz et al. 2015). Increased levels of hepatic lipids may also promote hepatic
insulin resistance (Schwarz et al. 2015), possibly by increasing levels of diacylglycerol (DAG),
which activates protein kinase C epsilon (PKCε) leading to increased serine rather than tyrosine
Fructokinase C
Liver a
Intestine
m Uric acid
Fructose Unregulated hepatic uptake/
metabolism of fructose
p b n
Visceral d
adiposity q DNL o
c Fatty acid
oxidation
r d
s Liver lipid VLDL production/

e secretion
Inflammatory ( DAG nPKC serine P
factors of insulin receptor) f
i ( ApoCIII, Dyslipidemia
ApoB,
MTP activity) g
t k
h j CVD risk
Muscle lipid accumulation

( DAG nPKC serine P of insulin receptor)
Hepatic
l
insulin
resistance Whole body insulin resistance
FIGURE 14.1 Potential mechanisms by which consumption of fructose promotes the development of metabolic
syndrome: The initial phosphorylation of dietary fructose in the liver is largely catalyzed by fructokinase C (a),
which is not regulated by hepatic energy status. This results in unregulated fructose uptake and metabolism by the
liver. The excess substrate leads to increased DNL (b). DNL increases the intrahepatic lipid supply directly via
synthesis of fatty acids (c) and indirectly by inhibiting fatty acid oxidation (d). Increased levels of intrahepatic lipid
content promote very low–density lipoprotein (VLDL) production and secretion (e). This leads to increased levels
of circulating TG and low-density lipoprotein cholesterol (dyslipidemia [f]), risk factors for CVD (g). Increased
levels of hepatic lipid may also promote hepatic insulin resistance by increasing levels of diacylglycerol, which may
activate novel protein kinase C (nPKC) and lead to serine phosphorylation (serine P) of the insulin receptor and
insulin receptor substrate 1 (IRS-1) and impaired insulin action (h). Due to selective insulin resistance, DNL is even
more strongly activated in the insulin-resistant liver DNL (i), which has the potential to generate a vicious cycle
(circular arrows). This cycle would be expected to further exacerbate VLDL production and secretion via increased
intrahepatic lipid supply. Hepatic insulin resistance also exacerbates VLDL production/secretion (j) by increas-
ing apolipoprotein (apo)B availability and apoCIII synthesis and by upregulating microsomal triglyceride transfer
protein expression. This exacerbates and sustains exposure to circulating TG, leading to muscle lipid accumulation
(k), impaired insulin signaling, and whole-body insulin resistance (l). The fructokinase-catalyzed phosphorylation
of fructose to fructose-1-phosphate, which results in the conversion of ATP to AMP and a depletion of inorganic
phosphate, leads to uric acid production via the purine degradation pathway (m). High levels of uric acid are associ-
ated and may contribute to increased risk for development of fatty liver (n), CVD (o), and MetS. Fructose exposure
in the intestine (p) and liver (q) and fructose-induced increases of visceral adipose (r) may promote inflammatory
responses that further promote liver lipid accumulation (s) and/or impair hepatic insulin signaling (t). (Reprinted
from Stanhope, K.L. and Havel, P.J., Mechanisms by which dietary sugars influence lipid metabolism, circulat-
ing lipids and lipoproteins, and cardiovascular risk, in M. Goran and L. Tappy, Eds., Dietary Sugars and Health,
Taylor & Francis Group, LLC., Boca Raton, FL, pp. 267–282, 2014. With permission.)
phosphorylation of the insulin receptor and insulin receptor substrate 1 (IRS-1) and, thus, impaired
insulin action (Jornayvaz and Shulman 2012). Due to selective insulin resistance, DNL is even more
strongly activated in the insulin-resistant liver (Lewis et al. 2002). This has the potential to generate
a vicious cycle: DNL increases liver lipid, which increases hepatic insulin resistance, which further
increases DNL (Figure 14.1—circular arrows).
Very Low–Density Lipoprotein Production and Secretion

Increased intrahepatic lipid content promotes very low–density lipoprotein 1 (VLDL1) production
and secretion (Adiels et al. 2006), which leads to increased circulating levels of postprandial tri-
glyceride (TG) and dyslipidemia (Adiels et al. 2008). Our short-term studies demonstrate that post-
prandial levels of TG are increased within 1 day of fructose, HFCS or sucrose consumption at 25%
Ereq (Teff et al. 2004, 2009, Stanhope and Havel 2008a). The vicious cycle described above would
be expected to further exacerbate VLDL production and secretion by increasing the hepatic lipid
supply (Adiels et al. 2006). Hepatic insulin resistance, however, may also indirectly increase VLDL
production and secretion by (1) increasing the availability of apolipoprotein B (apoB) (Fisher 2012,
Christian, Sacco, and Adeli 2013), the protein component of VLDL; (2) upregulating microsomal
triglyceride transfer protein expression (Lewis et al. 2002), which catalyzes the assembly of TG and
apoB into VLDL; and (3) increasing the production of apolipoprotein CIII (apoCIII) (Yao and Wang
2012). There is evidence to suggest that apoCIII plays a critical role in promoting the second-step
incorporation of lipid into VLDL, which converts VLDL2 (smaller, TG-poor particles) into larger,
TG-rich VLDL1 particles (Sundaram et al. 2010, Qin et al. 2011). Both fructose-fed rhesus mon-
keys (Bremer et al. 2014) and young adults consuming HFCS-sweetened beverages (Stanhope et al.
2015) exhibit increased levels of fasting and/or postprandial apoCIII.
Dyslipidemia
The overproduction of VLDL1 has been described as the underlying defect that leads to the dyslip-
idemia that is characteristic of patients with T2D and MetS: high blood concentrations of TG and
small dense LDL and low blood concentrations of HDL (Adiels et al. 2008). A possible mecha-
nism for this relationship is that as levels of VLDL1 increase, this lipoprotein subclass becomes
the preferred substrate of cholesteryl ester transfer protein (CETP) (Chapman et al. 2010). CETP
exchanges the cholesterol in LDL and HDL with the TG in VLDL1. The resulting TG-enriched LDL
can undergo hepatic lipase-mediated lipolysis, yielding smaller, denser LDL. Small dense LDL is
considered to be more atherogenic than larger, more buoyant LDL in part due to its reduced affinity
for the LDL receptor, thus increasing its retention time in the circulation (Diffenderfer and Schaefer
2014). The TG-enriched HDL resulting from CETP remodeling is more likely to be degraded by the
kidney rather than continue participation in reverse cholesterol transport (Packard 2003, Chapman
et al. 2010). This scenario is proatherogenic as opposed to the antiatherogenic scenario that occurs
when VLDL1 levels are low and LDL and HDL are the preferred substrates of CETP. In the anti-
atherogenic scenario, CETP catalyzes the transfer of cholesterol from HDL to LDL. The choles-
terol-enriched LDL is then cleared by hepatic LDL receptors, and the cholesterol-depleted HDL
continues participation in reverse cholesterol transport (Chapman et al. 2010).
It has also been suggested that apolipoprotein CIII (apoCIII) is the primary mediator of the
relationship between VLDL and small dense LDL (Sacks 2015) and CVD (Wyler von Ballmoos,
Haring, and Sacks 2015). VLDL can contain apoE or apoCIII or both. ApoCIII inhibits the clearance
of TG-rich lipoproteins from the circulation (Boren et al. 2015), while apoE binds to high-affinity
hepatic receptors and allows for clearance of lipoproteins (Sacks 2015). In hypertriglyceridemia,
excessive production of a VLDL subspecies that does not contain apoE, but is relatively enriched in
apoCIII, results in an accumulation of TG-rich VLDL that is metabolized by lipolytic enzymes to
small dense LDL (Sacks 2015).
Intramyocellular Lipid Accumulation and Whole-Body Insulin Resistance
Increased exposure of skeletal muscle to high circulating postprandial TG concentrations, along
with decreased fat oxidation (Cox et al. 2012a) during fructose consumption, may lead to intramyo-
cellular lipid accumulation (Maersk et al. 2012). Intramyocellular lipid concentrations are correlated
with reduced whole-body insulin sensitivity in humans (Krssak et al. 1999). It is possible, but not
definitive (Watt and Hoy 2012), that this relationship is mediated by the same mechanism described
for the development of hepatic insulin resistance: DAG-mediated activation of PKCε resulting in
serine phosphorylation of the insulin receptor, IRS-1, and other downstream insulin signaling pro-
teins (Samuel and Shulman 2012). It is also possible that other factors such as inflammation and
oxidative stress (Anderson et al. 2009) contribute to muscle insulin resistance (Coen and Goodpaster
2012). While the mechanisms that lead to muscle insulin resistance are not established, two short-
term (9 and 21 days) studies in which subjects consuming fructose exhibited reduced hepatic insulin
sensitivity during hyperinsulinemic euglycemic clamps, but not reduced whole-body insulin sensi-
tivity (Aeberli et al. 2013, Schwarz et al. 2015), suggest that whole-body insulin resistance occurs
subsequently to hepatic insulin resistance.
Hyperuricemia
The unregulated hepatic uptake and metabolism of fructose also leads to increased concentrations of cir-
culating uric acid (Cox et al. 2012b, Bruun et al. 2015, Stanhope et al. 2015). The fructokinase-catalyzed
phosphorylation of fructose to fructose 1-phosphate, which results in the conversion of adenosine tri-
phosphate (ATP) to adenosine monophosphate (AMP) and a depletion of inorganic phosphate, leads
to uric acid production via the purine degradation pathway (Mayes 1993). Uric acid is a potential
mediator of metabolic disease, with most but not all (Zalawadiya et al. 2015) recent studies reporting
that it is strongly associated with and predictive of MetS, fatty liver, and CVD (Cai et al. 2013, Billiet
et al. 2014, Viazzi et al. 2014). High uric acid levels resulting from fructose consumption may also
explain the association between sugar consumption and increased blood pressure (Johnson et al. 2015).
Studies performed in rodents suggest that the mechanisms involve the uric acid–dependent activation
of the rennin–angiotensin system via the induction of oxidative stress and development of endothelial
dysfunction due to reduction in endothelial nitric oxide levels (Feig et al. 2013).
Visceral Adipose Accumulation

There is little evidence in human subjects upon which to base a mechanism by which fructose may
preferentially promote fat accumulation into visceral adipose tissue (VAT) over subcutaneous adipose
tissue (SAT). We have hypothesized that reduced lipoprotein lipase (LPL) activity during consump-
tion of fructose may be involved (Stanhope et al. 2009, Stanhope 2012). LPL activity is an important
determinant of TG storage in the adipocyte (Eckel 1989). However, the LPL in SAT is markedly more
sensitive to activation by insulin than the LPL in VAT (Fried et al. 1993), which in the insulin-sensitive
individual would favor the storage of TG in SAT instead of VAT. Fructose consumption, however,
reduces insulin sensitivity and also postmeal insulin responses, unlike glucose or starch consumption
(Stanhope et al. 2011b), thus lowering LPL activity (Stanhope et al. 2009). This would preferentially
lower TG uptake by SAT, thereby increasing the availability of TG for uptake into VAT. Recently, it
has been reported that VAT accumulation is positively associated with the amount of apoCIII in apoB-
containing lipoproteins (Wang et al. 2014). While the author suggests that VAT accumulation increases
the amount of apoCIII in apoB-containing lipoproteins, it is interesting to speculate whether apoCIII
may be involved in mediating fructose-induced VAT accumulation. Circulating apoCIII concentrations
are increased by fructose consumption (Bremer et al. 2014, Stanhope et al. 2015), and apoCIII has
been demonstrated to inhibit LPL in vitro (Wang et al. 1985).
Inflammation
A chronic state of inflammation may also be an important contributor to the pathophysiology under-
lying MetS (Khodabandeloo et al. 2016, Welty, Alfaddagh, and Elajami 2016). Fructose may pro-
mote inflammatory responses by several pathways.
Fructose exposure, compared with glucose exposure, results in the activation of c-jun NH2-
terminal kinase (JNK) in isolated hepatocytes (Wei et al. 2007). In nonparenchymal liver cells, such
as hepatic macrophages and hepatic stellate cells, JNK is involved in inflammation and fibrosis
(Seki, Brenner, and Karin 2012). JNK activation may also be involved in fructose-induced hepatic
insulin resistance via increased serine phosphorylation of IRS-1 and reduced insulin-stimulated
tyrosine phosphorylation of IRS-1 and IRS-2 (Wei et al. 2007, 2013).
Studies in mice and nonhuman primates show that direct exposure of the intestine to fructose increases
the intestinal translocation of endotoxin, which appears to produce hepatic toxicity (Bergheim et al. 2008,
Kavanagh et al. 2013). Specifically, in nonhuman primates fed weight-maintaining diets with fructose
for 6 weeks, biochemical indicators of liver injury and inflammation were markedly increased com-
pared with control diet, and these changes were highly correlated to indices of microbial translocation
(Kavanagh et al. 2013). In mice consuming fructose, administration of antibiotics prevented endotoxemia
and reduced lipid accumulation and peroxidation in the liver (Bergheim et al. 2008).
The increase in visceral fat resulting from overconsumption of fructose (Stanhope et al. 2009,
Maersk et al. 2012) could also lead to chronic, low-grade inflammation that can alter lipolysis,
insulin sensitivity, fibrinolysis, and possibly macrophage infiltration (Tchernof and Despres 2013).
Fructose Overload versus the Established Paradigm

As discussed in detail above, the delivery of elevated amounts of fructose to the intestine and liver
results in a cascade of events that promote the development of MetS. This sequence of events differs
from the more established paradigm described for the development of MetS (Asrih and Jornayvaz
2015). In this scenario, VAT accumulation due to overnutrition and a sedentary lifestyle leads to
large, insulin-resistant, and more lipolytic adipocytes and increased circulating levels of free fatty
acids (FFA). In the liver, the FFA promote liver lipid accumulation and VLDL production/secretion.
In muscle, the FFA interfere with insulin signaling. We have suggested that this pathway (Asrih and
Jornayvaz 2015) does not explain the metabolic dysregulation resulting from excess consumption
of fructose and fructose-containing sugars (Stanhope and Havel 2008b, Stanhope 2012). Subjects
consuming fructose for 10 weeks had significantly lower 24-hour circulating FFA concentrations
than subjects consuming glucose (Stanhope et al. 2009). Overall, the results from this study (Stanhope
et al. 2009) support the hypothesis that in a setting of positive energy balance (~1.5 kg weight gain in
8 weeks), the hepatic substrate overload and upregulation of DNL induced by fructose consumption
represents a much faster route to metabolic dysregulation than the modest increases of circulating
FFA induced by glucose consumption. Furthermore, the results of Schwarz et al. demonstrate that
fructose can induce metabolic dysregulation even in the absence of weight gain (Schwarz et al.
2015). In this study, men consuming 25% Ereq from fructose-sweetened beverages as part of an
energy-balanced diet exhibited increased DNL, inhibition of whole-body fat oxidation, increased
liver lipid content, and decreased liver insulin sensitivity in only 9 days, without an increase of body
weight (Schwarz et al. 2015).
DIETARY SUGAR CONSUMPTION AS A MODIFIABLE

RISK FACTOR FOR METS: SCIENTIFIC EVIDENCE
Results from both observational and dietary intervention studies support the suggestion that added
sugars consumption is a modifiable risk factor for MetS.
Observational Studies
Epidemiological studies report positive associations between the consumption of added sugars
or SSB and MetS in both children (Bremer, Auinger, and Byrd 2009, Wang et al. 2013, Eloranta
et al. 2014, Mirmiran et al. 2015) and adults (Dhingra et al. 2007, Denova-Gutierrez et al. 2010,
Hostmark 2010, Mattei et al. 2012, Barrio-Lopez et al. 2013, Green et al. 2014, Ejtahed et al. 2015).
Interestingly, in the Framingham Heart Study Offspring and Third Generation cohorts, the odds
ratios for having at least one MetS risk factor (excluding waist circumference) in SSB consum-
ers versus non-SSB consumers were comparable in normal-weight (1.9 odd ratio, CI: 1.4–2.6),
overweight (2.0, 1.4–2.9), and obese individuals (1.9, 1.1–3.4) [85], suggesting the effects of SSB
consumption on MetS risk factors is not mainly dependent on body weight/adiposity. It was recently
reported that SSB consumption increased the risk of MetS in Korean women (n = 8540) but not in
men (n = 5432) (Chung et al. 2015). However, less than 25% of these participants consumed ≥1
SSB/week. The total added sugars consumption of the SSB consumers averaged 9% and 12% of
daily energy for men and women, respectively (Chung et al. 2015), which is below the 13%–15%
estimates for all U.S. adults (Ervin and Ogden 2013, Yang et al. 2014).
The formally defined individual components of MetS: hypertriglyceridemia (Duffey et al. 2010,
Welsh et al. 2010, 2011, de Koning et al. 2012, Ambrosini et al. 2013), low HDL (Welsh et al.
2010, 2011, de Koning et al. 2012, Kosova, Auinger, and Bremer 2013, Hert et al. 2014), increased
waist circumference/VAT (Collison et al. 2010, Odegaard et al. 2012, Pollock et al. 2012, Kosova,
Auinger, and Bremer 2013, Chan et al. 2014, Ma et al. 2014, Mollard et al. 2014), elevated blood
pressure (Brown et al. 2011, Cohen, Curhan, and Forman 2012, Kell et al. 2014, Xi et al. 2015),
and high fasting glucose/homeostatic model assessment of insulin resistance (HOMA-IR) (Bremer,
Auinger, and Byrd 2009, Perichart-Perera et al. 2010, Wang et al. 2013, Lana, Rodriguez-Artalejo,
and Lopez-Garcia 2014, Wang 2014, Santiago-Torres et al. 2016) have all been shown to be associ-
ated with sugar consumption in observational studies. Some of the components of MetS that are not
formally defined have also been reported to be associated with sugar consumption: insulin resistance
(indexed by parameters other than HOMA-IR) (Yoshida et al. 2007, Wang 2014), fatty liver (Zelber-
Sagi et al. 2007, Assy et al. 2008, Ouyang et al. 2008, Ma et al. 2015), and elevated circulating uric
acid (Choi et al. 2008, Bomback et al. 2010, Chang 2011, Zgaga et al. 2012, Lin et al. 2013), small
dense LDL (Aeberli et al. 2007), C-reactive protein (Kosova, Auinger, and Bremer 2013, Hert et al.
2014, Gonzalez-Gil et al. 2015), and fibrinogen (Miura et al. 2006) concentrations.
Dietary Intervention Studies

Numerous studies in which added sugars have been added to or removed from the diets of human
subjects show effects on the formally defined and not formally defined risk factors for MetS.
Effects of Sugar Consumption on the Formally Defined Components of MetS

TG, HDL, blood pressure, waist circumference/VAT, and fasting glucose concentrations.
TG
The diagnostic criterion for MetS is a fasting TG concentration ≥150 mg/dL. However, our laboratory
has conducted three dietary intervention studies in which participants were provided with beverages
sweetened with fructose or HFCS, and all three demonstrated that the effects of fructose-containing sug-
ars on fasting TG concentrations were minimal compared with the effects on circulating postprandial TG
profiles (Swarbrick et al. 2008, Stanhope et al. 2009, 2015). Fasting TG concentrations were unchanged
in the two 10-week studies in which subjects consumed 25% Ereq as fructose (Swarbrick et al. 2008,
Stanhope et al. 2009). In the 2-week dose–response study (Stanhope et al. 2015), we observed a modest
increase of fasting TG in subjects consuming beverages containing 25% Ereq as HFCS that was signifi-
cant compared with baseline (+11.2 ± 3.4 mg/dL (mean ± SEM), P < 0.01) but not compared with the
aspartame-consuming control group (P = 0.11). In contrast, consumption of these beverages induced
highly significant increases in postprandial TG, particularly 4–6 hours after dinner, the final meal of
the day (+65–70 mg/dL for 25% Ereq fructose-sweetened beverages [Swarbrick et al. 2008, Stanhope
et al. 2009], +37 ± 5 mg/dL for 25% Ereq HFCS-sweetened beverages [Stanhope et al. 2015]). Subjects
consuming the lowest dose of HFCS-sweetened beverages in the dose–response study, 10% Ereq, also
exhibited increased postprandial TG concentrations (+22 ± 8 mg/dL) that were significant compared with
baseline (P < 0.05) and with the aspartame-consuming control group (P < 0.001). The previously cited
study by Schwarz et al. also reported no effect on fasting TG (102 ± 7 vs 100 ± 7 mg/dL; P = 0.67) and a
marked effect on mean postprandial TG concentrations (172 ± 29 vs 140 ± 28 mg/dL; P = 0.002) when
men consumed energy-balanced, weight-maintaining diets that included fructose-sweetened beverages
compared with when they consumed isocaloric complex carbohydrate diets (Schwarz et al. 2015).
However, despite the less marked effects on fasting TG, a recent meta-analysis of 38 randomized
trials (total n of 1660) comparing the effects of altered sugar consumption (either di- or monosac-
charide) with a control arm on fasting TG concentrations reported an increase of 10 mg/dL (95%
CI: 6, 13 mg/dL; P < 0.0001) with high sugar consumption (Te Morenga et al. 2014). These stud-
ies (Te Morenga et al. 2014) and the studies conducted by our laboratory (Swarbrick et al. 2008,
Stanhope et al. 2009, 2011a, 2015) also support an effect of sugar to increase total cholesterol and
LDL-cholesterol.
HDL
The diagnostic criterion for MetS is a fasting HDL concentration <50 mg/dL for women and
<40 mg/dL for men. In our recently completed study of 187 men and women, age 18–40 years and
BMI 18–35 kg/m2, low HDL was the most prevalent MetS criteria at baseline, occurring in more
than 50% of the study group. High TG, the 2nd most prevalent MetS criterion, occurred in 15% of
the group. The published dietary intervention studies do not support a mechanism by which sugar-
induced overproduction of VLDL1 leads to TG enrichment of HDL, followed by degradation in
the kidneys and a lowering of circulating HDL levels. We have reported that fructose and/or HFCS
consumption do not affect HDL concentrations (Stanhope et al. 2011a, 2009, 2015, Swarbrick et al.
2008). Te Morenga et al. pooled the results of 29 studies (total n of 1515) and observed a small but
significant effect of dietary sugars, an increase in HDL by 0.8 mg/dL (95% CI: 0.0, 1.2 mg/dL; P =
0.02) (Te Morenga et al. 2014). However, in a recent 6-week crossover study, Maki et al. reported
a lowering of HDL concentrations when subjects consumed sucrose-sweetened beverage and des-
sert compared with when they consumed 2% fat milk and yogurt (Maki et al. 2015). It is also worth
noting that post hoc analyses of our previous 10-week study (Stanhope et al. 2009) and our recently
published 2-week study (Stanhope et al. 2015) show an HDL-lowering effect of sugar consumption
in subjects with higher compared with lower plasma HDL concentrations at baseline. Specifically,
when we divided 80 subjects who consumed 25% Ereq as fructose-, HFCS-, or sucrose-sweetened
beverages for 2 weeks into two groups based on baseline HDL concentrations ≥ or < than 45 mg/dL, the
subjects with the higher HDL levels exhibited a lowering of HDL (−3.4 ± 1.0 mg/dL) that was sig-
nificant compared with their baseline levels (P = 0.002) and compared with the change in subjects
with lower HDL concentrations (+0.5 ± 0.5 mg/dL, P = 0.0006 higher vs lower HDL at baseline).
Research is needed to examine HDL function and HDL subspecies in response to sugar consump-
tion. Interestingly, it has recently been suggested (Taskinen and Boren 2015) that low HDL-C may
simply be a long-term marker of persistently elevated circulating TG and therefore not directly
involved in the pathophysiology of CVD.
Blood Pressure
Either systolic blood pressure ≥130 mm Hg or diastolic blood pressure ≥85 mm Hg represents
a diagnostic criterion for MetS. While none of our intervention studies with dietary sugars have
demonstrated an effect of sugar consumption on blood pressure (Swarbrick et al. 2008, Stanhope
et al. 2009, 2011a, 2015), Te Morenga et al. reported that sugar consumption increased diastolic
blood pressure by 1.4 mm Hg (95% CI: 0.3, 2.5 mm Hg; P < 0.02) in 324 subjects pooled from
12 published clinical studies (Te Morenga et al. 2014). There was a significant interaction between
study length and the change of blood pressure, with diastolic blood pressure increasing by 5.6 mm
Hg (95% CI: 2.5, 8.8 mm Hg; P = 0.0005) in subjects pooled from three trials lasting longer than
8 weeks (Te Morenga et al. 2014). It is important to note, however, that in these three longer trials,
subjects consumed sucrose (+20% of daily calories) with ad libitum diets and their change in body
weight was 1.8–4.0 kg higher than the control groups provided with starch, aspartame-sweetened
snacks, and/or beverages. In the eight trials in which energy-balanced diets were provided or recom-
mended, diastolic blood pressure increased by 0.65 (95% CI: −0.31, 1.61 mm Hg; P = 0.18). Marked
reductions of blood pressure have been reported when the intervention consisted of reducing sugar
consumption. Diastolic blood pressure decreased by 5 mm Hg (95% CI: −8.1 to −1.8 mm
Hg; P = 0.003) in only 9 days in 43 Latino and African-American children with MetS receiving con-
trolled, energy-balanced diets that decreased their added sugars consumption from their usual 27%
to 10% Ereq (Lustig et al. 2016). In another study, children with nonalcoholic fatty liver disease
(NAFLD) who reduced consumption of fructose from beverages and refined foods exhibited a sig-
nificant lowering of systolic blood pressure by approximately 12 mm Hg at 3 and 6 months (Mager
et al. 2015). These blood pressure reductions, however, could have been mediated by decreased
body weight in the 9-day study (−0.9 ± 0.2 kg; P < 0.001) (Lustig et al. 2016) and decreased body fat
in the 6-month study (36.4 ± 6.4 vs 29.9 ± 8.4%; P = 0.04) (Mager et al. 2015). Thus, studies that
show a weight- or body fat–independent effect of increased or reduced sugar consumption on blood
pressure are lacking.
Waist Circumference/VAT
The Cardiometabolic Think Tank states that visceral adiposity is fundamental to the pathophys-
iology of MetS (Sperling et al. 2015); however, the diagnostic criterion for visceral adiposity
is waist circumference. Waist circumference is easy and inexpensive to measure in both clini-
cal and research settings. However, unlike assessment of VAT and SAT by magnetic resonance
or computed tomography imaging, it does not distinguish between intra- and extra-abdominal
fat. Three studies of varying length, 6 months (Maersk et al. 2012), 10 weeks (Stanhope et al.
2009), and 4 weeks (Silbernagel et al. 2011), have assessed the effects of sugar consump-
tion on VAT and SAT accumulation with imaging of the abdominal region. The 6-month study
was conducted in Denmark and subjects consumed 1 L/day of sucrose-sweetened cola (~20%
Ereq), isocaloric amounts of low-fat milk, 1 L/day aspartame-sweetened beverages, or 1 L/day
water for 6 months (Maersk et al. 2012). Body weight at the end of the intervention period
was not significantly different from the baseline in any group. Subjects consuming sucrose
exhibited increased VAT, while the other three groups did not. The increase of VAT in sub-
jects consuming sucrose was significantly greater than in the subjects who consumed low-fat
milk, despite comparable changes of body weight (Sucrose: +1.3%; Milk: +1.4%) (Maersk
et al. 2012). The 10-week study conducted by our laboratory reported that, despite comparable
increases of body weight (~1.5 kg; P = 0.47, between group difference) and waist circumfer-
ence (~1.8 cm; P = 0.86, between group difference), subjects consuming fructose-sweetened
beverages preferentially accumulated fat in VAT, while subjects consuming glucose exhibited
a significant increase of fat in SAT (Stanhope et al. 2009). While the fructose-induced increase
in VAT (P < 0.01 compared with baseline) was nearly statistically significant compared to that
of glucose (P = 0.059 between group difference), it was significant in men consuming fructose
compared with men consuming glucose (P < 0.05) (Stanhope et al. 2009). In contrast, a 4-week
study found no differences in VAT and SAT accumulation in subjects consuming high levels
of fructose or glucose (Silbernagel et al. 2011). This may be related to the shorter duration,
but it may also be due to the nearly significant difference in body weight gain between the two
groups (P = 0.056). Glucose consumption resulted in weight gain (+1.7 ± 0.4 kg, P = 0.001
within group), and fructose consumption did not (+0.2 kg ± 0.6 kg, P = 0.40). More studies that
(1) utilize imaging to differentiate SAT and VAT, (2) utilize intervention periods longer than
4 weeks, and (3) are not confounded by differential weight gain between groups are needed to
clarify the effects of added sugar intake on visceral adiposity.
Fasting Glucose Concentrations

The final MetS diagnostic criterion is impaired fasting glucose, defined as a fasting blood glucose
concentration ≥100 mg/dL. High fasting blood glucose was the least prevalent (5%) MetS criterion
in our recent study population of 187 young men and women. Impaired fasting glucose is associated
with increased risk of future T2D and is indicative of insulin resistance, specifically hepatic insulin
resistance (Abdul-Ghani and DeFronzo 2009). Impaired glucose tolerance, indexed as a 2-hour
glucose concentration between 140 and 199 mg/dL during an oral glucose tolerance test (OGTT),
is indicative of whole-body insulin resistance, and is an even stronger predictor of future T2D
(Abdul-Ghani and DeFronzo 2009). In two separate studies, we observed increases of fasting glu-
cose concentrations in older, overweight adults consuming 25% Ereq fructose-sweetened beverages
for 10 weeks (Swarbrick et al. 2008, Stanhope et al. 2009). Reiser et al. reported increases of fast-
ing glucose and impaired glucose tolerance when insulin-resistant subjects consumed standardized
6-week crossover diets containing 18% or 33% Ereq as sucrose compared with a diet providing 5%
Ereq sucrose (Reiser et al. 1981). In contrast to these studies, fasting glucose concentrations were
not changed in subjects consuming fructose- or HFCS-sweetened beverages for 2 weeks (Stanhope
et al. 2011a) or sucrose-sweetened beverages for 6 months (Maersk et al. 2012). Fasting glucose was
reduced (−5.4 mg/dL, 95% CI: −7.2, −3.6 mm Hg; P < 0.001), and glucose tolerance was improved
in the 43 Latino and African-American children with MetS who lost weight when consuming con-
trolled, energy-balanced meals that decreased their consumption of added sugars from their usual
27% to 10% Ereq for 9 days (Lustig et al. 2016). Fasting glucose concentrations were also decreased
in overweight/obese adults who were provided with four servings of water/day as replacements for
caloric beverages for 6 months (Tate et al. 2012). They exhibited reduced fasting glucose concentra-
tions (~ −3 mg/dL) compared with their baseline levels (P = 0.0027) and compared with the control
group who received general dietary instructions and no water (P = 0.019). During the 6-month
intervention, both the group consuming water and the control group lost significant amounts of body
weight (−2.0% ± 0.4% and −1.8% ± 0.4%, respectively) (Tate et al. 2012). Effects of sugar con-
sumption on fasting blood glucose may prove to be more discernable and consistent in intervention
studies that reduce sugar consumption rather than increase it.
Effects of Sugar Consumption on Components of MetS That Are Not Formally Defined:
Liver Lipid Accumulation, Insulin Resistance, and other Biomarkers of MetS.
Liver Lipid Accumulation
NAFLD has been described as an important comorbidity of MetS (Marchesini et al. 2001), and it
has been suggested that it be included as a criterion for the diagnosis of MetS (Tarantino and Finelli
2013). Fatty liver also plays a prominent role in the mechanism by which we propose that consump-
tion of added sugars promotes the development of MetS, particularly insulin resistance and dyslip-
idemia. Measuring hepatic lipid content noninvasively via imaging (magnetic resonance imaging
and magnetic resonance spectroscopy) is a fairly new technique. Accordingly, there are only a few
intervention studies that have reported the effect of sugar consumption on liver fat content. In the
previously discussed 6-month intervention, subjects consuming 1 L/day sucrose-sweetened cola
exhibited increased liver fat compared with those consuming isocaloric amounts of milk, or iso-
volumetric amounts of water or aspartame-sweetened beverages (Maersk et al. 2012). Consumption
of 25% Ereq as fructose-sweetened beverages increased liver fat in 9 days compared with isoca-
loric complex carbohydrate in men consuming energy-balanced diets that prevented weight gain
(Schwarz et al. 2015). In another study, 14 overweight adults who normally consumed about three
cans of soda per day were provided and asked to replace the soda with aspartame-beverages for
12 weeks (Campos et al. 2015). The reduction in liver fat content in the subjects who switched to
consuming aspartame was 53% greater than that of the control group that continued to consume the
SSBs (P < 0.05). Importantly, the eight subjects with higher levels of liver fat (>5%) exhibited more
marked decreases of hepatic lipid content than the six subjects with lower amounts of liver fat prior
to intervention (−57% vs −17%, P < 0.05) (Campos et al. 2015).
Insulin Resistance
Insulin resistance is a critical feature of MetS and has been proposed to be an important link between
liver fat accumulation and MetS (Asrih and Jornayvaz 2015). As previously stated, it is not a diag-
nostic criterion for MetS because it is not formally defined or easy to measure in clinical prac-
tice. Numerous methods have been employed to assess insulin sensitivity in research studies, and
the advantages and limitations of these methods have been recently described (Dube et al. 2013).
The easiest and least expensive method is HOMA-IR, which requires only the measurement of
fasting glucose and insulin. Its main advantage is its utility for large population studies (Dube et al.
2013); however, it is frequently used in intervention studies with limited sample sizes. Shaibi et al.
compared the sensitivity of HOMA-IR to detect changes of insulin sensitivity to more laborious
methods in adolescents participating in two separate lifestyle interventions (Shaibi et al. 2011).
Boys in a 16-week exercise intervention study exhibited a 45% improvement of insulin sensitiv-
ity measured by frequently sampled intravenous glucose tolerance tests that was not detected by
HOMA-IR. Young females participating in a 12-week study testing a nutrition education program
exhibited a 34% improvement in an index of whole-body insulin sensitivity assessed during an
OGTT with multiple sampling that failed to be detected by HOMA-IR (Shaibi et al. 2011).
Unfortunately, most of the dietary intervention studies testing the effects of sugar consump-
tion on insulin sensitivity have utilized HOMA-IR. While Johnston et al. did observe an increase
in HOMA-IR in overweight men consuming an isocaloric diet containing 25% Ereq as fructose-
sweetened beverage compared with men consuming the same diet with glucose-sweetened bever-
age (Johnston et al. 2013), most studies have failed to detect effects on HOMA-IR. For example,
as noted above, subjects consuming 1 L of sucrose-sweetened beverage/day exhibited increased
liver fat compared to all three control groups and also an increase in intramyocellular fat compared
with baseline levels (Maersk et al. 2012). These changes would be expected to be associated with a
decrease of insulin sensitivity; however, the changes in insulin sensitivity as assessed by HOMA-IR
were highly variable (i.e., −5.0 ± 24.0 for the group consuming diet cola; 13.7 ± 23.5 for group
consuming water [mean ± SEM]) and not different among the four groups. Subjects who consumed
aspartame-sweetened beverages in place of SSB also exhibited significant decreases of liver fat;
however, insulin sensitivity assessed by HOMA-IR was not significantly affected (Campos et al.
2015). In contrast, Schwarz and colleagues measured both hepatic and whole-body insulin sensitiv-
ity with hyperinsulinemic euglycemic clamps, along with an assessment of endogenous glucose pro-
duction (Schwarz et al. 2015). In addition to significant increases of DNL and liver fat content, they
also observed a significant decrease of hepatic insulin sensitivity (nonfasting endogenous glucose
production rate 30% higher during fructose consumption compared with complex carbohydrate con-
sumption, P < 0.01), while whole-body sensitivity was unchanged (Schwarz et al. 2015). Hepatic
insulin sensitivity, assessed by endogenous glucose production, was also decreased during 3 weeks
of moderate (80 g/day, ~13% Ereq) fructose consumption compared with glucose consumption,
while whole-body insulin sensitivity was not different (Aeberli et al. 2013). As already stated, these
two short-term studies support the sequence of events described in our proposed mechanism, in
which whole-body insulin resistance occurs downstream of hepatic insulin resistance. In our longer
10-week study, we observed a 17% decrease of whole-body insulin sensitivity in subjects consum-
ing fructose using deuterated glucose to measure glucose disposal through the glycolytic pathway
during an OGTT (Stanhope et al. 2009). Studies assessing the changes in insulin sensitivity during
sucrose and HFCS consumption utilizing techniques more sensitive than HOMA-IR are needed.
Other Biomarkers of MetS

Several studies have demonstrated that consumption of fructose and fructose-containing sugars
increase circulating uric acid (Cox et al. 2012b, Johnston et al. 2013, Bruun et al. 2015, Stanhope
et al. 2015), apolipoprotein B (Swarbrick et al. 2008, Stanhope et al. 2009, 2011a, 2015), and small
dense LDL (Stanhope et al. 2009, 2011a, Aeberli et al. 2011). Our recent data (Stanhope et al.
2015) also demonstrate that apoCIII is increased by HFCS consumption and that both uric acid
and apoCIII are strong biomarkers, and possibly mediators, of independent pathways by which
consumption of HFCS increases risk factors for CVD. C-reactive protein (CRP), a general marker
of inflammation, was increased in young healthy men after both fructose and glucose consumption
(40 or 80 g/day for 3 weeks) (Aeberli et al. 2011) and in older subjects with impaired glucose toler-
ance after sucrose, HFCS, or honey consumption (50 g/day for 2 weeks) (Raatz, Johnson, and Picklo
2015). Our group has not detected increases of CRP during sugar consumption in humans but have
observed increases of monocyte chemotactic protein-1 (MCP-1) and plasminogen activator inhibi-
tor-1 (Cox et al. 2011). In rhesus monkeys consuming fructose-sweetened beverages for 1 year,
MCP-1 and CRP were increased after 6 and 12 months (Bremer et al. 2011).
CONCLUSION
Evidence from epidemiological studies suggest that consumption of fructose-containing sugars
is associated with the prevalence and/or development of MetS and all of the established compo-
nents of the MetS, plus features of MetS that are not formally defined, such as insulin resistance,
fatty liver, and hyperuricemia. Results from dietary intervention studies showing that many of
these components and features are adversely affected when sugar intake is increased and benefi-
cially affected when sugar intake is decreased corroborate the epidemiological evidence. There
are plausible and interconnected mechanisms by which consumption of fructose-containing sug-
ars may lead to the development of the clustered, interrelated risk factors that constitute MetS.
Therefore, we believe that consumption of added sugars should be considered a modifiable risk
factor for MetS. The recommendation of the Cardiometabolic Think Tank to emphasize dietary
patterns such as the DASH and Mediterranean diets (Sperling et al. 2015) is sound. However,
specific recommendations to reduce consumption of added sugars to the levels recommended by
the 2015–2020 Dietary Guidelines for American (United States Department of Health and Human
Services and U.S. Department of Agriculture 2015) or to the even lower levels recommended by
the American Heart Association (Johnson et al. 2009) may be beneficial in the prevention and
management of MetS.
ACKNOWLEDGMENTS
The studies conducted by Drs. Havel and Stanhope’s research group were supported with funding
from NIH grants R01 HL-075675, 1R01 HL-091333, and 1R01 HL-107256 and a multicampus
award from the University of California, Office of the President (UCOP #142691). These projects
also received support from Grant Number UL1 RR024146 from the National Center for Research
Resources (NCRR), a component of the National Institutes of Health (NIH), and NIH Roadmap
for Medical Research. Dr. Stanhope is UC Davis School of Medicine Dean’s Scholar in Women’s
Health supported by the Office of the Dean, UC Davis School of Medicine.
CONFLICTS OF INTEREST
Drs. Stanhope and Havel have no conflicts of interest to report.
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15 Dietary Carbohydrate
Restriction in the
Management of NAFLD
and Metabolic Syndrome
Grace Marie Jones, Kathleen Mulligan,
and Jean-Marc Schwarz
CONTENTS
Introduction.....................................................................................................................................276
Scope of Disease.............................................................................................................................276
Hepatic Triglyceride Accumulation...........................................................................................276
Risk Factors and Etiology..........................................................................................................278
Epidemiology.............................................................................................................................279
NAFLD in Children...................................................................................................................280
Treatment...................................................................................................................................280
Effect of Carbohydrate Restriction on NAFLD..............................................................................281
Low-Carbohydrate Diets in NAFLD..........................................................................................281
Carbohydrate Quality.................................................................................................................285
Recommendations......................................................................................................................286
Other Dietary Treatments................................................................................................................286
Antioxidants...............................................................................................................................286
Probiotics and Prebiotics............................................................................................................287
Pharmaceutical Approaches to the Management of NAFLD..........................................................287
Insulin-Sensitizing Agents.........................................................................................................287
Bile Acid Analogs......................................................................................................................288
Pentoxifylline.............................................................................................................................288
Summary.........................................................................................................................................289
References.......................................................................................................................................289
ABSTRACT
Nonalcoholic fatty liver disease (NAFLD) and metabolic syndrome (MetS), which are influenced by
diet and genetics, contribute individually to the increased risk for cardiovascular disease and type 2
diabetes. Understanding the etiology of these diseases is paramount to the development of effective
preventive steps and treatments. Here, we focus on NAFLD as MetS has been covered elsewhere.
NAFLD is a cluster of diseases that ranges from accumulation of fat in the liver to inflammation
and to cirrhosis of the liver. Currently, the worldwide incidence of NAFLD is 24% and affects both
adults and children. The specific mechanisms by which the liver accumulates fat are unknown but
involve an imbalance in the flow of lipids entering and exiting the liver. Obesity plays a central
role, as adipose tissue is involved in many pathways that directly impact the pathology of NAFLD.
Interestingly, risk factors for NAFLD and its progression have been linked to specific polymorphisms
275
in genes that encode for proteins involved in the metabolism of hepatic lipids. Currently, the primary
treatment for NAFLD is weight loss and lifestyle changes. A hypocaloric diet and reductions in
carbohydrate intake have been shown to reduce the accumulation of hepatic fat in NAFLD patients.
Our recommendations for treating this patient population include not only the restriction of carbo-
hydrates but also a specific reduction in the intake of simple sugars. Pharmacological agents such
as vitamins, bile acid analogs, and probiotics and prebiotics are often prescribed in the presence of
other comorbidities such as type 2 diabetes.
INTRODUCTION
Nonalcoholic steatohepatitis (NASH) was first described in 20 patients at the Mayo Clinic in 1980
(Ludwig, Viggiano et al. 1980). Since then, our understanding of nonalcoholic fatty liver disease
(NAFLD) and NASH has evolved, and many efforts have gone into elucidating its etiology, diagnosis,
progression, and treatment. NAFLD is an umbrella term that describes a full spectrum of diseases
ranging from hepatic fat accumulation to cirrhosis of the liver (Kleiner, Brunt et al. 2005). NAFLD
is defined by histological analysis of liver biopsies showing fat infiltration in ≥5% hepatic cells
(Figure 15.1) of people consuming no more than one or two servings of alcohol per day in women and
men, respectively (Chalasani, Younossi et al. 2012). NAFLD is associated with other prevalent chronic
diseases such as obesity and metabolic syndrome (MetS) (Kang, Greenson et al. 2006, Masuoka and
Chalasani 2013), the symptoms of which include elevated fasting blood glucose, hypertension, dyslip-
idemia, and increased waist circumference. Together, these comorbid conditions increase the risk for
cardiovascular disease and type 2 diabetes (Targher, Bertolini et al. 2007, Siddiqui, Fuchs et al. 2015).
In this chapter, we focus on the effectiveness of carbohydrate restriction as a dietary intervention for
NAFLD. Additionally, we briefly discuss the progression of NAFLD, theories of etiology, risk factors,
and treatments, followed by dietary recommendations for NAFLD treatment.
SCOPE OF DISEASE
Hepatic Triglyceride Accumulation
The liver plays a central role in lipid metabolism, and hepatic triglyceride accumulation is the result
of an imbalance between the hepatic influx and efflux of fatty acids (FAs). On one hand, the liver
takes up circulating nonesterified fatty acids (NEFAs) and fat from triglyceride-rich lipoprotein
remnants and synthesizes FAs (de novo lipogenesis [DNL]) from carbohydrates, while on the other
hand, it secretes FAs as triglycerides in very-low-density lipoproteins (VLDL) and produces energy
from FAs through β-oxidation (Figure 15.2) (Liu, Bengmark et al. 2010). Triglyceride accumula-
tion in the liver is a disruption in the delicate balance of the uptake, synthesis, and export of FAs as
VLDL or hepatic oxidation (Fabbrini, Sullivan et al. 2010).
A proposed mechanism of NAFLD or ectopic fat accumulation is the presence of a positive
energy balance and limited adipose tissue storage capacity, leading to increased circulating NEFAs
and spillover into ectopic tissues including the liver (Mittendorfer, Magkos et al. 2009). In this
situation of large FA input, the liver is unable to oxidize or secrete the increased FAs and lipid
droplets accumulate. Obesity with adipose tissue insulin resistance can also increase lipolysis owing
to diminished suppression of hormone-sensitive lipase by insulin, thus resulting in increased free
FA flux to the liver. In addition, acute hyperinsulinemia inhibits the formation and secretion of
VLDL (Lewis, Uffelman et al. 1995), while the stimulation of DNL by insulin continues to pro-
duce new fat from carbohydrate. Together, these factors can lead to FA accumulation in the liver
(Tessari, Coracina et al. 2009). A 2005 study reported that DNL accounted for 26% of the FAs
present in the livers of individuals with NAFLD (Donnelly, Smith et al. 2005). Additionally, it has
been demonstrated that DNL is significantly elevated in insulin-resistant states (Tappy, Schwarz
et al. 1998, Tappy, Berger et al. 1999, Schwarz, Chiolero et al. 2000, Lo, Mulligan et al. 2001,
Dietary Carbohydrate Restriction in the Management of NAFLD and Metabolic Syndrome 277
(a) (b)
I S
(c) (d)
(e)
FIGURE 15.1 (See color insert.) Histological features of NAFLD. (a) Marked steatosis without inflam-
mation, hepatocytes injury (ballooning), or fibrosis. Steatosis is concentrated in acinar zone 3, the microcir-
culatory unit through which blood exists the liver around the terminal hepatic venule (in circle) and shows
sparing of the periportal, zone 1 hepatocytes, the microcirculatory unit through which portal and systemic
blood enter and mix. This is the adult pattern of nonalcoholic fatty liver disease (NAFLD) (trichrome staining).
(b) Steatohepatitis with marked steatosis (S), ballooning (B), lobular and portal inflammation (I) and extensive
bridging fibrosis (hematoxylin and eosin staining). (c) Fibrosis in the perisinusoidal spaces of zone 3 is detected
by trichrome stain; bridging fibrosis (arrow) is noted between two central veins. Hepatocytes with steatosis are
seen, but no ballooned hepatocytes are present. (d) Nonalcoholic steatohepatisis (NASH) with cirrhosis, but no
active lesions remain. One would only know this was a case of NASH-related cirrhosis by having had a prior
biopsy with the diagnosis of active NASH. (e) Hepatocellular carcinoma after the development of NASH and
cirrhosis. (Reprinted by permission from Macmillian Publishers Ltd.: Nat. Rev. Dis. Primers, (Brunt 2015)
Copyright, 2015.)
Lipolysis
(60%)
Triglycerides
Fatty
Dietary fat acids
(10%–15%)
Carbohydrate and DNL Oxidation

Hyperinsulinemia
(25%–30%)
VLDL secretion
FIGURE 15.2 (See color insert.) Contributions of various metabolic pathways to liver steatosis in humans.
Reductions in fatty acid oxidation and triglyceride export seem to have only minor roles in hepatic triglyceride
deposition. By contrast, the increased availability of fatty acids from the adipose tissue through unabated lipol-
ysis and de novo lipogenesis (DNL) from glucose are major providers of lipids in steatotic livers. (Reprinted
with permission from Taskinen, M.R. and Boren, J., Atherosclerosis, 239(2), 483, 2015.)
Schwarz, Mulligan et al. 2002, Schwarz, Linfoot et al. 2003) and in NAFLD (Lambert, Ramos-
Roman et al. 2014). The sterol regulatory element binding protein 1c (SREBP-1c), a transcription
factor essential for the expression of proteins involved in glycolysis, and carbohydrate response
element binding protein are normally expressed in response to insulin and glucose, respectively.
People with NAFLD overexpress SREBP-1c along with the genes encoding fatty acid synthase and
acetyl-CoA carboxylase, two key lipogenic enzymes (Higuchi, Kato et al. 2008, Lima-Cabello,
Garcia-Mediavilla et al. 2011). Importantly, expression of ACC leads to increased malonyl-CoA lev-
els and inhibition of carnitine palmitoyltransferase-1 (CPT-1), effectively reducing the liver’s ability
to dispose of lipids via mitochondrial β-oxidation. Thus, DNL impacts NAFLD by both increasing
intrahepatic de novo FA synthesis and decreasing hepatic FA oxidation. DNL is driven by both
carbohydrate consumption and hyperinsulinemia and may be a significant contributor to fatty liver.
The amount of liver fat, inflammation, and scar tissue determines the stage of NAFLD, as shown
in Figure 15.1 (Nalbantoglu and Brunt 2014, Brunt 2015). NAFLD can be divided into two catego-
ries: (1) steatosis or steatosis with inflammation (approximately 70%–75% of patients), and
(2) NASH, which is defined by the combination of steatosis with inflammation and cellular bal-
looning (~25%–30% of patients). Nearly 20% of patients with NASH progress to cirrhosis (Rinella
2015) (Figure 15.1d). NASH can also progress to fibrosis (Figure 15.1c). Hepatocellular carcinoma
can also occur in patients diagnosed with NASH or cirrhosis. The beginning stages of NAFLD are
asymptomatic; the initial diagnosis can be dependent on abnormal liver function tests, specifically
alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels (Maximos, Bril et al.
2015); histological analysis of a liver biopsy sample, which is the standard for NAFLD diagnosis
(Nalbantoglu and Brunt 2014); or radiologic imaging (Charatcharoenwitthaya and Lindor 2007).
Radiologic imaging includes (1) ultrasound (Dasarathy, Dasarathy et al. 2009), (2) noncontrast com-
puted tomography (CT) (Rofsky and Fleishaker 1995), and (3) magnetic resonance spectroscopy
(MRS) and/or imaging (MRI) (Reeder, Cruite et al. 2011).
Risk Factors and Etiology

Factors that play a role in the development and progression of NAFLD include metabolic and genetic
influences (Romeo, Kozlitina et al. 2008, Makkonen, Pietilainen et al. 2009), nutrition, inflammatory
bowel disease, HIV status (Cassol, Misra et al. 2013), infection with the hepatitis C virus (Patel and
Harrison 2012), the gut microbiota (Machado and Cortez-Pinto 2012, Mouzaki, Comelli et al. 2013),
adipose tissue, and the immune system. NAFLD is a multifactorial disease that has many mechanisms
that regulate its establishment and progression. A central factor is obesity (Hsiao, Kuo et al. 2007),
which is impacted by diet, genetic predisposition, level of physical activity, and the gut microbiota
(Greenhill 2015). Moreover, adipose tissue plays a role in increased circulation of free FAs (Donnelly,
Smith et al. 2005), insulin resistance, and proinflammatory mediators (Catalan, Gomez-Ambrosi et al.
2015). Each of these factors has separate downstream effects that directly impact the entire spectrum
of NAFLD from steatosis to fibrosis to cirrhosis and hepatocellular cancer. Additionally, disruptions in
the intestinal lining, caused by small intestinal bacterial overgrowth, can lead to the presence of endo-
toxins in the bloodstream, which enter the liver via the portal vein, contributing to the inflammatory
response and steatohepatitis (Cani, Amar et al. 2007, Miele, Valenza et al. 2009).
Genome-wide association studies have identified loss-of-function gene variants that are involved in
hepatic lipid metabolism and play a major role in the pathogenesis of NAFLD (Dongiovanni, Romeo
et al. 2015). The I148M variant of the patatin-like phospholipase domain-containing 3 gene, PNPLA3
or adiponutrin, a lipase involved in the hydrolysis of triglycerides (Pingitore, Pirazzi et al. 2014), was
strongly associated with increased fat levels and inflammation in the liver. Interestingly, Hispanics as a
group are more susceptible to NAFLD and are more likely to have the I148M variant than African- or
European-Americans (Romeo, Kozlitina et al. 2008). A second variant, E167K of the transmembrane 6
superfamily member 2 gene, was found in two separate studies (Holmen, Zhang et al. 2014, Kozlitina,
Smagris et al. 2014) and has been shown to regulate hepatic lipid metabolism via VLDL secretion
(Mahdessian, Taxiarchis et al. 2014). Additionally, the glucokinase regulator P446L polymorphism has
a significant association with NAFLD and has been shown to increase DNL, liver fibrosis, and tri-
glyceride levels (Speliotes, Yerges-Armstrong et al. 2011, Wu, Lemaitre et al. 2013, Petta, Miele et al.
2014, Aguilar-Olivos, Almeda-Valdes et al. 2015, Santoro, Caprio et al. 2015). Other genes involved in
hepatic lipid metabolism, including FATP5, LYPLAL1, NCAN, PPAR, PPP1R3B, LPIN1, TRIB1, and
UCP2,* have been implicated in the pathogenesis of NAFLD. However, further research is needed to
clarify their suspected roles (Speliotes, Yerges-Armstrong et al. 2011, Kitamoto, Kitamoto et al. 2014).
A handful of clinical studies on NAFLD and the gut microbiota have shown a change in the
abundances of particular phyla in people with NAFLD and NASH when compared with healthy
controls. While a direct role of the gut microbiota has not been demonstrated, evidence for its role
in insulin resistance and obesity, two risk factors for NAFLD, has been suggested (Ley, Turnbaugh
et al. 2006, Cani and Delzenne 2009, Caricilli and Saad 2013). Emerging data will elucidate the role
of the gut microbiota and its interaction with diet in the development of CVD, obesity, type 2 diabe-
tes, and NAFLD. Other comorbid conditions in which NAFLD has been described include polycys-
tic ovary syndrome (Karoli, Fatima et al. 2013, Vassilatou, Vassiliadi et al. 2015), hypothyroidism
(Sert, Pirgon et al. 2013, Ludwig, Holzner et al. 2015), obstructive sleep apnea (Chou, Liang et al.
2015, Qi, Huang et al. 2015), hypopituitarism and hypogonadism (Hazlehurst and Tomlinson 2013),
Hepatitis C (Bondini and Younossi 2006), and HIV (Guaraldi, Squillace et al. 2008).
Epidemiology
A recent meta-analysis reported that the global prevalence of NAFLD was 24% (Zhu, Dai et al. 2015).
This number, however, might be misleadingly low in that the reported prevalence of NAFLD varies widely
and is highly dependent on the method of diagnosis and the population studied. Estimated prevalence is
20%–30% in Western countries and 5%–18% in Asia (Satapathy and Sanyal 2015). The prevalence of
NAFLD in different studies in Asian countries has ranged from 27% in China (264 of 922 randomly
selected subjects via MRS; [Wong, Chu et al. 2012]), 18% in Japan (in 47 of 263 living liver donors via
* FATP5, fatty acid transport protein 5; LYPLAL1, lysophospholipase-like 1; NCAN, neurocan; PPAR, peroxisome prolifera-
tor-activated receptor; PPP1R3B, protein phosphatase 1 regulatory subunit 3B; LPIN1, lipin 1; TRIB1, tribbles homologue
1; and UCP2, uncoupling protein 2.
biopsy) (Yamamoto, Takada et al. 2007), 51% in Korea (303 of 589 living liver donors via biopsy, ultra-
sound, and/or CT) (Lee, Kim et al. 2007), and in India, depending on the region, between 9% and 35% via
ultrasound and/or CT (Singh, Nayak et al. 2004, Amarapurkar, Kamani et al. 2007, Mohan, Farooq et al.
2009, Das, Das et al. 2010). Conversely, studies performed in western countries have reported the preva-
lence of NAFLD to be 20%–25% in Italy (57 of 287 subjects without suspected liver diseases versus 78
of 311 subjects with suspected liver disease via ultrasound) (Bedogni, Miglioli et al. 2005), 26% in Spain
(198 of 766 subjects in a cross-sectional population study via ultrasound) (Caballeria, Pera et al. 2010),
and 26% in the United Kingdom (295 of 1118 primary care patients with abnormal liver function tests
via ultrasound) (Armstrong, Houlihan et al. 2012). In the large U.S. Dallas Heart Study that used MRS,
the prevalence of hepatic steatosis was 34% (Browning, Szczepaniak et al. 2004). The prevalence in this
study varied based on ethnicity: 45% in Hispanics, 33% in Whites, and 24% in Blacks. There were no
differences between males and females among Hispanics or Blacks; however, the prevalence of steatosis
in white men was greater than that in white women (42% versus 24%, respectively).
NAFLD in Children
The prevalence of NAFLD in the pediatric population is increasing in parallel with the rise in child-
hood obesity. Children are susceptible to the same risk factors for NAFLD as adults, including influ-
ences of race/ethnicity, family history, and genetic and environmental factors. One environmental
factor unique to this population is the feeding regimen during infancy. A 2009 study showed that
children breastfed 0.5–17 months (median = 8 months, n = 91) were less likely to develop NASH
and fibrosis compared to formula-fed children (Nobili, Bedogni et al. 2009). Furthermore, longer
duration of breastfeeding, up to 17 months, was associated with lower risk of NASH. Presently, the
treatment of NAFLD in children is focused on reducing obesity through weight loss and negative-
energy balance/energy restriction diets (Nobili, Alkhouri et al. 2015).
Treatment
Presently, weight loss and lifestyle changes are the primary NAFLD-specific treatments recommended.
Other approaches, which will be discussed later, include use of insulin-sensitizing agents, antioxidants,
and other agents. Current weight loss methods used to treat NAFLD/NASH can be categorized as
follows: (1) behavior therapy/lifestyle modification, (2) anti-obesity drugs, and (3) weight loss sur-
gery. The magnitude of weight loss has a proportional relationship with disease improvement (Promrat,
Kleiner et al. 2010). Behavioral therapy (Centis, Marzocchi et al. 2010, Moscatiello, Di Luzio et al.
2011), increased physical activity (Bae, Suh et al. 2012), and dietary recommendations should lead to a
decrease in caloric intake, increase in energy expenditure, and consequent weight reduction (Chalasani,
Younossi et al. 2012). While specific exercises and diets have not been identified to treat NAFLD, high-
intensity exercise (Oh, Shida et al. 2015) and carbohydrate restriction (40% CHO versus 60% CHO)
(Ryan, Abbasi et al. 2007, Browning, Baker et al. 2011) (<10% CHO versus low-fat diet, 45%–65%
CHO) (Ryan, Abbasi et al. 2007, Browning, Baker et al. 2011) when compared to other diets with simi-
lar weight loss have been shown to significantly reduce the level of hepatic fat. Interestingly, a recent
meta-analysis of 28 randomized trials (n = 1644 randomized patients) showed that exercise alone was
associated with reductions of hepatic lipids (standardized mean difference, −0.69), AST and ALT levels,
and weighted mean difference, −4.85 IU/L and −3.30 IU/L, respectively (Orci, Gariani et al. 2016).
Additionally, Orci et al. found that subjects with a higher baseline BMI benefitted from physical activity
with respect to lowering liver fat. Orlistat, a proven weight loss aid, functions to reduce dietary fat absorp-
tion and is used to manage people with obesity (Rucker, Padwal et al. 2007). However, it is currently not
recommended as a stand-alone treatment for NAFLD as studies have shown no histologic improvements
with its use (Chalasani, Younossi et al. 2012). Finally, bariatric surgery, which leads to significant weight
loss, includes Roux-en-Y gastric bypass, laparoscopic adjustable banding, sleeve gastrectomy, or bilio-
pancreatic diversion with duodenal switch, has been shown to improve AST and ALT levels and liver
histology (Bower, Toma et al. 2015). However, controlled studies have not evaluated bariatric surgery as
a treatment for NAFLD, and therefore, this procedure is not a primary treatment (Chalasani, Younossi
et al. 2012, Aguilar-Olivos, Almeda-Valdes et al. 2015).
EFFECT OF CARBOHYDRATE RESTRICTION ON NAFLD

A central factor in the pathophysiology of NAFLD is obesity, and therefore the primary component
of NAFLD treatment is through weight loss and lifestyle modification, including diet and physical
activity. As described earlier, many studies have shown weight loss to promote the reduction of
hepatic fat in NAFLD patients. Furthermore, the association of NAFLD with other chronic diseases,
such as type 2 diabetes and cardiovascular disease, has led to investigations that include the redis-
tribution of macronutrients, increased macronutrient quality, or specific dietary patterns
(i.e., Mediterranean diet). Here we focus on the studies that sought to assess the effect of carbohy-
drate (CHO) restriction, with or without weight loss, on NAFLD.
Low-Carbohydrate Diets in NAFLD (See Table 15.1)

Based on the USDA guidelines, the current recommended distribution of macronutrient intake ranges
from 45% to 65% of calories from carbohydrates, 5% to 35% of calories from protein, and 20%
to 35% of calories from fat (U.S. Department of Health and Human Services and U.S. Department
of Agriculture 2015). A low-carbohydrate or carbohydrate-restricted diet would typically limit the
calories from carbohydrate to less than 35% (Sedlacek, Playdon et al. 2011, Tay, Luscombe-Marsh
et al. 2015), whereas a high-carbohydrate diet generally consists of a minimum of 45% of calories
derived from carbohydrates (Krebs, Elley et al. 2012, Masters, Aarabi et al. 2012). Several studies
have examined the effect of carbohydrate restriction on NAFLD and other associated risk factors
such as weight, liver enzyme levels, and glucose metabolism (Table 15.1). Table 15.2 summarizes
the macronutrient content in these studies.
In 2005, Huang et al. conducted a pilot study that investigated the impact of intense 1-year
nutritional counseling and moderate carbohydrate intake in overweight and obese people with
liver biopsy–diagnosed NASH. They found that 60% of participants responded favorably to the
intervention. In patients with an improved histological score, there were significant mean reduc-
tions in weight (7%), BMI (−2.25 kg/m2), waist circumference (−6.94 cm), and aminotransferase
levels (−15.44 IU/L AST and −33.11 IU/L ALT) (Huang, Greenson et al. 2005). Additionally,
these individuals demonstrated reductions in liver fat and total NASH score. This study sug-
gested that intense nutritional education is an important aspect of weight loss and improvement
in NASH scoring. However, they did not address how the modification of macronutrient con-
tent, in conjunction with nutritional education, affected features of NAFLD/NASH as there was
no control group. To that end, two groups examined the effect of dietary counseling combined
with either a hypocaloric low-carbohydrate diet (<90 g CHO) versus a hypocaloric low-fat diet
(Haufe, Engeli et al. 2011) or an isocaloric low-fat, high-carbohydrate diet versus an ad libitum
Mediterranean diet (Ryan, Itsiopoulos et al. 2013). The study by Haufe et al. aimed to assess
whether reducing overall caloric intake by 30% decreased liver fat or whether the specific reduc-
tion of either carbohydrate or fat calories caused a decline in ectopic liver fat. In this randomized
study of overweight and obese subjects, some with a high liver fat content (assessed by MRS),
both the low-carbohydrate and low-fat diets had similar effects on weight loss and reductions
in liver fat. The authors concluded that these improvements were due to the reduction in caloric
intake and not to a shift in relative percentages of macronutrients (Haufe, Engeli et al. 2011).
Ryan et al. utilized a randomized crossover intervention study design to evaluate how insulin
sensitivity and liver fat are impacted by a low-fat, high-carbohydrate diet (30% calories from fat,
50% from carbohydrate, and 20% from protein) versus the Mediterranean diet (40% calories from
fat, 40% from carbohydrate, and 20% from protein) in subjects with NAFLD. This study found
282
TABLE 15.1
Characteristics and Results of Dietary Intervention Studies in Subjects with NAFLD, NASH, or High Liver Fat
First Author, Measure
Pub Year Subjects n BMI (kg/m2) Assessments Liver Fat Intervention/Protocol Results
Browning NAFLD (MRS) 18 35.7 ± 7 Hepatic TG, weight MRS 2 weeks: <20 CHO g/day Both diets: ↓ liver fat
et al. (2011) 9-CRD (CRD) or 1200–1500 (42%) and ↓ weight
9-CaloricRest. calories/day (caloric rest.) (4.3%), greater loss with
CRD diet.
Duarte et al. NAFLD 48 31.8 ± 4.7 AST, ALT, BMI, lipids, N/A 75 days: high-protein, Improvements in lipid
(2014) (biopsy), HOMA, HbA1c. insulin, hypocaloric diet, 25% kcal profile, glucose
overweight or body composition Fat. 35% protein. 40% CHO. homeostasis, and liver
obese 20 g fiber/day enzymes.
Haufe et al. Overweight and 102 28–40.6 Liver fat, SAT, VAT MRS 6 months: weekly group Both diets resulted in
(2011) obese, some Lipids, Glucose meetings + hypocaloric diet similar ↓ liver fat, ↓SAT,
with high liver metabolism, ALT, AST, (reduced by 3 0%): ≤90 g ↓VAT, ↓ weight loss.
fat content biochemical parameters CHO, 0.8 g protein/kg Low CHO greater ↑
weight, ≥3 0% fat (Low insulin sensitivity, ↓
CHO) or ≤20% fat,0.8 g total and LDL
protein/kg weight, remaining cholesterol, ↓ TGF-β1.
kcal from CHO (low fat)
Huang et al. Overweight/ 15 34 ± 7 Hepatichistological Biopsy 1 year: 40%–45%CHO Improvements in HOMA,
(2005) obese with improvement, insulin (complex + Fiber), liver histology ↓ weight,
NASH resistance, SAT, VAT, 35%−40% fat ( MUFAs and ↓ BMI, ↓ TAT, ↓ VAT, ↓
(biopsy) PUFAs), and 15%–20% AST, ↓ ALT (reductions
protein not significant).
(Continued)
Characteristics and Results of Dietary Intervention Studies in Subjects with NAFLD, NASH, or High Liver Fat
First Author, Measure
Pub Year Subjects n BMI (kg/m2) Assessments Liver Fat Intervention/Protocol Results
Kani et al. NAFLD 45 low kcal, 15, 45.6 ± 2.6 low kca1, Liver enzymes, N/A 8 weeks: low kcal diet, 55% All three groups
(2014) (sonography) low kcal- CHO, 49.3 ± 3.5 low coagulating factors, lip CHO: 15% protein: 30% fat improved, greatest
15, low kcal kcal- CHO,48.5 ± id profiles, EMI, weight, or low kcalCHO, 45%CHO: improvement of
CHO, soy, 15 3.7 low kcal-CHO, 20% protein: 35% Fat, 30 g assessments in Low
soy red meat or Low kcal-CHO, kcal-CHO with 30 g soy
45% CHO: 20% protein: nut group.
35% Fat with 30 g soy nut
Perez- Overweight 14 36.5 ± 0.54 Steatosis, weight, BMI, Ultrasound 12 weeks: Spanich Ketogenic All subjects cured of MS,
Guisado with metabolic LDLc, ALT, AST, Mediterranean Diet (SKMD): significant reduction in all
et al. (2011a) syndrome glycemia, blood unlimited kcal, ≤30 green measured assessments.
(MetS) and pressure, TAGs vegetable CHO, ≥30 mL
NAFLD virgin olive oil, 200–300 mL
wine
Ryan et al. NAFLD 12 32.0 ± 4.2 Hepatic steatosis and 1
H-MRS 6 weeks: dietary counseling, LF-HCD: ↓, liver fat
(2013) (biopsy), Insulin sensitivity low-fat, high-CHO (7%), no change in
nondiabetic (LF-HCD) and 5 weeks: insulin sensitivity, MD:
dietary counseling, ↓ liver fat (39%), ↑
Mediterranean diet(MD) insulin Sensitivity
Dietary Carbohydrate Restriction in the Management of NAFLD and Metabolic Syndrome
283
TABLE 15.2
Macronutrient Composition of Dietary Intervention Studies in Subjects with NAFLD,
NASH, or High Liver Fat
1st Author, Pub Year Macronutrient Composition
Browning et al. (2011) 50% carbohydrate: 16% protein: 34% fat versus
8% carbohydrate: 33% protein: 59% fat
Duarte et al. (2014) 40% carbohydrate: 35% protein: 25% fat plus 20 g fiber/day
Haufe et al. (2011) ≤90 g carbohydrates, 0.8 g protein per kg body weight, and a minim urn of 30% fat versus
Fat content of ≤20% of total energy intake, 0.8 g protein per kg body weight, and the
remaining energy content provided by carbohydrates
Huang et al. (2005) 40%−45% of daily calories from carbohydrates with an emphasis on complex carbohydrates
with fiber, 35%−40% fat (emphasized mono-and polyunsaturated fats), and 15%–20% protein
Kani et al. (2014) 55% carbohydrate:15% protein: 30% Fat versus
45% carbohydrate: 20% protein: 35% Fat, 30 g red meat versus
45% carbohydrate: 20% protein: 35% Fat with 30 g soy nut
Perez-Guisado et al. (2011) <30 g of carbohydrates in the form of green vegetables and salad, a minimum of 30 mL of
virgin olive oil, 200–400 mL of red wine, unlimited protein
Ryan et al. (2013) 40% carbohydrate: 40% fat (mono- and polyunsaturated fats): 20% protein versus
50% carbohydrate: 30% fat: 20% protein
that the low-fat, high-carbohydrate diet reduced liver fat by 7% (assessed by MRS) and did not
change insulin sensitivity (assessed by hyperinsulinemic-euglycemic clamp), whereas the moder-
ate carbohydrate restriction diet (Mediterranean diet) resulted in a 39% decrease in liver fat and
increased insulin sensitivity, both without weight loss (Ryan, Itsiopoulos et al. 2013). Together,
these studies show that while nutritional education is important (education included group and
individual nutrition counseling emphasizing healthy choices by a nutritionist and the promotion
of gradual weight loss by a registered dietician), two of the three studies show that macronutrient
content, particularly the reduced carbohydrate, results in improved NAFLD features. However,
one study suggested that the reduction of calories plays a greater role in the reduction of liver fat
than the reduction of carbohydrate.
This latter issue was addressed in part by Browning et al. (Browning, Baker et al. 2011) and
Kani et al. (Kani, Alavian et al. 2014) by evaluating whether carbohydrate restriction or caloric
restriction reduces hepatic triglycerides and whether a low-carbohydrate, low-calorie diet has a
beneficial role in NAFLD. The Browning study found both a 2-week very low–carbohydrate diet
(8% calories from CHO) and a caloric restriction diet (1200–1500 kcal/day, 50% calories from
CHO) resulted in a 4.3% weight loss and a 42% reduction of hepatic triglycerides as measured by
MRS. However, when the effects on liver fat, measured by MRS, were examined for each diet, the
reduction of liver fat was considerably greater with the CHO-restricted diet (55% versus 28% for
the caloric restriction diet) (Browning, Baker et al. 2011). The second study controlled for caloric
intake, with a 200–500 kcal/day reduction of required intake, and randomized subjects to: (1) low-
calorie diet, 55% CHO:15% protein:30% fat, or (2) a low-calorie, high-carbohydrate diet, 45%
CHO:20% protein:35% fat, or (3) low-calorie, low-carbohydrate diet + soy, 45% CHO:20% pro-
tein:35% fat plus 30 g soy protein and 30 g red meat. This 8-week study found that a low-calorie,
high-carbohydrate, soy-containing diet significantly reduced ALT levels and serum fibrinogen
levels, markers of liver function and inflammation, respectively, as compared to a low-calorie diet
or a low-calorie, moderate-carbohydrate diet (Kani, Alavian et al. 2014). Taken together, these
studies show that hypocaloric diets improved features of NAFLD. However, a hypocaloric diet
combined with a CHO-restricted diet resulted in greater improvements in a short-term study of
both 2 and 8 weeks.
Interestingly, Kani et al. found that a moderately high protein intake of 20% of calories supple-
mented with or without 30 g of soy was beneficial to weight reduction and lipid and metabolic
markers. While the inclusion of soy in the diet tended to be associated with greater improvements,
the differences were not statistically significant compared to the diet without soy. The researchers
hypothesized that the components of soy might reduce NAFLD-induced inflammation. It is unclear
whether these effects resulted from higher protein intake per se or reduction in carbohydrate calories
by substitution of protein. In the liver, protein is important for hepatocyte regeneration, lipopro-
tein assembly, and lipid export. While research on the effect of high-protein diets and NAFLD has
not been done, early studies demonstrate a positive effect. Duarte et al. showed that a hypocaloric
high-protein, low-carbohydrate diet (35% calories from protein and 40% calories from CHO) in
NAFLD patients resulted in improvements in lipid profile, glucose homeostasis, and liver enzymes
compared to baseline (Bezerra Duarte, Faintuch et al. 2014). A prospective study that investigated
the effect of the Spanish Ketogenic Mediterranean Diet (SKMD), a very low–carbohydrate (≤30 g
of carbohydrate), high-protein diet, in overweight Spanish men found that the 12-week interven-
tion caused significant reductions in body weight and aminotransferase levels (Perez-Guisado and
Munoz-Serrano 2011a). Importantly, 21.4% of subjects experienced total regression of fatty liver as
measured by abdominal ultrasonography (Perez-Guisado and Munoz-Serrano 2011a). The SKMD
has also been used to successfully treat risk factors for NAFLD, such as MetS and obesity (Perez-
Guisado, Munoz-Serrano et al. 2008, Perez-Guisado and Munoz-Serrano 2011b). Although further
studies are needed to verify these results, a high-protein, low-carbohydrate diet may be beneficial
to NAFLD patients.
Carbohydrate Quality
Recent studies have suggested that not only carbohydrate quantity but also carbohydrate quality (simple
versus complex) can play a role in influencing liver fat content. In a pilot study, eight healthy adults
hospitalized in a metabolic ward for 19 days were given two weight-maintaining isocaloric diets with
the same macronutrient distribution (15% protein, 35% fat, 50% carbohydrate) for consecutive 9-day
periods. During one dietary period, 25% of energy came from fructose; during the other periods, complex
carbohydrate was substituted for fructose (Schwarz, Noworolski et al. 2015). While weight was stable on
both diets, the high-fructose diet resulted in higher levels of DNL (18.6% ± 1.4% versus 11.0% ± 1.4%,
complex carbohydrate; P = 0.001) and higher liver fat, measured by MRS, in all participants (137%
of values with complex carbohydrate). In an outpatient feeding study in obese children who reported
high levels of sugar intake (>15% sugar and >5% fructose of daily caloric intake), 9 days of fructose
restriction with isocaloric substitution of complex carbohydrate resulted in reductions in fasting glucose,
insulin, and lipids (Lustig, Mulligan et al. 2016). Importantly, these same subjects had a 56% decrease in
DNL-Area Under the Curve (n = 40) and 22% reduction in liver fat (n = 36) (Schwarz, Noworolski et al.
2017). Although the individuals in each of these studies were not selected for liver fat levels (≥5%), these
carefully controlled dietary studies suggest that carbohydrate quality, simple carbohydrates, specifically
fructose, plays an important role in liver fat accumulation and that DNL is a contributor to liver fat.
The relationship between fructose, lipogenesis, and liver fat is notable. Fructose is a potent stimu-
lator of DNL in that its metabolism bypasses the first regulatory enzyme of the glycolytic pathway.
Because the enzymes for fructose metabolism are entirely hepatic, it is essentially metabolized in
the liver and provides lipogenic precursors such as acetyl-CoA for DNL. Animal studies have shown
that fructose is converted to FAs at rates of up to 18.9 times greater than glucose (Mayes and Laker
1986) and produces increased hepatic triglyceride levels (Thorburn, Storlien et al. 1989). A 2005
study in healthy humans showed that fructose is a potent lipogenic substrate and that it significantly
increases hepatic triglyceride levels (Faeh, Minehira et al. 2005). Therefore, an increase in fructose
providing lipogenic precursors and an increase in DNL can contribute to hepatic FA accumulation or
NAFLD (Fabbrini, Sullivan et al. 2010); conversely, a reduction in dietary fructose intake can lead
to decreases in liver fat content.
Recommendations
Many studies have evaluated the effects of low-carbohydrate versus low-fat diets for the prevention
of cardiovascular disease, type 2 diabetes, and MetS on liver function but not specifically in the
context of NAFLD. A meta-analysis of randomized controlled clinical trials found that both diets
reduced body weight and other risk factors for NAFLD (Hu, Mills et al. 2012). Based on human
studies and the known mechanisms that promote accumulation of liver fat, we recommend the
following nutritional measures to reduce the risk for cardiovascular disease, type 2 diabetes, and
hyperlipidemia in NAFLD patients:
• Intense nutritional education and lifestyle support group.

• If weight loss is indicated, a hypocaloric diet, to improve aminotransferase levels and pro-
mote weight reduction (Andersen, Gluud et al. 1991, Petersen, Dufour et al. 2005, Ryan,
Abbasi et al. 2007).
• A diet with a total carbohydrate intake of less than 45% (Huang, Greenson et al. 2005, Browning,
Baker et al. 2011, Haufe, Engeli et al. 2011, Perez-Guisado and Munoz-Serrano 2011, Ryan,
Itsiopoulos et al. 2013, Bezerra Duarte, Faintuch et al. 2014, Kani, Alavian et al. 2014).
• A diet with reduced simple sugar intake, especially fructose, to less than 10% of caloric
intake (Volynets, Machann et al. 2013, Schwarz, Noworolski et al. 2015, Gugliucci, Lustig
et al. 2016, Lustig, Mulligan et al. 2016).
• Dietary fats should be high in mono- and polyunsaturated FAs (Bjermo, Iggman et al.
2012, Bozzetto, Prinster et al. 2012, Rosqvist, Iggman et al. 2014).
OTHER DIETARY TREATMENTS

Antioxidants
Oxidative stress is thought to play a key role in the development of NAFLD (Satapati, Kucejova
et al. 2016). Because of this, antioxidants, specifically vitamin E, a free-radical scavenger, have
been the focus of several randomized clinical trials. The results of the adult Pioglitazone ver-
sus Vitamin E versus Placebo for the Treatment of Non-diabetic Patients with Nonalcoholic
Steatohepatitis (PIVENS) trial, which investigated the potential therapeutic effect of vitamin E
(800 IU/day) or pioglitazone (30 mg/day) or placebo over 96 weeks, showed that treatment with
vitamin E resulted in an improvement in histologic scoring compared to placebo and pioglitazone
(Sanyal, Chalasani et al. 2010). Both vitamin E and pioglitazone resulted in significant reduc-
tions in AST and ALT levels compared to placebo. Additionally in a meta-analysis of nine trials
(n = 119), vitamin E was found to reduce the levels of AST and ALT, markers of disease status
in patients with NAFLD, NASH, and chronic hepatitis C (Ji, Sun et al. 2014). Interestingly, the
results of the Treatment of Nonalcoholic Fatty Liver Disease in Children study, in which Vitamin
E, metformin, and placebo were tested, significant reductions in both hepatocellular ballooning
and NAFLD activity score* were observed in all test groups (Lavine, Schwimmer et al. 2011),
similar to the adult PIVENS study that tested pioglitazone, vitamin E, and placebo. Additionally,
there was no difference between the groups in ALT levels. While vitamin E continues to be con-
sidered a NAFLD/NASH therapeutic, its exact mechanism and benefit need further investigation
(Chalasani, Younossi et al. 2012).
Polyphenols are naturally occurring antioxidants found in a range of foods including onions,
broccoli, apples, grapes, milk thistle, and curry (Salomone, Godos et al. 2015). Several random-
ized clinical trials in people with NAFLD examined the efficacy of polyphenols and have shown
* The NAFLD activity score assessed on a scale of 0–8, with higher scores indicating more disease. The components of this
measure includes steatosis (0–3), lobular inflammation (0–3), and hepatocellular ballooning (0–2) (Lavine, Schwimmer
et al. 2011).
beneficial effects such as significant reductions in ALT, AST, and insulin resistance with resveratrol,
found in red grapes, mulberries, peanuts, and cocoa (Faghihzadeh, Adibi et al. 2014, Chen, Zhao
et al. 2015). Additionally, silymarin alone, a component of milk thistle, resulted in reductions in
AST and ALT levels (Solhi, Ghahremani et al. 2014). While these results are promising, further
clinical trials are needed to understand dosage, bioavailability, and potential toxicities, as well as
the impact of polyphenols on inflammation and fibrosis, and the role of polyphenols in NAFLD
treatment.
Probiotics and Prebiotics

The gut microbiota has recently gained much attention due to new techniques that allow for its char-
acterization and investigations into its modulation. The liver is directly linked to the intestines by
the portal vein and is therefore influenced by the nature of the bacterial population, such as LPS, or
by its effects, increased gut permeability (Cani, Amar et al. 2007). Together, these might contribute
to the pathogenesis of NAFLD. There is much interest in the potential of probiotics and prebiotics
in the management of NAFLD. However, pending clinical trials in humans and the need to identify
a clear mechanism of action leave many questions unanswered (Aller, De Luis et al. 2011, Kirpich,
Marsano et al. 2015, Lambert, Parnell et al. 2015).
PHARMACEUTICAL APPROACHES TO THE MANAGEMENT OF NAFLD

Currently, there are no approved pharmacological treatments for the management of NAFLD.
However, there are emerging agents that have promise, including insulin-sensitizing agents, gluca-
gon-like peptide-1 (GLP-1) agonists, and bile acid analogs.
Insulin-Sensitizing Agents
Insulin resistance is thought to play a critical role in the pathogenesis of NAFLD and is strongly
associated with an increased risk for type 2 diabetes and cardiovascular disease (Eguchi, Eguchi
et al. 2006). Accordingly, many have studied the effectiveness of the biguanide metformin and
thiazolidinediones such as pioglitazone, two classes of insulin sensitizers, for the management of
NAFLD. Metformin, an antidiabetic drug, is believed to function primarily by reducing hepatic
gluconeogenesis, stimulating glucose uptake by muscle, and increasing FA oxidation in both
skeletal muscle and liver to increase insulin sensitivity. Several trials that examined the effec-
tiveness of metformin in NAFLD showed reductions in ALT and insulin resistance (Marchesini,
Brizi et al. 2001, Schwimmer, Behling et al. 2005, Nobili, Marcellini et al. 2006, Duseja, Das
et al. 2007, Loomba, Lutchman et al. 2009, Nadeau, Ehlers et al. 2009); some have also shown
histologic improvements and lower AST levels (Nair, Diehl et al. 2004, Bugianesi, Gentilcore
et al. 2005). However, a meta-analysis (Rakoski, Singal et al. 2010) of three controlled trials
(n = 96) in which aminotransferase levels and/or histology were measured demonstrated that
there were no differences between the metformin versus control groups (Uygun, Kadayifci et al.
2004, Haukeland, Konopski et al. 2009). As such, metformin is not currently recommended as
a NAFLD-specific treatment but is used for patients with concurrent diabetes and insulin resis-
tance (Chalasani, Younossi et al. 2012, Spengler and Loomba 2015). The thiazolidinedione drug
class, to which Pioglitazone belongs, binds peroxisome proliferator-activated receptor gamma
(PPARγ), a transcription factor that activates genes involved in regulating FA storage and glucose
metabolism. Pioglitazone acts as an insulin sensitizer by promoting adipocyte differentiation and
the redistribution of liver and muscle fat to adipose tissue (Miyazaki, Mahankali et al. 2002).
The aforementioned PIVENS study (Sanyal, Chalasani et al. 2010) and Belfort et al. (Belfort,
Harrison et al. 2006) showed pioglitazone improved AST and ALT serum levels, steatosis, and
steatohepatitis (assessed by biopsy and MRS). While long-term use is required, the safety and
efficacy of pioglitazone has not been evaluated. Finally, the double-blind, randomized liraglutide
safety and efficacy in patients with non-alcoholic steatohepatitis (LEAN) study tested the effect
of daily liraglutide injections for 48 weeks in overweight patients with biopsy-proven NASH
(Armstrong, Gaunt et al. 2016). The LEAN study found that 39% (n = 9/23) of patients taking
liraglutide showed resolution of NASH compared to 9% (n = 2/22) of the control group. Overall,
the liraglutide group was more likely to have improvements in steatosis, 83% versus 45% of the
control group. Interestingly, the patients that received liraglutide had significant reductions in
weight, BMI, and HbA1c levels as compared to the placebo group. Together, these improvements
show the effectiveness of liraglutide and the need for longer and larger randomized controlled
trials. Other classes of drugs that need further investigation include dipeptidyl peptidase-4 inhibi-
tors, other GLP-1 receptor agonists (Li, Zhao et al. 2015), and renal sodium glucose transporter
blockers (Jung, Jang et al. 2014).
Bile Acid Analogs

Obeticholic acid, a synthetic variant of chenodeoxycholic acid, functions to activate the farnesoid
X receptor (Cariou, van Harmelen et al. 2006, Porez, Prawitt et al. 2012). Activation of this receptor
results in improved insulin sensitivity and decreases in hepatic gluconeogenesis and plasma triglyc-
eride levels (Cariou, van Harmelen et al. 2006, Porez, Prawitt et al. 2012). A randomized controlled
study evaluated the efficacy of obeticholic acid in 283 diabetic and nondiabetic volunteers with NASH
(farnesoid X nuclear receptor ligand obeticholic acid for non-cirrhotic, non-alcoholic steatohepatitis
study). After 72 weeks, twice the number of subjects treated with obeticholic acid had histological
improvements compared to the placebo group (n = 50 [45%] versus n = 23 [21%]) (Neuschwander-
Tetri, Loomba et al. 2015). Additionally, the NAFLD activity score and ALT were both reduced and
significantly different between the two groups, favoring the treated group. The results of this study
show obeticholic acid to be a promising treatment for NASH. However, 23% (n = 33 of 141) of the
treatment group suffered from pruritus as compared to 6% (n = 9 of 142) in the placebo-controlled
group and an increase in LDL and total cholesterol in the treatment group as compared to the control
group. The long-term safety profile and efficacy of the drug requires further study.
A second synthetic molecule, aramchol, composed of cholic acid, a bile acid, and arachidic
acid, a saturated FA, functions to inhibit stearoyl coenzyme A desaturase 1 (SCD1). SCD1 is an
enzyme involved in the biosynthesis of FAs, and preclinical studies have shown that its inhibition
decreases the synthesis of FAs and increases β-oxidation in the liver and brown adipose tissue
(Dobrzyn, Dobrzyn et al. 2004, Dobrzyn and Ntambi 2005). Recently, a randomized 3-month
trial demonstrated the efficacy and short-term safety of aramchol in people with NAFLD (n = 20)
(Safadi, Konikoff et al. 2014). Specifically, there was an average 12.6% decrease in liver fat in
those randomized to aramchol compared to a 6.4% increase in liver fat in the control group. While
these results are promising, the long-term effects of aramchol have not yet been evaluated.
Pentoxifylline
The efficacy of pentoxifylline, a nonspecific TNFα inhibitor, as an NAFLD therapeutic has been
studied in several trials yielding mixed results. Adams et al. and Satapathy et al. reported improve-
ments in AST and ALT levels (Adams, Zein et al. 2004, Satapathy, Garg et al. 2004). Additionally,
Satapathy et al. showed significant serum TNFα reduction and improved insulin resistance, mea-
sured by the homeostatic metabolic assessment insulin resistance index. Conversely, in other pla-
cebo-controlled studies pentoxifylline treatment resulted in no change in aminotransferase levels
(Van Wagner, Koppe et al. 2011) or improved histology (Zein, Yerian et al. 2011). Interestingly,
two independent meta-analyses with overlap in datasets, (1) three randomized studies and two
prospective studies, n = 147 and (2) five randomized placebo-controlled trials, n = 147, showed
pentoxifylline not only reduced aminotransferase levels but also reduced body weight, BMI, blood
glucose, lobular inflammation, TNFα, and fibrosis (Du, Ma et al. 2014, Zeng, Zhang et al. 2014).
At this time, further studies are necessary to clarify the potential of pentoxifylline as a therapeutic.
SUMMARY
Currently, NAFLD is a hidden epidemic. Although the disease is asymptomatic, the risk factors,
such as MetS, central obesity, hypertension, dyslipidemia, or type 2 diabetes (Rinella 2015),
can be readily determined and are used to screen patients that are suspected to have NAFLD. As
of now, there is no standard of treatment besides moderate weight loss. While multiple clinical
trials have evaluated the effectiveness of pharmaceutical and dietary treatments on the improve-
ment of steatosis, standard recommendations promote weight loss through diet and lifestyle
changes. Successful weight loss and improvement in liver histology have been demonstrated
in subjects that reduced carbohydrate intake to less than 45% of total caloric intake. Attention
toward carbohydrate quality as well as quantity is recommended, with particular attention to
reducing simple carbohydrate consumption. Dietary fat intake should consist primarily of mono-
and polyunsaturated fats.
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13-Oxo 9,10
9-Oxo
ODE DiHOME
ODE
12,13
PGB2 DiHOME
15k- 9- 13- sEH
PGF2α HODE HODE
sEH
9,10
EpOME
PGC2 12,13
Plasma membrane alox15
EpOME
PGF2αv
PGA2 pla2g4a cyp2j fam 5,6-
Linoleic acid
cyp2c fam diHETrE
PGE2
16- sEH
HETE
TXB2 8,9-
ptges 1/2/3 5,6- diHETrE
EET
TXA2 PGG2 ptgs 1/2 Arachidonic acid cyp2j fam sEH

ptgs 1/2
cyp2c fam 8,9-
tbxas 1 PGH2
cyp2b fam EET
ptgs 1/2 ptgis alox5 alox15 alox8 alox12 11,12-

EET
14,15-
EET sEH
PGD2 PGB2 5- 15- 8- 12- 11-
HPETE HPETE HPETE HPETE HETE
sEH 11,12-
diHETrE
15k-
PGJ2 5- 8- 12-
PGF2a 15- 14,15-
HETE HETE HETE
HETE diHETrE
LTA4 alox5
15d-
PGJ2 alox12 LXA4 HXA3 HXA3
Ita4h Itc4s
LXB4
LTB4 LTC4
LTD4
LTE4
(a)
FIGURE 10.2 An overview of metabolites of omega-6 FAs (a). (Continued)

13- 9-
HOTrE HOTrE
22- 21-
HDoHE HDoHE
PD1
alox5 alox5
20- 10S, 17S-

5- HDoHE DiHDoHE
Epoxide
HEPE
Linolenic acid hydrolase
22- RvD1
21-
8- HpDHA
HpDHA
HEPE 20- 17-
HpDHA HDoHE
alox5 RvD2
9- 17-
HEPE EPA DHA alox5 HpDHA RvD5
16-
HpDHA alox5 HpDHA
16-
alox5 alox5 HDoHE
11-
14-
HEPE
13- HpDHA
18- HpDHA 14-
12- HEPE 4- 7- HDoHE
HEPE HpDHA HpDHA
8- 11-
10- Maresin
HpDHA HpDHA
15- HpDHA
HEPE
11-
RvE1 4-
7- HDoHE
HDoHE
HDoHE 13-
8- 10- HDoHE
HDoHE HDoHE
(b)
FIGURE 10.2 (Continued) An overview of metabolites of omega-3 FAs (b). Boxes show enzymes responsible for production of metabolites (brown boxes designate
processes that can be accomplished nonenzymatically). (Taken from Tam, V.C., Semin. Immunol., 25(3), 240, 2013. With permission; Abbreviations given in the original
paper.)
(a) (b)
I S
(c) (d)
(e)
FIGURE 15.1 Histological features of NAFLD. (a) Marked steatosis without inflammation, hepatocytes
injury (ballooning), or fibrosis. Steatosis is concentrated in acinar zone 3, the microcirculatory unit through
which blood exists the liver around the terminal hepatic venule (in circle) and shows sparing of the periportal,
zone 1 hepatocytes, the microcirculatory unit through which portal and systemic blood enter and mix. This is the
adult pattern of nonalcoholic fatty liver disease (NAFLD) (trichrome staining). (b) Steatohepatitis with marked
steatosis (S), ballooning (B), lobular and portal inflammation (I) and extensive bridging fibrosis (hematoxylin
and eosin staining). (c) Fibrosis in the perisinusoidal spaces of zone 3 is detected by trichrome stain; bridg-
ing fibrosis (arrow) is noted between two central veins. Hepatocytes with steatosis are seen, but no ballooned
hepatocytes are present. (d) Nonalcoholic steatohepatisis (NASH) with cirrhosis, but no active lesions remain.
One would only know this was a case of NASH-related cirrhosis by having had a prior biopsy with the d iagnosis
of active NASH. (e) Hepatocellular carcinoma after the development of NASH and cirrhosis. (Reprinted by
permission from Macmillian Publishers Ltd.: Nat. Rev. Dis. Primers, (Brunt 2015) Copyright, 2015.)
Lipolysis
(60%)
Triglycerides
Fatty
Dietary fat acids
(10%–15%)
Carbohydrate and DNL Oxidation

Hyperinsulinemia
(25%–30%)
VLDL secretion
FIGURE 15.2 Contributions of various metabolic pathways to liver steatosis in humans. Reductions in fatty
acid oxidation and triglyceride export seem to have only minor roles in hepatic triglyceride deposition. By
contrast, the increased availability of fatty acids from the adipose tissue through unabated lipolysis and de novo
lipogenesis (DNL) from glucose are major providers of lipids in steatotic livers. (Reprinted with permission
from Taskinen, M.R. and Boren, J., Atherosclerosis, 239(2), 483, 2015.)
Food
Generation of microbial-
derived metabolites
Local effects
on intestine Microbiota
Direct/indirect
effects on CVD risk
Dyslipidemia
Insulin resistance Systemic inflammation
Obesity
LPS
FIGURE 30.1 Potential mechanisms by which the gut microbiota affects cardiovascular risk. Changes in
microbial community structure can change gut permeability and allow microbes to enter the bloodstream
(Leaky gut syndrome). Lps or other bacterial products can induce low-grade systemic inflammation.
Alternatively, the microbiota can metabolize nutrients in food to specific metabolites that either increase or
decrease susceptibility to cardiometabolic disease.
16 Effects on Cardiometabolic Risk
Dietary Starches and Grains
Nathalie Bergeron and Ronald M. Krauss
CONTENTS
Introduction.................................................................................................................................... 297
Carbohydrate Quantity................................................................................................................... 298
Lipid and Lipoprotein Risk Factors.......................................................................................... 298
CVD Risk.................................................................................................................................. 299
Glycemic Control...................................................................................................................... 300
Diabetes Risk............................................................................................................................ 301
Glycemic Index.............................................................................................................................. 301
Inflammation............................................................................................................................. 302
CVD Outcomes......................................................................................................................... 303
Glucose Homeostasis and Glycemic Control............................................................................ 304
Diabetes Risk............................................................................................................................ 305
Whole Grains................................................................................................................................. 306
CVD Risk.................................................................................................................................. 307
Glycemic Control...................................................................................................................... 307
Diabetes Risk............................................................................................................................ 308
Conclusion..................................................................................................................................... 308
References...................................................................................................................................... 308
ABSTRACT
The 2015 Dietary Guidelines Advisory Committee is the first to recommend an upper limit for added
sugars, based on adverse effects of excess intake on adiposity and cardiometabolic risk. Although there
has been continued guidance to replace added sugars with healthy carbohydrates, no specific recom-
mendations regarding intake of dietary starch and total carbohydrates have been made. In this chapter, we
review evidence from randomized controlled trials and prospective cohort studies of the effects of dietary
carbohydrates on cardiometabolic health, taking into consideration the quantity of dietary carbohydrates
consumed and their quality described in terms of their glycemic index/glycemic load and their degree of
processing (e.g., whole vs. refined grains). Replacement of refined grains with whole grain products has
been shown to improve features of atherogenic dyslipidemia and glycemic control. Overall, the evidence
to date suggests that limiting total carbohydrate intake and emphasizing minimally or unprocessed whole
grains (~3 servings/day) is associated with reduced risk for CVD and type 2 diabetes.
INTRODUCTION
Dietary carbohydrates are the main energy (E) source in the diet, representing an average of 48%
and 51% of daily calories in U.S. males and females, respectively (DGAC 2015), and ~63% of
daily E intake worldwide (ChartsBin.com 2016). In the United States, it is estimated that ~15%
297
of daily calories are derived from added sugars (Yang et al. 2014), with the remainder of carbo-
hydrates (33%E–36%E) consumed as naturally occurring sugars and starches. As we continue to
contend with obesity, insulin resistance, and their associated risk for type 2 diabetes (T2DM) and
cardiovascular disease (CVD), concerns over the amount and quality of carbohydrates that are con-
sumed have come to the forefront of nutrition policy documents, in recognition of the view that
higher-carbohydrate diets may contribute to deterioration of metabolic variables associated with
cardiometabolic risk.
Carbohydrates are commonly categorized as sugars, oligosaccharides (indigestible carbohydrates
comprising 2–10 monosaccharide units), and polysaccharides (i.e., starches) on the basis of their
chemical structure. More relevant to cardiometabolic health are properties that take into account
how carbohydrates are digested and absorbed in the small intestine and how they affect glycemia,
insulinemia, and ensuing metabolic responses. When categorized in this way, carbohydrates that are
not digested and absorbed in the human small intestine, namely, dietary fiber and oligosaccharides,
are separated from “glycemic/available” carbohydrates. As such, carbohydrate-rich foods are often
classified on the basis of their blood glucose-raising effects in comparison to glucose or white bread
(glycemic index, GI) and their glycemic load (GL), which considers both the GI of a food and its
total carbohydrate content, thus reflecting both carbohydrate quality and quantity (Jenkins et al.
1981, Salmerón et al. 1997b). Carbohydrate-rich foods can also be classified based on their degree
of processing (whole vs. refined grains). While a standard definition is still lacking, whole grain
foods are generally referred to as those made from the entire grain and consisting of the endosperm,
germ, and bran, the outer layer of the whole grain that protects the starchy endosperm from diges-
tive enzymes (this definition, however, remains imperfect as it fails to take into consideration the
extent of processing of the product, i.e., milled vs. whole kernel grains). Conversely, in refined grain
products, only the endosperm remains after removal of the bran and germ.
In this chapter, we review evidence from randomized controlled trials and prospective cohort
studies, focused mainly on the effects of complex carbohydrates (i.e., starches) on risk factors and
clinical outcomes for CVD and diabetes. We take into consideration the quantity of dietary car-
bohydrates consumed and, importantly, their quality described in terms of their GI/GL and their
degree of processing (e.g., whole vs. refined grains). These properties affect the rate of digestion of
carbohydrate-containing foods and are more strongly related to measures of cardiometabolic health
than is categorization based on the chemical structure of carbohydrates (Mozaffarian 2016). Readers
are referred to Chapters 13 through 15 for comprehensive reviews of the effects of added sugars and
the value of their restriction on cardiometabolic health.
CARBOHYDRATE QUANTITY
Lipid and Lipoprotein Risk Factors
In 1977, the Senate Select Committee on Nutrition and Human Needs issued dietary goals for the
American people that recommended reduction in total fat intake to no more than 30%E, reduction
of saturated fat to 10%E, and an increase in dietary carbohydrate consumption to 58%E (48%E as
complex carbohydrate and naturally occurring sugars; 10%E as refined/processed sugars) to reduce
CVD risk, and for prevention of overweight, obesity, and diabetes. Less often appreciated is the rec-
ognition that Americans have generally followed the nutrition advice promulgated by the USDA and
the American Heart Association, with a recent report showing a reduction in intake of total fat from
45%E to 34%E, saturated fat from 13.5%E to 10.7%E, and a reciprocal increase in total carbohydrate
intake from 39%E to 51%E from 1971 to 2011 (Cohen et al. 2015). While restriction of saturated fat
is a well-established and effective strategy for LDL-cholesterol (C) lowering, it is increasingly rec-
ognized that the concurrent increase in carbohydrates that has accompanied the adoption of low-fat
diets (Cohen et al. 2015) promotes features of atherogenic dyslipidemia including elevated plasma
triglycerides (TG), reduced HDL-C, and increased concentrations of small dense LDL particles (Dreon et al.
Dietary Starches and Grains 299
1994, 1997, 1999), an effect that is independent of starch quality (Bergeron et al. 2016, Sacks et al.
2014). Notably, whereas hepatic lipogenesis has been implicated in the triglyceride-raising effects of
added sugars/fructose (see Chapters 14 and 15), we and others have shown that high carbohydrate
intake increases levels of apoCIII in apoB-containing lipoproteins (Furtado et al. 2008, Shin and
Krauss 2010). This may contribute to the atherogenic effects of these particles both by retarding their
plasma clearance and directly promoting inflammation (Sacks 2015).
The earlier findings are generally consistent with numerous clinical trials comparing effects
on lipids and lipoproteins of diets varying in carbohydrate amount, namely, low-carbohydrate vs.
low-fat diets, without consideration for carbohydrate quality. In a meta-analysis of 23 longer-term
(6–24 months) randomized controlled trials (N = 2788) in which lower-carbohydrate (≤45%E;
weighted mean 23%E) and low-fat higher-carbohydrate diets (fat ≤30%E; weighted mean 26%E;
carbohydrates ≥46%E) were compared, the lower-carbohydrate diets resulted in a greater decrease
in plasma TG (pooled mean net change, −14 mg/dL; 95% CI −19.4, −8.7) and greater increase
in HDL-C (3.3 mg/dL; 95% CI 1.9, 4.7) from baseline, but a lesser reduction in LDL-C levels
(3.7 mg/dL; 95% CI 1.0, 6.4) compared to higher-carbohydrate low-fat diets (Hu et al. 2012). While
these effects occurred in the context of comparable reductions in body weight across diets, one must
consider the likelihood that changes in plasma lipids may, at least in part, have been mediated by
weight loss. In an earlier meta-analysis of 30 short-term feeding studies (2–12 weeks) conducted
under weight maintenance conditions (N = 1213), low-fat high-carbohydrate diets (fat, 18%E–30%E;
carbohydrates, 53%E–65%E) were compared with moderate-fat diets (fat, 34%E–50%E; carbohy-
drates, 35%E–50%E) in which carbohydrates were partially replaced with monounsaturated fats,
whereas saturated fat was on average comparable across diets (8%E–9%E) (Cao et al. 2009). Under
these isoenergetic conditions, there was a significant increase in HDL-C (2.28 mg/dL) and a reduc-
tion in plasma TG (−9.36 mg/dL) with the moderate-fat lower-carbohydrate diets compared to low-
fat diets, and similar reductions in LDL-C across diets. Based on these lipid changes, predicted CVD
risk was calculated and estimated to be 6.4% lower in men and 9.3% lower in women consuming
moderate-fat lower-carbohydrate diets compared to low-fat diets (Cao et al. 2009).
Although LDL-C remains the clinical benchmark for assessing efficacy of lipid-lowering ther-
apy, information about LDL particle subclass concentrations, particularly small vs. larger LDL,
may more accurately reflect effects on CVD risk (Krauss 2010, 2014). In a 13-week dietary inter-
vention in moderately overweight men, a low-carbohydrate diet (26%E) reduced small dense LDL
(P = 0.03) and increased LDL peak particle diameter (P = 0.007), a measure of the diameter of the
most abundant class of LDL particles, compared to a higher-carbohydrate diet (54%E) (Krauss et al.
2006), also suggesting a benefit of carbohydrate restriction on measures of CVD risk. A very-low-
carbohydrate weight maintenance ketogenic diet (8%E) consumed for 6 weeks has also been shown
to significantly increase LDL peak particle diameter and the percentage of larger LDL-I in compari-
son to a habitual lower-fat (32%E) higher-carbohydrate (47%E) diet (Sharman et al. 2002).
CVD Risk
Because evidence from randomized trials with clinical outcome data is lacking, we rely on epi-
demiological evidence to study the relationship of diet composition to CVD risk. Several large
prospective cohort studies have evaluated the association of low-carbohydrate diets with CVD risk
and mortality. In most instances, dietary intake was assessed with food frequency questionnaires
and translated into an overall low-carbohydrate diet score that also considered energy from protein and
fat (Fung et al. 2010, Halton et al. 2006, Nakamura et al. 2014) or only protein (Lagiou et al. 2012,
Trichopoulou et al. 2007). (Note that the lowest decile low-carbohydrate diet score reflected higher
carbohydrate intake and, conversely, the highest decile score reflected lower carbohydrate intake.)
Comparison of the highest vs. lowest deciles of the low-carbohydrate diet score showed no associa-
tion with coronary heart disease (CHD) risk (relative risk [RR] 0.94, 95% CI 0.74–1.19) (Halton
et al. 2006) or CVD mortality (HR 1.00, 95% CI 0.84–1.2) (Fung et al. 2010) in women from the
Nurses’ Health Study (NHS; N > 82,000 women followed for 20–26 years). However, in men from
the Health Professionals Follow-up Study cohort (HPFS; N = 44,548; 20 years follow-up), a low-
carbohydrate diet was associated with an increase in CVD mortality (HR 1.15, 95% CI 0.96–1.37)
which was largely attributed to concurrent intake of animal-based foods. By contrast, low-carbo-
hydrate vegetable-based diets were associated with reduced CVD mortality in both cohorts (pooled
HR 0.77, 95% CI 0.68–0.87) (Fung et al. 2010). Together the earlier findings differ from what
has been reported for Swedish women (N ≥ 42,000; mean follow-up 12–15.7 years) (Lagiou et al.
2007, 2012), and Greek men and women of the European Prospective Investigation into Cancer and
Nutrition (EPIC) study (N = 22,944; follow-up 10 years), in whom a low-carbohydrate high-protein
diet was associated with increased cardiovascular mortality (Lagiou et al. 2007, Trichopoulou et al.
2007) and increased incidence of cardiovascular events (Lagiou et al. 2012).
The discrepancies in findings across cohorts, that is, showing an increase in CVD risk and mor-
tality with low carbohydrate intake in cohorts from Sweden and Greece but no effect in U.S. women,
may reflect differences in the quality of carbohydrate-containing foods that are displaced with car-
bohydrate restriction. It has indeed been argued that in Swedish women, replacement of mostly
whole grains with protein may have had detrimental effects on measures of CVD, whereas among
U.S. women (NHS), replacement of mostly refined starches and sugars with any other nutrient
would be less likely to adversely affect risk (Willett 2007). Notably, in the NHS cohort (Halton et al.
2006), subgroup analysis of the relationship of individual macronutrients revealed that whereas
total carbohydrate intake was marginally associated with CVD risk (RR 1.22, 95% CI 0.95–1.56),
glycemic load (the product of GI and the amount of carbohydrate in a given food) was significantly
associated with increased risk (RR 1.90, 95% CI 1.15–3.15), emphasizing the importance of consid-
ering both the quantity and quality of carbohydrates consumed.
Glycemic Control
Acute postprandial glucose and insulin responses to test meals are commonly used as indicators of
whole-body insulin sensitivity. It is generally believed that over time, chronically high levels of glucose
and, thereby, insulin can lead to pancreatic ß-cell dysfunction and the development of insulin resistance,
a predisposing condition to T2DM and increased CVD risk (Blaak et al. 2012). Because carbohydrate is
the main macronutrient affecting insulin secretion and ensuing blood glucose levels, many studies have
focused on either dietary carbohydrate restriction or the manipulation of carbohydrate quality and the
relationship of these dietary strategies to measures of glycemic control and diabetes risk.
Many randomized controlled trials testing effects of carbohydrate quantity on measures of gly-
cemic control have, by design, been conducted under weight loss conditions, precluding inference
to weight stable conditions. In the earlier referenced meta-analysis of 23 longer-term randomized
controlled trials (N = 2788) that compared low-carbohydrate diets to low-fat diets (Hu et al. 2012),
pooled mean net changes from baseline in fasting blood glucose did not differ for low-carbohydrate
(−10.4 mg/dL) vs. low-fat diets (−10.1 mg/dL), likely due to weight loss, which was comparable
across diets (−6.1 kg and −5.0 kg, respectively). Notably, however, a series of small studies con-
ducted in eight men with untreated T2DM showed that improvements in glycemic control through
dietary modification can occur under conditions where body weight is unchanged. In these 5–10-week
trials, a low-carbohydrate diet (achieved primarily by restriction of dietary starches and providing
30%E carbohydrate, 30% protein, 40% fat) in replacement for a higher-carbohydrate lower-fat diet
(55%E carbohydrate, 15% protein, 30% fat) reduced fasting glucose levels and 24 h total glucose
area by ≥28% and ≥35%, respectively, with no change in fasting insulin levels (Nuttall et al. 2008,
Gannon et al. 2010). Mean glycated hemoglobin (HbA1c, a measure of glycemic control over a 2 to
3-month period) was also decreased by 25% after 10 weeks (Gannon et al. 2010), in overall agree-
ment with a 12-month study in obese patients (80% men) that showed a greater reduction in glycated
hemoglobin (−0.7%) in a subgroup of 54 diabetic patients assigned to a low-carbohydrate diet vs.
low-fat diet, even after adjustment for weight loss (Stern et al. 2004). While the earlier findings
suggest a beneficial effect of carbohydrate restriction per se on glycemic control, larger studies in
more diverse patient populations are warranted before definitive conclusions can be drawn.
Diabetes Risk
Data linking total carbohydrate intake to risk of T2DM is derived largely from cohorts where low
carbohydrate scores were used to characterize dietary intake. During 20 years of follow-up of the
NHS and HPFS cohorts, there was no association of extreme deciles of the low-carbohydrate diet
score with diabetes risk among women (Halton et al. 2008), but there was an increased risk asso-
ciated with reduced carbohydrate intake among men, the latter driven largely by concurrent high
intake of red and processed meat (de Koning et al. 2011); after adjustment for these dietary vari-
ables, the association of the low carbohydrate score with diabetes was no longer significant. In a
Japanese cohort of 64,674 men and women (5 years follow-up), the risk of T2DM was reduced for
the lowest vs. highest quintile of carbohydrate intake among women (RR 0.63, 95% CI 0.46–0.84),
but this effect became nonsignificant after adjustment for GL, illustrating the importance of carbo-
hydrate quality in diabetes risk (Nanri et al. 2015). A number of epidemiological studies found no
association of total carbohydrate intake with predicted risk of diabetes (Hu et al. 2001). These obser-
vations are in general agreement with a meta-analysis of 22 cohort studies that showed a greater
association with diabetes risk for glucose (RR 1.77, 95% CI 1.06–2.65) and fructose (RR 1.68, 95%
CI 1.01–2.59) than for total carbohydrate intake (RR 1.23, 95% CI 1.09–1.39) in trials ≥10 years of
follow-up (Alhazmi et al. 2012).
In summary, observational studies among U.S., European, and Asian cohorts using “low-
carbohydrate diet scores” to qualify dietary consumption show mostly no association of total car-
bohydrate intake per se with CVD or diabetes risk. Evidence that low-carbohydrate diets appear to
be protective when consumed in association with plant-based foods, but are detrimental in animal-
based diets, are consistent with the notion that effects of carbohydrates, and their restriction, on
cardiometabolic risk are to some extent modulated by the nutrients and foods that replace them and
the overall dietary context in which carbohydrates are consumed.
In the absence of randomized controlled trials of low-carbohydrate diets on clinical outcomes,
we rely on trials with surrogate measures of CVD and diabetes risk. Such studies show benefits
of restricting total carbohydrate intake, manifest most notably as ameliorations in features of ath-
erogenic dyslipidemia, namely, plasma TG, HDL-C, and small dense LDL particles, along with
improvement in glycemic control and glycated hemoglobin in individuals with T2DM.
GLYCEMIC INDEX
As mentioned earlier, the glycemic index (GI) is a measure of carbohydrate quality and represents
the postprandial blood glucose response to 50 g of carbohydrates from a test food, expressed as a
percentage of the blood glucose response to the same amount of carbohydrate from a reference food,
namely, glucose or white bread (Wolever et al. 1991). It is used to categorize foods on the basis of
their rate of digestion and absorption, with high-GI foods having a GI ≥ 70, whereas low-GI foods
have a GI ≤ 55 (Augustin et al. 2015). Also as noted earlier, the concept of GL has been developed
to take into consideration both carbohydrate quality and quantity, and is the product of a foods’ GI
and the amount of carbohydrate in a typical serving of that food. Hence, foods with a higher GL
generally elicit more rapid and larger blood glucose and insulin responses.

The effects of GI and GL on plasma lipids and lipoproteins are inconsistent. Several cross-sectional
studies (Levitan et al. 2008, Liu et al. 2001, McKeown et al. 2009, Shikany et al. 2010) have shown
reduced plasma TG and increased HDL-C levels with lower GL or GI diets and have suggested that
the differential lipid effects may be more pronounced with increasing BMI (Liu et al. 2001, Shikany
et al. 2010). Notably, the positive association with TG and inverse association with HDL-C appeared
to be stronger for GL than GI (Liu et al. 2001). In the earlier studies, lowest vs. highest quartiles/
quintiles ranged from 68 to 81 for GI (Levitan et al. 2008, Liu et al. 2001, McKeown et al. 2009,
Shikany et al. 2010) and from 50 to 180 for GL (Levitan et al. 2008, Liu et al. 2001, McKeown et al.
2009, Shikany et al. 2010). Evidence from dietary intervention studies, however, is mixed. A
recent meta-analysis of 28 randomized controlled feeding trials ≥4 weeks (N = 1272) reported no
effect of GI on plasma TG and HDL-C, but reduced LDL-C (−6.2 mg/dL, 95% CI −9.3 to −3.1) with
low-GI diets. Notably, subgroup analysis revealed that improvements in LDL-C occurred only when
low-GI diets were also high in fiber, but not when fiber was matched across diets (Goff et al. 2013).
The observation that low- vs. high-GI diets did not differentially affect plasma TG and HDL-C in
this meta-analysis (Goff et al. 2013) suggests that GL, which captures both carbohydrate quality and
quantity, and/or carbohydrate quantity as such, may be more important determinants of these bio-
markers of CVD risk than GI, in agreement with an earlier meta-analysis that reported lowering of
plasma TG with low-GL diets (Livesey et al. 2008). Alternatively, it is possible that benefits of low-
GI diets on plasma TG and HDL-C may be attenuated if the diet has an abundance of low-GI foods
that are high in fructose, a simple sugar shown to promote atherogenic dyslipidemia (Chapters 14
and 15).
The OmniCarb study is the most rigorous clinical trial to date to evaluate the effect of GI on CVD
risk factors. It compared high-GI (65) vs. low-GI (40) diets in the context of DASH-like dietary
patterns with high (58%E) or moderate (40%E) carbohydrate intake (Sacks et al. 2014) in 163 over-
weight and obese adults. In this 5-week intervention study, carbohydrate amount influenced lipid
risk factors to a greater extent than GI. Low vs. high GI levels had no effect on HDL-C and apoB
in the context of either moderate or high carbohydrate intake but resulted in a modest reduction in
TG (5 mg/dL) on the moderate-carbohydrate diet. In keeping with the documented effect of carbo-
hydrates on atherogenic dyslipidemia, the high- vs. moderate-carbohydrate diet increased plasma
TG (18%–20%) at both high and low GI levels and reduced HDL-C (4%) at the high GI level. The
authors concluded that in an otherwise healthy dietary setting, lowering the GI may not improve
cardiovascular risk factors. The RISCK study tested the effects of higher GI (GI, 64) vs. lower GI
(GI, 51–53) in the context of either high monounsaturated fat diets or low-fat diets, in comparison to
a high-GI high saturated fat control diet, in a 24-week intervention in 548 men and women at high
risk of metabolic syndrome (Jebb et al. 2010). Replacing saturated fat with either carbohydrate or
monounsaturated fat reduced total cholesterol and LDL-C, but the most significant reductions were
achieved for diets with low-GI carbohydrates. No other differences in plasma lipids were noted
between low- and high-GI diets, in overall agreement with the Diogenes study in 932 overweight
adults (Gogebakan et al. 2011), and a meta-analysis of 14 longer-term randomized controlled trials
(≥6 months) that reported no differential effect of GI or GL on plasma lipids (Schwingshackl and
Hoffmann 2013). Discrepancies in findings between cross-sectional studies and randomized con-
trolled trials may reflect differences in populations studied, duration of interventions, inherent limi-
tations associated with the variability of GI measurements (Matthan et al. 2016), and the potential
for misclassification of low- vs. high-GI foods in food frequency questionnaires. It is also possible
that in cross-sectional studies, GI/GL may be a reflection of dietary patterns including other bioac-
tive components that favorably affect biomarkers of CVD risk.
Inflammation
The modest effects of GI/GL on plasma lipids as noted earlier suggest that other pathways, such as
those impacting inflammation, may be implicated in the association of low-GI/low-GL diets with
reduced CVD risk, as reviewed in the following text. A recent systematic review suggests that the
anti-inflammatory benefits of low-GI diets documented in epidemiological studies are supported by
several dietary intervention studies (Buyken et al. 2014). In cross-sectional analyses among healthy
middle-aged women (WHS, N = 18,137) (Levitan et al. 2008), middle-aged women with T2DM
(NHS, N = 891) (Qi et al. 2006), and smaller cohorts at high risk for CVD (PREDIMED, N = 511)
(Bulló et al. 2013), or with generally impaired glucose tolerance (Dutch cohort, N = 786) (Du et al.
2008), a high dietary GI was associated with increases in two biomarkers of inflammation that are
strongly associated with CVD risk (Ridker et al. 2000): CRP (Du et al. 2008, Levitan et al. 2008,
Qi et al. 2006) and IL-6 (Bulló et al. 2013). The associations of dietary GL with inflammatory markers
in observational studies are less consistent (Buyken et al. 2014).
The Diogenes study, involving 932 overweight adults from eight European countries, is the larg-
est clinical trial to date to evaluate the effect of GI on inflammatory markers. After 26 weeks of
weight maintenance, the low-GI diet (GI, ~56) led to a significantly greater reduction in CRP (−0.46
mg/L; 95% CI 0.79–0.13) than the high-GI diet (GI, ~61) (Gogebakan et al. 2011), in agreement
with results from a 1-year Canadian study in 162 individuals with T2DM where CRP levels were
30% lower with the low-GI diet (GI, 55) than the high-GI diet (GI, 63) (CRP 1.95 vs. 2.75 mg/L,
respectively; P = 0.0078) (Wolever et al. 2008). While smaller dietary trials have failed to reproduce
these findings (Buyken et al. 2014), results from larger intervention studies suggest that low-GI diets
may reduce low-grade inflammation in higher-risk individuals.
CVD Outcomes
There is evidence that chronically elevated fasting and postprandial glycemia and insulinemia, by
promoting insulin resistance and oxidative stress as well as inflammation, are implicated in the
development of both T2DM and CVD (Blaak et al. 2012). Numerous epidemiological studies have
investigated the relationship of GI/GL to chronic disease risk among different cohorts and popula-
tions. In the NHS including 75,521 women and 761 cases of CHD during a 10-year follow-up, high
GL and, to a lesser degree, high GI were associated with an increased risk of CHD (Liu et al. 2000b).
When stratified by BMI, the increased risk of CHD with GL was most pronounced among women
with BMI > 23, consistent with a recent meta-analysis in which GL was associated with increased
CHD risk among men and women with a higher BMI (RR 1.49, 95% CI 1.27–1.76) but not among
those with lower BMI (RR 1.03, 95% CI 0.86–1.23) (Fan et al. 2012). In a large Italian cohort of the
EPICOR trial (N = 47, 749 men and women; 7.9 years follow-up and 463 CHD cases), a significant
increase in the risk of CHD was observed across categories of GL (RR 2.24, 95% CI 1.26–3.98) and
carbohydrates from high-GI foods (RR 1.68, 95% CI 1.01–2.75) in women, but not in men. High
carbohydrate intake from low-GI foods, starch, or sugar was not associated with CHD risk in either
gender (Sieri et al. 2010). Similar associations of high GL with increased CHD risk were recently
reported in the EPIC Greek cohort study (20,275 men and women; 10.4 years follow-up) (Turati
et al. 2015). Notably, in this cohort, high adherence to a Mediterranean diet with low/moderate GL
(tertiles I and II of GL) was associated with a 40% lower risk of CHD compared with a high GL/
high adherence to a Mediterranean diet, suggesting a benefit to reducing GL even in an otherwise
healthy dietary pattern.
Several meta-analyses of prospective cohort studies examining the relation between GI/GL and
CVD were published in 2012, with generally comparable conclusions. A meta-analysis of eight
prospective cohort studies (N = 220,050 participants; 4,826 incident cases) (Dong et al. 2012), and
another based on 10 cohort studies (eight overlapping with the Dong meta-analysis; N = 240,936
participants; 6,940 incident cases) (Mirrahimi et al. 2012) showed significant associations of GL
and GI with CHD risk among women, but not men. Notably, GL was more strongly associated
with CHD risk (55%–69% increase) than GI (26% increase). Results of another meta-analysis
of 14 cohorts (90% overlap with the Mirrahimi meta-analysis, with an additional five cohorts,
and extending CHD outcomes to stroke and heart failure) (N = 229,213 participants; 11,363
cases) indicated an 18% increase in risk for cardiac events per 50-unit increment of GL among
Caucasians (Ma et al. 2012). Yet another meta-analysis (12 cohorts, 438,073 participants [100%
overlap with Mirrahimi meta-analysis, with two additional cohorts], and 9,424 incident CHD cases
and 2,123 stroke cases) (Fan et al. 2012) concluded that high GL, but not GI, was associated with
an increased risk for stroke as well as CHD. In a dose–response analysis, there were 5% and 3%
increases in risk of CHD and stroke, respectively, per 50-unit increment in GL level (Fan et al.
2012). A subsequent meta-analysis showing a significant association of high GL, but not of GI or
total carbohydrate, to increased risk of stroke suggests that this relationship is independent of total
carbohydrate intake (Cai et al. 2015).
Glucose Homeostasis and Glycemic Control

In the context of cardiometabolic health, low-GI diets are most studied for their effects on measures
of glycemic control for the management of diabetes. In a meta-analysis of 14 controlled trials lasting
12 days to 12 months (N = 356 individuals with type 1 and type 2 diabetes), low-GI diets (average
GI = 65) reduced HbA1c by 0.34% points and plasma fructosamine (a measure of glycosylated
blood proteins that reflects glycemic control over the preceding 2–3 weeks) by 0.2 mmol/L more
than that achieved with high-GI diets (average GI = 83) (Brand-Miller et al. 2003). When data
from HbA1c and fructosamine were combined and the difference between the low- and high-GI
diets was expressed in percentage terms, the low-GI diet reduced values by 7.4%, an effect esti-
mated by the authors to translate into clinically meaningful reductions in diabetes-related endpoints
based on data from the UKPDS trial (UKPDS 1998). In a later meta-analysis of 45 controlled diet
intervention trials (N = 972/treatment arm) ≥1 week duration and including individuals who were
healthy, had type 1 or type 2 diabetes, or were at risk of CHD, low-GI diets reduced fasting glucose
and glycated proteins and improved measures of insulin sensitivity assessed by clamp, frequently
sampled IV glucose tolerance test, or insulin tolerance test (Livesey et al. 2008). These effects were
of greater magnitude in individuals with blood glucose levels >5 mmol/L (90 mg/dL) and, notably,
were independent of unavailable carbohydrate (i.e., indigestible oligosaccharides and fiber) intake.
In individuals with type 1 and type 2 diabetes (N = 612), the improvement in glycemic control
manifest as reductions in HbA1c (0.4%) or fructosamine (0.23 mmol/L) with low- vs. high-GI diets
was confirmed in a meta-analysis of 12 randomized controlled trials of ≥4 weeks duration (Thomas
and Elliott 2010). A recent meta-analysis of longer-term studies (≥6 months, 16 trials, N = 3073)
compared the effectiveness of popular dietary approaches (i.e., low-carbohydrate, low-GI, vegetar-
ian, vegan, Mediterranean, and high-protein) vs. control diets on measures of glycemic control and
confirmed that low-GI vs. control diets (3 trials, N = 357) improved HbA1c. However, the magni-
tude of the effect was small (−0.14%) and of questionable clinical relevance. Low-carbohydrate,
Mediterranean, and high-protein diets were also effective at reducing HbA1c, with the greatest
effect attributed to Mediterranean diets (−0.47%) (Ajala et al. 2013). The smaller effect sizes of low-
GI diets in these longer-term studies, as compared to those reported in earlier meta-analyses (Brand-
Miller et al. 2003, Livesey et al. 2008), raise the possibility of metabolic adaptation or reduced
compliance over time with such restrictive regimens.
While clinical data generally suggest benefits of low-GI/low-GL diets for glycemic control in
diabetes, the effectiveness of this approach in nondiabetics is questionable. In the 5-week OmniCarb
trial in 163 overweight and obese adults discussed earlier, DASH-like low-GI diets did not improve
glycemic control compared to high-GI diets and, in the context of high carbohydrate intake, actually
reduced insulin sensitivity determined as areas under the curve (AUC) for glucose and insulin after
a glucose tolerance test (Sacks et al. 2014). It was speculated that the low-GI diet may have pro-
moted morning insulin resistance as a means of maintaining adequate blood glucose levels. Similar
observations were reported in a controlled 28-day feeding study involving 89 healthy adults who
were either normal weight or overweight/obese, and where low- and high-GL diets resulted in com-
parable measures of insulin sensitivity (estimated as HOMA-IR) (Runchey et al. 2012). Acutely,
high-GL meals induced higher postprandial insulin and glucose responses, but the ratio of iAUC
insulin/iAUC glucose was comparable for both high- and low-GL test meals, suggesting no detri-
mental effect of GL on ß-cell function.
Diabetes Risk
While a mechanistic relationship between GI and risk of T2DM remains uncertain, it is suggested
that the increase in postprandial glucose resulting from high-GI diets raises insulin demand which,
over time, may compromise ß-cell function and eventually lead to higher diabetes risk (Ludwig
2002). As is the case for CVD risk, numerous longitudinal studies have also investigated the rela-
tionship of GI/GL to risk of T2DM among different cohorts. This was first reported in the NHS in
65,173 women (40–65 years old) (Salmerón et al. 1997b) and HPFS in 42,759 men (40–75 years old)
(Salmerón et al. 1997a) and later among younger women in NHSII (N = 91,249; 24–44 years old)
(Schulze et al. 2004) without NIDDM or CVD at onset. At 6–8 years of follow-up, the relative risk
of diabetes was increased 37%–59% in the highest vs. lowest quintile of GI (Salmerón et al. 1997a,b,
Schulze et al. 2004), and 25%–47% in the highest vs. lowest quintile of GL, in models adjusted for
cereal fiber intake (Salmerón et al. 1997a,b). When the effects of GL and cereal fiber were exam-
ined jointly, the risk for diabetes was even higher for the combination of high GL (>165) and
low cereal fiber (<3.2 g/day) vs. low GL (<143) and higher cereal fiber intake (>5.8 g/day) (RR
= 2.17–2.5) (Salmerón et al. 1997a,b). In an updated analysis of the combined NHS, NHSII, and
HPFS cohorts (N = 205,157; follow-up 3,800,618 person-years), individuals in the highest vs. low-
est quintiles of energy-adjusted GI and GL had a 33% and 10% higher risk of T2DM, respectively,
suggesting that GI is more strongly associated with diabetes risk than GL (Bhupathiraju et al. 2014).
When high-GI or high-GL diets were combined with low cereal fiber intake, the risks of T2DM
were respectively 59% and 47% higher compared to low-GI/low-GL high-fiber diets, underscoring
the importance of highly processed carbohydrate-containing foods in this relationship (Bhupathiraju
et al. 2014, Salmerón et al. 1997a,b). The possible association between processed carbohydrates and
diabetes risk was also highlighted in a subsequent analysis of NHSII (AlEssa et al. 2015) in which
the starch-to-cereal fiber ratio was found to have the strongest association with diabetes risk (RR =
1.39; CI 1.27, 1.53; P-trend < 0.0001) vs. RRs of 1.28 (CI 1.17, 1.39; P-trend < 0.0001), 1.12 (CI
1.02, 1.23; P-trend = 0.03), and 1.09 (CI 1.00, 1.20; P-trend = 0.04) for carbohydrate-to-cereal fiber,
starch-to-total fiber, and carbohydrate-to-total fiber ratios, respectively.
In addition to the findings from the NHS and HPFS cohorts, a relationship of GI and/or GL
to diabetes risk has been corroborated in the Dutch EPIC cohort (Sluijs et al. 2010), the Black
Women’s Health Study (N = 59,000) (Krishnan et al. 2007), and in a cohort of Chinese women (N =
64,227) (Villegas et al. 2007), but not in the Women’s Health Study (Meyer et al. 2000) or other pro-
spective studies (Mosdol et al. 2007, Sahyoun et al. 2008, Simila et al. 2011). These inconsistences
may reflect population/ethnic differences, the subjectivity of assigning GI values to mixed meals
and local foods leading to possible misclassification, and the fact that in some studies assessment of
dietary intake occurred only at baseline rather than at regular intervals during follow-up, thus failing
to account for changes in dietary intake over time.
Despite inconsistencies across prospective cohort studies, several meta-analyses conducted since
2008 report a positive relationship between high-GI or high-GL diets and diabetes risk (Barclay et al.
2008, Dong et al. 2011, Greenwood et al. 2013, Livesey et al. 2013). In a meta-analysis of nine pro-
spective cohort studies (N = 422,224) investigating the relationship of GL/GL to the risk of T2DM
among mostly female participants (Barclay et al. 2008), comparison between the highest and low-
est quintiles of GI/GL showed GI to be more strongly associated with diabetes risk (40% increase)
than GL (27% increase), independent of dietary fiber intake. Positive associations between GI/GL
and risk of T2DM were confirmed in a subsequent meta-analysis of 13 prospective cohort studies
(75% overlap with cohorts from the Barclay meta-analysis, with an additional seven cohorts) con-
ducted between 1997 and 2010 (N = 530,875; 4–14 years follow-up) (Dong et al. 2011). In the most
recent dose–response meta-analysis reporting on 24 studies (N = 757,984; 7.5 million person-years
follow-up), there was a 45% increase in diabetes risk per 100 g increment in GL in the fully adjusted
model (Livesey et al. 2013). The relationship between GL and diabetes risk was found to be stronger
among females than males, in European Americans vs. other ethnic groups (albeit the latter were not
well represented), and in studies using dietary instruments of greater validity. Whereas the associa-
tion of GL with diabetes risk was observed over a wide range of GL values (62–279 g/2000 kcal),
it was significant only at GL > 95 g/2000 kcal, leading the authors to recommend a daily target of
less than 100 g GL. In Americans consuming 48%–51% daily energy from carbohydrates (DGAC
2015), this GL would correspond to ~250 g carbohydrates with a GI = 40, but may more practically
be achieved by reducing carbohydrate intake to 180 g (≅35% daily kcal) and selecting foods in the
upper range of what are considered low-GI foods (<55) (Augustin et al. 2015).
WHOLE GRAINS
In the United States, an emphasis on consumption of grain products dates back to the 1992 USDA
food pyramid, which advocated consumption of 6–11 servings each day of bread, cereal, rice, and
pasta, with only slight consideration for the quality of foods that constituted these grain servings. In
its most recent iteration, the Dietary Guidelines for Americans 2015–2020 recommends the selec-
tion of whole grains as staple items of healthy food patterns. Yet, it is estimated that the majority
of the U.S. population does not meet recommendations for 3–4 oz/day whole grains and >70% of
Americans exceed recommended intakes for refined grains (DGAC 2015). The absence of a uni-
versally accepted definition for what constitutes a whole grain product (Ferruzzi et al. 2014) makes
identification of such foods challenging. Designations range from ≥25% to ≥51% whole grain or
bran by weight. A ratio of total carbohydrate to fiber (in g/serving) ≤10:1 has also been proposed as
a means of identifying whole grain foods (Lloyd-Jones et al. 2010). It is important to recognize that
even if a standard definition for whole grain foods were in place, characteristics such as the structure
and degree of processing of grains (e.g., the extent to which they are milled vs. cracked vs. intact)
will modulate physiological responses. Yet such characteristics are rarely described or reported in
observational and randomized controlled trials (Ross et al. 2015), making it difficult to evaluate the
association of “whole grains” to cardiometabolic health.

The cholesterol-lowering properties of whole grains have, in part, been ascribed to their content of
soluble fibers, which can reduce bile acid reabsorption and favor their fecal excretion. The ensuing
hepatic conversion of cholesterol to bile acids promotes LDL-receptor upregulation and increased
hepatic uptake of LDL particles (Surampudi et al. 2016). In a recent meta-analysis of 24 randomized
controlled trials in which effects of whole grains on plasma lipids were evaluated by type of whole
grain product, total whole grain intake reduced LDL-cholesterol vs. control (−0.09 mmol/L, 95% CI
−0.03, −0.15), but the magnitude of effect was greatest with whole grain oat (−0.17 mmol/L, 95% CI
−0.10, −0.25) (Hollaender et al. 2015). In contrast, whole grain wheat or mixed whole grains derived
mostly from wheat products showed no effect on plasma lipids, likely because wheat contains mostly
insoluble fiber. The number of trials in which rye, barley, or rice were compared to control foods were
too few to arrive at meaningful conclusions regarding their cholesterol-lowering potential. Notably,
in a meta-regression to test whether dose could predict changes in plasma lipids, there was a nega-
tive association between whole grains and total cholesterol and LDL-C for studies in which intake
was <100 g/day, but the association was positive when the entire range of intakes (28–213 g/day)
was included (Hollaender et al. 2015). Together, these findings suggest possibly undesirable plasma
lipid effects at higher levels of whole grain intake, possibly reflecting an effect of total carbohydrate
load. This observation is consistent with results from our own study in which provision of diets high
in resistant starch, a form of dietary starch which undergoes limited digestion by α-amylases in the
small intestine, did not lower plasma and LDL-C compared to low-resistant-starch diets (Bergeron
et al. 2016). Notably, we found that independent of the resistant starch content of the diet, high vs. low
carbohydrate intake increased plasma TG and large VLDL particles, in keeping with the notion that
total carbohydrate load may be an important determinant of atherogenic dyslipidemia.
To date, evidence from randomized controlled trials fails to support the notion that the protective
effect of whole grains on CVD risk is due to anti-inflammatory effects (Buyken et al. 2014).
CVD Risk
Studies consistently report inverse associations between whole grain intake and the risks of CVD
(Jensen et al. 2004, Mellen et al. 2008, Wu et al. 2015a, Ye et al. 2012). However, and as referred
to earlier, interpretation of such studies has been complicated by the lack of a universal definition
of what constitutes a whole grain food (Cho et al. 2013). In addition, most whole grain products
contain dietary fiber that may or may not account for their health benefits. For example, the
HPFS (Jensen et al. 2004) reported that the significant inverse association between whole grain
intake and CVD risk (6% reduction in CVD risk for each 20 g increment in whole grain intake)
was eliminated after adjustment for dietary fiber and other diet factors, suggesting that dietary
fiber accounts, at least in part, for the protective effects of whole grain products. An earlier meta-
analysis of 10 prospective cohort studies found that the highest category of whole grain intake
(48–80 g/day, ≅3–5 servings) was associated with a 21% reduction in CVD risk when compared
to rare or no intake, but it is unclear whether the multivariable adjustment model accounted for
dietary fiber (Ye et al. 2012). An updated analysis that combined the NHS and the HPFS showed a
9% reduction in total mortality and 15% reduction in CVD mortality for the highest (33 g/day) vs.
lowest quintiles (4.2 g/day) of whole grain intake when adjusted for the “healthy eating index,”
but adjustment was not made for dietary fiber per se. It was estimated that every serving of whole
grain (28 g) was associated with a 9% lower CVD mortality (Wu et al. 2015a). These findings
concur with an updated meta-analysis of 14 cohort studies (N = 786,076; 23,957 CVD deaths) in
which the highest vs. lowest categories of whole grain intake were associated with an 18% reduc-
tion in CVD mortality; in a dose–response analysis, each 16 g increase in whole grain intake (~1
serving/day) was associated with a 9% lower risk of CVD mortality (RR 0.91, 95% CI 0.90–0.93)
(Zong et al. 2016). Hence, consuming ~3–4 servings/day whole grain products (50–70 g/day,
equivalent to 3–4 slices of whole grain bread), as recommended in the Dietary Guidelines for
Americans, was associated with 19%–23% reduction in CVD mortality, compared with no whole
grain consumption (Zong et al. 2016). Collectively, the earlier studies suggest a cardioprotective
role of whole grains, likely mediated in part by the fiber (Wu et al. 2015b) and phytonutrients
inherent in these products (Ras et al. 2014). In contrast, no association with CVD or CHD has
been reported for total refined grain, white bread, refined breakfast cereal, or total rice intake
(Aune et al. 2016, Mellen et al. 2008).
Glycemic Control
Soluble fibers in whole grain products have been proposed to improve glycemic control by delay-
ing gastric emptying and slowing the rate of glucose absorption, thereby attenuating the postpran-
dial excursion of blood glucose after carbohydrate-containing meals (Kaline et al. 2007). In a
meta-analysis of 14 observational studies (~48,000 individuals of European descent), each serving
of whole grain foods (assessed by food frequency questionnaires, 24 h recalls and food diaries)
reduced fasting glucose by −0.019 mmol/L and fasting insulin by −0.021 pmol/L (Nettleton et al.
2010). While relatively few randomized controlled dietary intervention trials have examined the
association of whole grains to glycemic control, the meta-analysis of Ye et al. (2012, 11 studies)
showed that increased intake of whole grains vs. a control diet was associated with a reduction in
fasting glucose (95% CI −1.65, −0.21), whereas reduction in plasma insulin levels was not signifi-
cant (Ye et al. 2012). These findings should be interpreted with caution given the substantial het-
erogeneity noted across trials, likely reflecting differences in type of whole grains tested, method
of estimating whole grain intake, nature of the control interventions, study duration, and health
status of study participants. Importantly, in randomized controlled trials, few studies have directly
compared whole vs. refined grains in otherwise comparable diets, making it difficult to determine
whether improvements in cardiometabolic parameters are the result of whole grains per se, or of
other bioactive components that typify the healthy dietary patterns in which whole grains are often
incorporated.
Diabetes Risk
The relationship of whole grain intake to diabetes risk was initially reported in the Women’s Health
Study (with similar findings in the NHS cohort [Liu et al. 2000a]), where the relative risk of diabetes
in the highest (median 20.5 servings/week) vs. lowest quintile (median 1 serving/week) of whole
grain intake was 0.79 (95% CI 0.65–0.96). Whereas refined grains, soluble fiber, and fiber from
fruits and vegetables were unrelated to risk, intake of cereal fiber and magnesium were associated
with reduced diabetes risk (RR 0.64, 95% CI 0.53–0.79 and RR 0.67, 95% CI 0.55–0.82, respectively)
suggesting that these constituents of whole grains confer protection from T2DM (Meyer et al. 2000).
In an earlier meta-analysis of six cohort studies (N = 286,125; 10,944 incident cases T2DM), it
was estimated that a 2 serving/day increment in whole grain intake would reduce the risk of T2DM
by 21% (95% CI 13%–28%) (de Munter et al. 2007). A subsequent dose–response meta-regression
analysis (with some overlapping studies and three additional cohorts) covering a broad range of
whole grain intake (2–154 g/day, the latter equivalent to ~5 servings/day) showed an absolute reduc-
tion of 0.3% in incidence of T2DM for each additional 10 g (~0.3 servings) of whole grains con-
sumed. Based on population prevalence of T2DM, it was estimated that increasing whole grain
intake from 7.5 to 45 g/day would lead to a 20% relative reduction in diabetes risk (Chanson-Rolle
et al. 2015), consistent with findings of de Munter et al. (2007). Total fiber intake has also been
found to be protective for diabetes risk (RR 0.81, 95% CI 0.73–0.90) in a meta-analysis of 17 studies
involving 488,293 participants (19,033 cases) (Yao et al. 2014). From this pooled analysis, it is esti-
mated that a daily intake of 30 g of dietary fiber (i.e., twofold more than what is currently consumed
by U.S. adults (McGill et al. 2015)) would be required to achieve comparable reductions in diabetes
risk than what is achieved with three servings of whole grain foods.
CONCLUSION
Dietary starches and grains are staple components of diets consumed worldwide. In assessing their
effects on cardiometabolic health, one must consider the quantity of carbohydrates consumed and,
importantly, the degree of processing of carbohydrate-containing foods. In randomized controlled
trials, higher-carbohydrate (≥45%E) diets are generally associated with increased plasma TG,
low HDL-C, increased abundance of small dense LDL particles, and worsened glycemic control
manifest as increased fasting glucose and hemoglobin A1C levels. While observational studies
indicate that total carbohydrate intake is marginally associated with the risk of cardiometabolic
disease, the importance of carbohydrate quality is emphasized by the stronger associations of GI
and GL (the product of GI and carbohydrate amount) with risk of T2DM and CVD, respectively.
Overall, the evidence to date suggests that the risk for these diseases is benefited by dietary patterns
that limit the intake of total carbohydrates and glycemic starches and include ~3 daily servings of
minimally or unprocessed whole grains.
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Section IV
Dietary Protein and
17 Interaction of Dietary
Protein and Energy Balance
Eveline A. Martens, Richard D. Mattes, and
Margriet S. Westerterp-Plantenga
CONTENTS
Introduction.................................................................................................................................... 318
Protein Intake in Negative Energy Balance................................................................................... 318
Protein Intake in Neutral Energy Balance...................................................................................... 319
Mechanisms behind Protein-Induced Appetite Control and Energy Expenditure......................... 319
Appetite..................................................................................................................................... 319
Energy Intake............................................................................................................................ 321
Energy Expenditure................................................................................................................... 322
Body Composition.................................................................................................................... 322
Protein Intake and Protein Turnover......................................................................................... 323
Protein Intake in Positive Energy Balance..................................................................................... 324
Reward Homeostasis Related to Dietary Protein........................................................................... 325
Protein Intake and Cardiovascular Diseases.................................................................................. 325
Adverse Effects of Protein Diets.................................................................................................... 326
Summary........................................................................................................................................ 326
Acknowledgment........................................................................................................................... 327
References...................................................................................................................................... 327
ABSTRACT
Nutrition plays a key role in the prevention of cardiovascular disease, acting in part, on the pre-
vention and treatment of obesity. Here the role of dietary protein in energy balance and weight
regulation with consequent implications for the prevention and management of cardiovascular dis-
eases is described. An energy-restricted high-protein diet has been shown to achieve body weight
loss and subsequent weight maintenance by reducing energy intake without changing appetite, and
maintaining energy expenditure by preserving FFM. Mechanisms behind protein-induced appetite
control and energy expenditure are direct and indirect effects of elevated plasma amino acid and
anorexigenic hormone concentrations, increased diet-induced thermogenesis (DIT), and a ketogenic
state. Mechanisms behind protein-induced energy expenditure and preservation of fat-free mass are
metabolic inefficiency, due to processing, protein turn-over, and possibly gluconeogenesis. In neu-
tral energy balance, a protein diet may prevent a positive energy balance due to the higher energy
expenditure. With respect to reward homeostasis, protein-induced food reward is limited, and may
affect compliance to a protein-modified diet. When applied within a range of 0.66–1.66 g/kgBW/d,
protein consumption has not been associated with increased health risks. Even in the elderly, benefi-
cial health effects of higher-protein intake might outweigh possible adverse effects.
In conclusion, higher-protein diets may reduce cardiovascular disease risk by decreasing the
susceptibility for the development of obesity.
317
INTRODUCTION
Cardiovascular diseases are important complications of obesity. The mechanism behind the devel-
opment of obesity is one or more uncompensated periods of positive energy balance. The energy
consumed in excess of need is stored in body reserves resulting in body weight gain (Westerterp
2013). Treatment of obesity entails loss of body weight, which requires a negative energy balance.
At a negative energy balance, energy is mobilized from body reserves (Westerterp 2013). The most
efficient and effective way to achieve a negative energy balance is with an energy-restricted diet
(Westerterp-Plantenga et al. 2009). However, a sudden decrease in energy intake from the habitual
diet results in increased feelings of hunger and desire to eat, and in a decrease of the feeling of
fullness. These changes in appetite make it difficult to sustain a lower energy intake. Furthermore,
body weight loss usually results in a reduction in energy expenditure, which is mainly caused by the
loss of fat-free mass (FFM). These conditions counteract the negative energy balance induced by
the energy-restricted diet, hampering body weight loss. Therefore, the aims of body weight loss are
to reduce energy intake without changing appetite and to maintain energy expenditure by preserving
FFM. Both goals can be achieved with an energy-restricted high-protein diet (Acheson 2013, Leidy
et al. 2015, Westerterp-Plantenga et al. 2012, Wycherley et al. 2012) (see Protein Intake in Negative
Energy Balance section).
A relevant question is whether the consumption of high-protein diets can also prevent the devel-
opment of a positive energy balance, thus preventing overweight and obesity. In this regard, longer-
term intervention studies assessing the effects of high-protein diets in normal-weight individuals
are evaluated (Protein Intake in Neutral Energy Balance section). This chapter also deals with the
mechanisms behind protein-induced appetite modulation and energy expenditure (Mechanisms
behind Protein-Induced Appetite Control and Energy Expenditure section). To complete the pic-
ture of the relation between dietary protein and energy balance, the effects of high-protein diets in
positive energy balance are described (Protein Intake in Positive Energy Balance section). Reward
homeostasis related to dietary protein, effects of dietary protein on cardiovascular diseases indepen-
dent of energy balance, and the adverse effects of protein diets are described in Reward Homeostasis
Related to Dietary Protein, Protein Intake and Cardiovascular Diseases, Adverse Effects of Protein
Diets sections. Throughout this chapter, a major theme is the distinction between short-term and
longer-term effects of protein diets.
PROTEIN INTAKE IN NEGATIVE ENERGY BALANCE

Achieving and maintaining a negative energy balance are necessary conditions for dietary
interventions to induce body weight loss. High-protein diets have the potential to maintain a negative
energy balance by sustaining satiety at the level of the original diet (Weigle et al. 2005, Westerterp-
Plantenga et al. 2006) and to limit the reduction of energy expenditure through sparing of FFM
(Martens and Westerterp-Plantenga 2014, Soenen et al. 2013). The strongest effects are documented
with diets restricted in carbohydrate and fat intake, but without restriction of protein intake. In this
case, the diet is typically relatively high in protein (as % of energy), while the protein intake (in g/day)
may be comparable to the original diet.
A few conditions apply for longer-term studies aimed at elucidating the effects of high-protein
diets on body weight. First, it is necessary to monitor and confirm compliance with the desig-
nated protein intake preferably with a quantitative, objective biomarker, such as urinary nitro-
gen. Second, differences in protein intake between the experimental and control diets must be
achieved and sustained to adequately test the efficacy of the high-protein diet (Soenen et al. 2013,
Westerterp-Plantenga et al. 2012, Wycherley et al. 2012). The conclusions of studies comparing
high-protein with normal-protein diets may differ from those testing high-protein and low-protein
diets (Westerterp-Plantenga et al. 2012). During energy restriction, sustaining protein intake at the
level of the minimal requirement (0.66 g/kg body weight/day) appears not to hinder body weight
Interaction of Dietary Protein and Energy Balance 319
loss and fat loss (Krieger et al. 2006, Soenen et al. 2013, Wycherley et al. 2012). An additional
increase of protein intake may not induce a larger loss of body weight, but can be effective in
maintaining a larger amount of FFM (Krieger et al. 2006, Soenen et al. 2013, Wycherley et al.
2012). For example, a 6-month energy-restricted diet with a daily protein intake just above the
minimal requirement (0.8 g/kg body weight) induces a comparable reduction in body weight to
an energy-restricted diet with a daily protein intake well above the minimal requirement (1.2 g/kg
body weight) (Soenen et al. 2013). However, a protein intake of 1.2 g/kg body weight/day results
in a greater decrease in fat mass and preservation of FFM (Soenen et al. 2013). Furthermore, high-
protein diets can be beneficial for weight maintenance after weight loss. Weight regain is less with
energy-restricted high-protein diets compared with normal-protein diets (Larsen et al. 2010, Soenen
et al. 2013). An energy-restricted high-protein diet in combination with exercise can even increase
muscle mass (Josse et al. 2011). Dietary protein intake below requirements could lead to less
weight loss and a higher risk for body weight regain (Acheson 2013).
Moreover, the effects described earlier may involve concurrent changes in protein, carbohydrate,
and/or fat intake. Increasing the relative protein content of a diet automatically results in a decrease in the
relative content of carbohydrate and/or fat. Nevertheless, a study by Soenen et al. demonstrated that the
effects of a high-protein intake on body weight loss and weight maintenance were present independent of
a low-carbohydrate intake (Soenen et al. 2012). Well-controlled studies comparing energy-restricted diets
with a high protein content and diets with a normal protein content, within a large range of fat contents,
also showed independent effects of a high-protein intake (Wycherley et al. 2012).
PROTEIN INTAKE IN NEUTRAL ENERGY BALANCE

If the protein-induced effects on appetite and energy expenditure observed during energy restriction
also hold under nonrestricted conditions, then increasing protein intake with a normal diet could
prevent overweight and obesity. Alternatively, the consumption of a diet with lower protein content
could increase the risk for body weight gain. Recently, a 12-week intervention study was performed
comparing high-protein (30% of energy from protein) and low-protein (5% of energy from protein)
diets, in weight stable individuals (Martens et al. 2014a). This was a randomized parallel group
study in 14 men and 18 women on diets containing 30/35/35 or 5/60/35 percentage of energy from
protein/carbohydrate/fat. Participants were able to sustain the high- and low-protein diets in this
field study, according to the biomarker urinary nitrogen. Nitrogen excretion increased significantly
in the high-protein diet group, from 11.8 ± 3.2 g/day at baseline to 21.7 ± 5.1 g/day during the
intervention, and decreased from 12.2 ± 3.4 g/day to 7.0 ± 2.4 g/day in the low-protein diet group
(Martens et al. 2014a). The low-protein diet indeed facilitated the development of a positive energy
balance, while the high-protein diet was beneficial to prevent this, showing a nonsignificant positive
energy balance (Martens et al. 2014b). The positive protein balance was not caused by a difference
in feelings of fullness or satiety yet by a difference in energy expenditure. Total energy expenditure
as measured in the respiratory chamber did not change with the high-protein diet over 12 weeks but
decreased with the low-protein diet (Martens et al. 2014b).
MECHANISMS BEHIND PROTEIN-INDUCED APPETITE CONTROL

AND ENERGY EXPENDITURE
Appetite
Short-term intervention studies using energy-balanced diets with large contrasts in relative protein
content have shown that high-protein diets are more satiating than diets lower in protein (Bendtsen
et al. 2013, Halton and Hu 2004, Leidy et al. 2010, Lejeune et al. 2006, Veldhorst et al. 2009b,d,
Westerterp-Plantenga et al. 2006). Furthermore, subjects consumed less food during an ad libitum
high-protein diet relative to baseline (Weigle et al. 2005). Despite this lower total energy intake,
subjects were similarly satiated and satisfied during the intervention and preintervention periods
(Martens et al. 2013, 2014c, Weigle et al. 2005).
Correspondingly, small increases in fullness and satiety ratings were observed as acute responses
to a high-protein diet in neutral energy balance (Martens et al. 2014b). After 1 week on the high-
protein diet, fullness scores were significantly increased, while on the low-protein diet, they were
decreased. In this situation, translation into large changes in energy intake was not possible, because
subjects had to maintain their body weight. After 12 weeks, appetite ratings were returned to the
level of the original diet, which suggests that the human body habituates to the satiating effects of
high-protein intake (Martens et al. 2014b). Thus, shifts in appetite may only appear as response to
changes in dietary protein content in the short term.
Protein-induced satiety is likely a combined expression with direct and indirect effects of
elevated plasma amino acids and anorexigenic hormone concentrations, increased diet-induced
thermogenesis (DIT), and a ketogenic state.
Elevated blood concentrations of amino acids stimulate satiety signaling in the brain (Acheson
et al. 2011, Fromentin et al. 2012, Hall et al. 2003, Morrison et al. 2012, Veldhorst et al. 2009a,d).
According to the “aminostatic theory,” serum amino acids that cannot be channeled into protein
synthesis directly serve as satiety signals (Mellinkoff et al. 1956). However, the aminostatic theory
failed to garner strong support because fasting circulating amino acid levels do not correlate with
appetitive sensations and there are noncongruent appetitive responses to protein sources varying in
the rate of amino acid appearance. Indirectly, dietary amino acids may act on satiety signaling via
receptors in the duodeno-intestinal and hepatoportal regions (Niijima et al. 2005). Depending on
the type of amino acid, they increase or decrease the activity of hepatic vagal afferent fibers, inner-
vating satiety centers in the brain (Niijima et al. 2005). The branched-chain amino acids leucine,
isoleucine, and valine reportedly play an important role in these mechanisms (Acheson et al. 2011,
Fromentin et al. 2012, Morrison et al. 2012, Niijima et al. 2005, Veldhorst et al. 2009a,d).
Furthermore, the satiety-stimulating effect of protein is related to increases in anorexigenic gut
hormones (Belza et al. 2013, Diepvens et al. 2008, Juvonen et al. 2011, Karhunen et al. 2008,
Maersk et al. 2012, Veldhorst et al. 2009a). Such hormones are produced in response to peripheral
and central detection of amino acids. They react to elevated protein intake from specific sources
and stimulate vagal activity in brain areas involved in the control of food intake (Davidenko et al.
2013, Leidy et al. 2013, Morrison et al. 2012, Veldhorst et al. 2009a). Concentrations of glucagon-
like peptide 1, cholecystokinin, and peptide YY consistently increase in response to high-protein
intakes (Belza et al. 2013, Diepvens et al. 2008, Juvonen et al. 2011, Karhunen et al. 2008, Maersk
et al. 2012, Veldhorst et al. 2009a). However, it should be emphasized that changes in appetite do
not consistently change in line with concentrations of amino acids or appetite hormones (Veldhorst
et al. 2009a,b,c).
Acute amino acid–related effects on appetite have been reported with several high-quality
proteins. A high-casein breakfast was more satiating than a normal-casein breakfast, coinciding with
prolonged elevated concentrations of plasma amino acids (Veldhorst et al. 2009d). Also, a high–soy
protein breakfast was more satiating than a normal–soy protein breakfast, which was related to
larger increases in plasma taurine concentrations (Veldhorst et al. 2009d). Taurine is synthesized
endogenously from cysteine, which in turn can be synthesized from methionine. Whey could be
more satiating than casein shortly after a meal as a result of fast digestion (Veldhorst et al. 2009a).
Theoretically, the digestion of fast proteins, such as whey, results in high and early rises of plasma
amino acids and appetite hormones. Casein, which is a slower protein, could have a more prolonged
satiety effect than whey (Boirie et al. 1997, Dangin et al. 2001). The slower digestion and absorption
rates of casein give more prolonged and maintained plasma amino acid and hormone concentrations
(Boirie et al. 1997, Dangin et al. 2001, Hall et al. 2003). However, no clear evidence exists for dif-
ferences in satiating capacity between different types of protein, at high concentrations (Acheson et al.
2011, Adechian et al. 2012, Bendtsen et al. 2013, Bowen et al. 2006, Hall et al. 2003, Juvonen et al.
2011, Lorenzen et al. 2012, Pal et al. 2014, Veldhorst et al. 2009a). Moreover, differences in satiety
responses between protein sources are scarcely observed in the longer term. It is hypothesized that
the concentrations of certain amino acids have to be above a particular threshold to promote a
relatively stronger hunger suppression or greater fullness (Veldhorst et al. 2009a). Certain dietary
proteins, mainly complete dietary proteins, reach these thresholds at lower concentrations than other
sources of protein. At high concentrations of dietary proteins, it may not be possible to discriminate
between complete proteins because the amino acid concentrations are above the threshold for all
sources. In the longer term, the amount of dietary protein intake rather than protein source may
determine the magnitude of satiety responses.
The theoretical basis of the relationship between protein-induced satiety and DIT may be that
increased energy expenditure at rest implies increases in oxygen consumption and body tem-
perature. The feeling of oxygen deprivation may be translated into satiety feelings (Westerterp-
Plantenga et al. 1999a,b). The presence of a positive relationship between the increase in satiety
and in 24 h DIT has been observed with an energy-balanced high-protein diet (Westerterp-
Plantenga et al. 1999b). The contribution of this mechanism to possible longer-term satiety
responses remains to be determined.
With respect to a ketogenic effect, fasting β-hydroxybutyrate concentrations increase in response
to a ketogenic high-protein diet that is concurrently a low-carb diet (Coleman and Nickols-Richardson
2005, Johnston et al. 2006, Veldhorst et al. 2009c). In a 1.5-day study, Veldhorst et al. observed that on
this low-carb high-protein diet, increased concentrations of β-hydroxybutyrate directly affected appe-
tite suppression (Veldhorst et al. 2010). A hyperketogenic state will not be reached with a common
high-protein diet when its carbohydrate content is high enough to prevent strong ketogenesis.
Nevertheless, the suggested contribution of ketogenesis to appetite suppression warrants further study
(Johnston et al. 2006, Laeger et al. 2010, Scharrer 1999, Veldhorst et al. 2010).
Energy Intake
The studies described earlier show that protein intake itself does not automatically determine the
magnitude of appetitive responses. Protein-induced satiety may depend on energy balance, and may
differ between the short-term and longer-term consumption of high-protein meals. At least in the
short term, ad libitum high-protein diets have been observed to sustain appetite at levels comparable
to the original diet, despite a lower energy intake. Energy-restricted, high-protein diets produce,
under some conditions, a sustained lower energy intake than diets with a lower protein content
(Gosby et al. 2011, Martens et al. 2013, 2014c). Again, this has occurred with appetite scores com-
parable to those elicited by the control diet (Gosby et al. 2011, Martens et al. 2013, 2014c). As a
result, individuals who consume a high-protein diet in combination with energy restriction are more
satiated and potentially less likely to consume additional calories from foods extraneous to dietary
prescription (Halton and Hu 2004).
The protein leverage hypothesis encompasses a geometrical model suggesting that energy intake
is adjusted to reach an individual-specific target protein amount (Simpson and Raubenheimer
2005). The regulation of protein intake may be stronger than the regulation of carbohydrate and fat
intake, and thus than that of total energy intake. However, evidence for a target for protein intake in
humans is equivocal (Gosby et al. 2011, Griffioen-Roose et al. 2012, Martens et al. 2013, 2014c).
The absence of complete protein leverage suggests that humans have a wide capacity to respond and
adapt to differences in protein intake. There may be stronger protein leveraging responses in various
animals than in humans (Raubenheimer and Simpson 1997, Shariatmadari and Forbes 1993, Tews
et al. 1992, Theall et al. 1984, Simpson and Raubenheimer 1997, Sorensen et al. 2008). Possible
species differences may reflect abilities to maintain protein intake for growth and reproduction on
low-protein diets and/or limitations on metabolism of excess protein from high-protein diets.
A recent intervention study demonstrated that humans are able to maintain high-protein (30% of
energy from protein) and low-protein diets (5% of energy from protein) and can respond and adapt
to a range of protein intakes for a period of at least 12 weeks (Martens et al. 2014b). In contrast,
the consumption of a diet extremely low in indispensable amino acids suppresses energy intake in
animals. The detection of reduced concentrations of indispensable amino acids in the brain affects
protein synthesis, subsequently leading to behavioral responses including underconsumption of
diets that lack a minimal amount of indispensable amino acids. Evidence for comparable behavioral
responses in humans is scarce. Consequently, the limits of adaptation to protein challenges acutely
and over the longer term remain to be clarified.
Energy Expenditure
In addition to the effects on appetite and energy intake, dietary protein may also modulate energy
expenditure. Most high-protein diet studies focusing on body weight loss have applied an energy-
restriction regimen to induce a negative energy balance. The decline in energy expenditure and
sleeping metabolic rate (SMR) as a result of body weight loss was less on a high-protein diet than
on a normal-protein diet (Whitehead et al. 1996). In addition, higher rates of energy expenditure
were observed as acute responses to energy-balanced high-protein diets (Mikkelsen et al. 2000,
Veldhorst et al. 2010). Longer-term studies of individuals in energy balance support a view that
higher-protein intake plays a role in the prevention of obesity via energy expenditure. In one trial,
energy expenditure was maintained on a high-protein diet during energy balance for 12 weeks
(Martens et al. 2014b; see also Protein Intake in Neutral Energy Balance section). In contrast,
the consumption of a low-protein diet resulted in a positive energy balance after 12 weeks. Thus,
at a constant body weight, a high-protein diet may protect against the development of a positive
energy balance. The consumption of a low-protein diet may increase the risk for the develop-
ment of a positive energy balance through adaptive thermogenesis (Martens et al. 2014b). The
observations on total energy expenditure were completely underscored by changes in SMR and
DIT (Martens et al. 2014b). Differential effects of dietary protein content on metabolic efficiency
may contribute to the explanation for changes in DIT in response to high- and low-protein diets.
The metabolic efficiency (the amount of MJ to ingest to increase 1 kg of body mass) is lower for
protein than for carbohydrate and fat. Increases in protein oxidation likely contribute to the small
increases in DIT in response to a high-protein diet. This may be beneficial to sustain total energy
expenditure in the longer term (Westerterp et al. 1999). It may be possible that a high-protein
intake induces a strong acute response on DIT, followed by a smaller but sustained effect in the
longer term (Luscombe et al. 2003).
Body Composition
During energy restriction, the decline in total energy expenditure and SMR as a result of body
weight loss is less on a high-protein diet than on a normal-protein diet (Whitehead et al. 1996).
This has been ascribed to the potential of high-protein diets to preserve FFM, the main determinant
of SMR. FFM only showed small increases and decreases after a 12-week intervention with high-
protein and low-protein diets in energy balance (Martens et al. 2014a). As a consequence, SMR did
not significantly change. Ultimately, a high-protein intake would be a strong stimulus for preserva-
tion of FFM.
Previous studies did observe an increase in FFM during a high-protein diet in negative (Josse et al.
2011) or neutral energy balance (Soenen et al. 2010). Likely, these changes could be explained by a
high-protein intake combined with physical activity.
Although changes in protein intake will not automatically result in marked changes in FFM,
this should not be interpreted to mean that metabolic function remains unaffected. Visceral adipose
tissue (VAT) volume has been linked to the metabolic disturbances associated with obesity, such
as diminished insulin sensitivity and dyslipidemia (Despres and Lemieux 2006). However, high
ectopic lipid content, especially intrahepatic triglyceride (IHTG) content, and not VAT volume, is
an independent risk factor for these metabolic disturbances (Fabbrini et al. 2009, Lettner and Roden
2008, Magkos et al. 2010). In general, weight loss improves metabolic function (Acheson 2013,
Wycherley et al. 2012), but a high-protein intake may modulate the IHTG content as well (Bortolotti
et al. 2009, 2011, Theytaz et al. 2012). In the context of prevention of metabolic disturbances, a
12-week intervention study determined the effects of high- and low-protein diets on the IHTG con-
tent in weight-stable individuals (Martens et al. 2014a). There was a trend for lower IHTG content
after the high-protein low-carbohydrate diet compared with the low-protein high-carbohydrate diet
(Martens et al. 2014a). This effect was caused by the difference in protein intake between the diets,
since the fat intakes were the same, and the carbohydrate intakes were within the normal ranges
(Martens et al. 2014a). This suggests that high-protein low-carbohydrate diets may be favorable for
the prevention of metabolic disturbances in healthy humans. High-protein intake seems to stimu-
late hepatic lipid oxidation because of the high energetic demand for amino acid catabolism and
ketogenesis (Veldhorst et al. 2009c, Westerterp-Plantenga et al. 2012). Furthermore, hepatic lipid
oxidation may be stimulated by an increased bile acid production, a process that may also inhibit
lipogenesis (Watanabe et al. 2004). Protein-induced glucagon secretion inhibits de novo lipogenesis
and stimulates hepatic ketogenesis (Gannon et al. 2001, Torres and Tovar 2007). Moreover, high-
protein intake may blunt the increase of very-low-density lipoprotein (VLDL)-TG concentrations
induced by carbohydrate intake (Hudgins et al. 1996, 2000, Schwarz et al. 1995). High VLDL-TG
concentrations may increase hepatic TG, and subsequently IHTG content (Schwarz et al. 1995).
Therefore, it is likely that the observed trend for a difference in the IHTG content between the diets
may be the result of combined effects involving changes in protein and carbohydrate intake.
Protein Intake and Protein Turnover

Acutely, high-protein intake stimulates protein synthesis and turnover and induces a small suppres-
sion of protein breakdown (Gilbert et al. 2011, Tang and Phillips 2009, van Loon 2012). However, it
could be speculated that prolonged low-protein intake leads to muscle loss due to the lack of precur-
sor amino acid availability for de novo muscle protein synthesis (Dideriksen et al. 2013, Symons
et al. 2009). Hursel et al. (2015) observed that protein turnover was also significantly higher after
a 12-week high-protein vs. low-protein diet, with significant increases in protein synthesis, protein
breakdown, and protein oxidation. Protein turnover was determined in the fasted state, but protein
balance was noted in the fasted as well as in the fed state (Hursel et al. 2015). Taking the fed state
into account, protein balance was positive with the high-protein diet, and negative with the low-pro-
tein diet (Hursel et al. 2015). Surprisingly, net protein balance was less negative after the low-protein
diet compared with the high-protein diet in the fasted state. Therefore, it is important to distinguish
protein turnover in the fasted state from that in the fed state.
Wolfe et al. discussed the role of protein synthesis and protein breakdown in FFM accretion
(Deutz and Wolfe 2013, Symons et al. 2009). The observed maximum response of protein synthesis
after a single serving of 20–30 g of dietary protein suggests that additional effects of protein intake
on FFM accretion are accounted for by the inhibition of protein breakdown. However, a beneficial
reduction of protein breakdown only occurs with acute ingestion of protein (Flakoll et al. 1989,
Greenhaff et al. 2008, Louard et al. 1995, Symons et al. 2009). Consequently, changes were not
apparent in the basal fasted state after prolonged high-protein intake (Hursel et al. 2015). The posi-
tive protein balance observed with a high-protein diet may be due to acute postprandial responses,
rather than the postabsorptive state.
Consumption of a low-protein diet for 12 weeks was not detrimental to young healthy individuals
who might have the ability to adapt acutely to this condition (Hursel et al. 2015). The adaptive meta-
bolic demand model developed by Millward may provide an explanation for the observation that
the human body is able to show physiological adaptations to changes in protein intake (Millward
2003). The model proposes that the metabolic demand for amino acids comprises a fixed component
and a variable adaptive component (Millward 2003). Short-term changes in protein intake are likely
within the adaptive range. Adaptations in protein and amino acid metabolism to changes in protein
intake largely occur via changes in whole-body protein turnover and amino acid oxidation (Tome
and Bos 2000). Changes in amino acid oxidation were reflected as decreased and increased nitrogen
excretion in response to the low- and high-protein diets, respectively. The activity of enzymes that
regulate (1) transamination, (2) the disposal of the carbon skeletons in intermediary metabolism,
and (3) the disposal of nitrogen through the urea cycle increased in response to high-protein intake
(Harper 1983, Harper et al. 1984). Nevertheless, a positive nitrogen balance following high-protein
intake (Garlick et al. 1999, Pannemans et al. 1995, Price et al. 1994, Tome and Bos 2000) does
not automatically reflect an increase in protein anabolism (Millward 2012a). The capacity of the
body to increase amino acid anabolism through an increase in lean body mass is limited (Millward
2012a). Only interventions using diets high in specific indispensable amino acids, such as leucine,
might be able to stimulate protein synthesis in specific target groups such as athletes or the elderly
(Millward 2012b, van Loon 2012). Therefore, transient retention or loss of body nitrogen because of
a labile pool of body nitrogen may contribute to adaptations in amino acid metabolism in response to
changes in protein intake (Munro 1964). Transient adaptive mechanisms may be distinguished from
mechanisms that maintain homeostasis in the body in the longer term.
PROTEIN INTAKE IN POSITIVE ENERGY BALANCE

To complete the picture of the relation between dietary protein and energy balance, this section
elaborates on the effects of high-protein diets in positive energy balance. Research participants are
able to consume a large amount of protein during overfeeding with a high-protein diet, in a con-
trolled setting, for 8 weeks (Bray et al. 2012). Nevertheless, it is likely that free-living individuals
more readily eat in excess of energy need on a low-protein diet compared with a high-protein diet
due to a lower satiating capacity of a low-protein diet. Still, support for protein leverage driving
energy intake on low-protein diets is scarce (Martens et al. 2013, 2014c).
It is hypothesized that the level of protein plays a major role in FFM deposition, and in the
stimulation of energy expenditure during overfeeding. In a randomized controlled trial, Bray et al.
investigated the effect of dietary protein content on weight gain, body composition, and energy
expenditure in young men and women in positive energy balance (Bray et al. 2012). Participants
were overfed with diets low in protein (5% of energy from protein), normal in protein (15% of
energy from protein), or high in protein (25% of energy from protein) for 8 weeks. Overeating pro-
duced significantly less weight gain in the low-protein diet group compared with the normal-protein
or the high-protein diet groups (Bray et al. 2012). Fat mass increased similarly in all three groups.
With the low-protein diet, the equivalent of more than 90% of the extra energy was stored as fat.
With the normal- and high-protein diets, the equivalent of only about 50% of the excess energy was
stored as fat. FFM only increased with the normal- and high-protein diets, and slightly decreased
with the low-protein diet (Bray et al. 2012).
The observed acute increase in total energy expenditure corresponds with data from short-term over-
feeding studies (Dallosso and James 1984, Flatt 1978, Garrow 1985, Krebs 1964). Acute increases in
urinary nitrogen production to remove excess protein may significantly contribute to the increase in
energy expenditure during overfeeding with normal- and high-protein diets. Total energy expenditure
did not differ between the high- and normal-protein diets but was higher than with the low-protein diet.
Correspondingly, resting energy expenditure (REE) increased with the normal- and high-protein diets,
but did not change in response to the low-protein diet (Bray et al. 2012). The accretion of FFM in the
normal- and high-protein groups was the principal contributor to the increase in REE (Bray et al. 2012).
Much of the increase in REE occurred within the first 2 weeks in normal- and high-protein diet groups
and was sustained throughout the remaining 6 weeks. This indicates that the acute responses were the
result of increased thermogenesis rather than of the increase in body weight (Bray et al. 2012, 2015).
Thus, from this study it is suggested that among persons living in a controlled setting, excess
energy intake alone may account for the increase in fat mass. Moreover, increases in energy expen-
diture and FFM may largely be predicted by protein intake (Bray et al. 2012).
REWARD HOMEOSTASIS RELATED TO DIETARY PROTEIN

Reward-driven eating behavior may dominate energy homeostasis (Born et al. 2013, Davidenko
et al. 2013, Fromentin et al. 2012, Journel et al. 2012, Lemmens et al. 2011). Several brain areas
that are involved in food reward might link high-protein intake with reduced food wanting and
thereby act as a mechanism involved in the reduced energy intake following high-protein intake
(Born et al. 2013, Davidenko et al. 2013, Fromentin et al. 2012, Journel et al. 2012, Lemmens et al.
2011). A hypothesized mechanism by which protein might act on brain reward centers involves
direct effects of certain amino acids as precursors of the neuropeptides, serotonin, and dopa-
mine (Davidenko et al. 2013, Journel et al. 2012). A high-protein, low-carbohydrate breakfast
vs. a n ormal-protein, high-carbohydrate breakfast led to reduced reward-related activation in the
hippocampus and parahippocampus before dinner (Fromentin et al. 2012).
Furthermore, acute food-choice compensation changed the macronutrient composition of a
subsequent meal to offset the protein intervention (Born et al. 2013). A compensatory increase in
carbohydrate intake was related to a decrease in liking and task-related signaling in the hypothala-
mus after a high-protein breakfast. After a lower-protein breakfast, an increase in wanting in the
hypothalamus was related to a relative increase in protein intake in a subsequent meal (Born et al.
2013). Protein intake may directly affect the rewarding value of this macronutrient (Born et al. 2013,
Griffioen-Roose et al. 2012). A limited protein-induced food-reward effect may affect compliance
to a protein-modified diet.
PROTEIN INTAKE AND CARDIOVASCULAR DISEASES

Although for prevention of cardiovascular diseases prevention of overweight and obesity are impor-
tant, nutrition also may play a role independently. In this respect, health effects of protein intake
have been studied in epidemiological studies.
One review provided evidence for an estimated average requirement of 0.66 g good quality pro-
tein/kg body weight/day based on nitrogen balance studies. Further, a relationship was proposed
between increased all-cause mortality risk and long-term low-carbohydrate high-protein diets; but
data were inconclusive for a relationship between all-cause mortality risk and protein intake per se.
Findings were also (1) suggestive of an inverse relationship between cardiovascular m ortality and
vegetable protein intake; (2) inconclusive for relationships between protein intake and cancer
and cancer-related mortality; (3) inconclusive for a relationship between cardiovascular diseases
and total protein intake; (4) suggestive for an inverse relationship between blood pressure and veg-
etable protein; (5) probable to convincing for an inverse relationship between soya protein intake
and LDL cholesterol; (6) inconclusive for a relationship between protein intake and bone health,
renal function, and risk of kidney stones; (7) suggestive for a relationship between increased risk
of type 2 diabetes and long-term low-carbohydrate high-protein high-fat diets; (8) inconclusive for
an impact of physical training on protein requirement; and (9) suggestive of an effect of physical
training on whole-body protein retention. The authors conclude that the evidence is inconclusive to
suggestive for protein intake and mortality and morbidity. Vegetable protein intake was associated
with decreased risk in many studies (Pedersen et al. 2013).
Another study reporting data on the relationship between dietary protein sources and the risk of
stroke in 84,010 women and 43,150 men during 26 and 22 years of follow-up suggests that stroke risk
may be reduced by replacing red meat with other dietary sources of protein. (Bernstein et al. 2012).
On the other hand, the atherosclerosis risk in communities study on dietary protein intake and
coronary heart disease did not find a relationship between type of dietary protein or major dietary
protein resources and risk for coronary heart disease (Haring et al. 2014).
Finally, Clifton (2011) describes, in a review paper, that meat protein is associated with an
increase in risk of heart disease. In the Nurses’ Health Study, diets low in red meat, containing
nuts, low-fat dairy, poultry, or fish, appeared to be associated with a 13%–30% lower risk of CHD
compared with diets high in meat. Low-carbohydrate diets high in animal protein were associated
with a 23% higher total mortality rate, whereas low-carbohydrate diets high in vegetable protein
were associated with a 20% lower total mortality rate. Recent soy interventions have been assessed
by the American Heart Association and found to be associated with only small reductions in LDL
cholesterol. Although dairy intake has been associated with a lower insulin resistance and metabolic
syndrome, the only long-term dairy intervention performed to date has shown no effects on these
outcomes (Clifton 2011).
Taken together, epidemiological studies on health effects of protein intake report inconclusive
results. It seems that consumption of red meat promotes the development of cardiovascular diseases,
while consumption of vegetable protein such as nuts, low-fat dairy, poultry, or fish may be protective
against cardiovascular diseases.
ADVERSE EFFECTS OF PROTEIN DIETS

There is a long-held view that high-protein intake might interfere with calcium homeostasis by
increasing the acid load. It is hypothesized that this could be partially buffered by bone, subse-
quently resulting in bone resorption and hypercalciuria (Calvez et al. 2012). In general, high-protein
intake does not seem to be associated with an impaired calcium balance. Clinically, large prospec-
tive epidemiologic studies have shown positive associations of protein intake with bone mineral
mass and reduced incidence of osteoporotic fracture (Bonjour 2005). Furthermore, nitrogen intake
seems to have a positive effect on calcium balance and consequent preservation of bone mineral
content (Westerterp 2002). With respect to renal issues, only patients with pre-existing dysfunction
reportedly have an increased risk for the development of kidney stones and renal diseases (Calvez
et al. 2012). In the elderly, beneficial health effects of higher-protein intake might outweigh the
adverse effects possibly because of the changes in protein metabolism with aging. In contrast, per-
sistent total protein and amino acid intake below requirements impairs bodily functions leading to
higher disease and mortality risks across the lifespan (Moughan 2012, Tome 2012). Taken together,
the application of relatively high-protein diets, whereby protein intake is sustained at the customary
level, does not seem to have any adverse effects in healthy individuals.
Although no clear recommendation exists that defines the safe upper limit of protein intake,
consumption of up to 1.66 g/kg body weight/day has not been associated with increased health risks
(Millward 2012a, WHO/FAO/UNU 2007). This means that sustaining or slightly increasing protein
intake during energy restriction likely poses no adverse effects in healthy individuals. However,
protein intake can exceed the suggested safe upper limit. The question arises whether and how
these high intakes of protein would negatively affect health. Recent studies applying medium-term,
high-protein interventions in neutral or positive energy balance did not report any adverse effects
(Bray et al. 2012, Martens et al. 2014a). However, the limits of adaptation to high-protein intake
over the longer term remain to be investigated.
SUMMARY
In summary, the effects of diets varying in protein differ according to energy balance. During energy
restriction, sustaining protein intake at the level of requirement appears to be sufficient to aid body
weight loss and fat loss. An additional increase of protein intake does not induce a larger loss of
body weight, but can be effective to maintain a larger amount of FFM. Protein-induced satiety is
likely a combined expression with direct and indirect effects of elevated plasma amino acid and
anorexigenic hormone concentrations, increased DIT, and a ketogenic state, which all feedback on
the central nervous system. In general, changes in appetite may only appear as short-term response
to changes in dietary protein content, because the human body may habituate to the satiating effects
of protein intake in the longer term.
The decline in energy expenditure and sleeping metabolic rate as a result of body weight loss is
less on a high-protein diet than on a normal-protein diet. In addition, higher rates of energy expen-
diture have been observed as acute responses to energy-balanced high-protein diets. In energy bal-
ance, high-protein diets may be beneficial to prevent the development of a positive energy balance,
whereas low-protein diets may facilitate this. Furthermore, high-protein, low-carbohydrate diets
may be favorable for the prevention of metabolic disturbances.
With respect to protein turnover, it is important to distinguish the fasted state from the fed state.
In the fed state, protein balance may be positive with high-protein diets, and negative with the
low-protein diets. Surprisingly, net protein balance may be less negative after low-protein diets
compared with high-protein diets in the fasted state. Therefore, a positive protein balance observed
at a high-protein diet may be due to acute postprandial responses. During positive energy balance,
excess energy intake alone may account for the increase in fat mass. Increases in energy expenditure
and FFM may largely be predicted by protein intake.
Regarding epidemiological studies on health effects of protein intake independent of energy bal-
ance and overweight, these appear to report inconclusive results. It seems that consumption of red
meat promotes the development of cardiovascular diseases, while consumption of vegetable protein
such as nuts, low-fat dairy, poultry, or fish may be protective against cardiovascular diseases.
In conclusion, higher-protein diets may reduce cardiovascular disease risk by decreasing the
susceptibility for the development of obesity. Results of epidemiological studies on health effects of
protein intake independent of energy balance and overweight appear to be inconclusive.
ACKNOWLEDGMENT
Eveline Martens has received funding for her research described in this chapter from the European
Union’s Seventh Framework Programme for research, technological development and demonstra-
tion under grant agreement 266408.
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18 A Protein-Centric
Perspective for Skeletal
Muscle Metabolism and
Donald K. Layman
CONTENTS
Abbreviations................................................................................................................................. 334
Introduction.................................................................................................................................... 334
Protein, Weight Loss, and Muscle Mass........................................................................................ 335
Developing an Amino Acid Perspective about Cardiometabolic Health....................................... 336
Optimizing Meal Pattern of Protein for Body Composition and Sarcopenia................................ 337
Leucine Defining a Meal Threshold for Dietary Protein............................................................... 338
Impact of Protein versus Carbohydrates on Cardiometabolic Risk Factors.................................. 341
Glycemic Regulations............................................................................................................... 341
Blood Lipids.............................................................................................................................. 342
Conclusions.................................................................................................................................... 343
References...................................................................................................................................... 344
ABSTRACT
High-protein diets have been shown to be beneficial for cardiometabolic health; however, the
specific effects of dietary protein are often confounded by parallel changes in energy or carbo-
hydrate intakes. In general, energy-restricted diets with higher protein and reduced carbohydrates
produce a greater loss of body weight and body fat while minimizing the loss of lean tissues and
lead to improvements in glycemic regulations and dyslipidemia. Specific roles of protein in cardio-
metabolic health may be most evident by focusing on amino acid metabolism in skeletal muscle.
Research studies in areas of obesity and weight loss, aging and sarcopenia, and exercise physiology
have cast new light on dietary protein needs and challenged the existing dietary guidelines as inad-
equate for long-term muscle health. These studies have identified a meal-based protein requirement,
in large part, associated with the unique roles of the essential amino acid leucine in the regulation of
skeletal muscle protein synthesis and energy metabolism. Postmeal increases in intracellular leucine
concentrations initiate a signaling cascade that triggers muscle protein synthesis and increases fatty
acid oxidation. These changes in muscle metabolism appear to explain the metabolic advantages of
dietary protein including protecting calorie-burning muscle tissue, increasing energy expenditure
characterized as diet-induced thermogenesis, and partitioning of weight loss to body fat. This review
will evaluate the meal-based protein-centric needs for skeletal muscle metabolism and their relation-
ship to cardiometabolic health.
333
ABBREVIATIONS
BCAA Branched-chain amino acids
BCKAD Branched-chain ketoacid dehydrogenase
EAA Essential amino acids
mPS Muscle protein synthesis
mTORC1 Mechanistic target of rapamycin complex 1
RDA Recommended Dietary Allowance
INTRODUCTION
Cardiometabolic disorders express a cluster of metabolic and physiological risk factors including
elevated body fat, abnormal blood triglycerides, and insulin resistance (Grundy et al., 2004). This
cluster of factors was originally termed “syndrome X” or the “metabolic syndrome.” Subsequent
research questioned if the metabolic syndrome was fundamentally a lipid disorder, dysfunction of
glycemic regulations, a consequence of obesity, or declining function of skeletal muscle (Reaven,
1996). These questions led to nutrition studies testing an array of diet combinations with higher or
lower amounts of fat, carbohydrates, and protein. Diets that reduce energy intake with higher protein
and lower carbohydrates have been shown to be beneficial for cardiometabolic health (Layman et al.,
2008). These diets produce a greater loss of body weight and body fat while minimizing the loss
of lean tissues and lead to improvements in glycemic regulations and dyslipidemia (Krieger et al.,
2006; Wycherley et al., 2012). In total, higher-protein diets appear to reverse many of the risk factors
associated with the metabolic syndrome. While the outcomes of these diets appear beneficial, defin-
ing the specific role of protein is often confounded by changes in total energy and carbohydrates.
The role of protein in cardiometabolic health can be viewed from different perspectives. Protein
can be viewed simply as a macronutrient competing with carbohydrates or fats as a source of calories.
Compared with carbohydrates or fats, protein provides greater satiety to reduce food intake and produces
greater thermogenesis to increase energy expenditure (Westerterp-Plantenga et al., 2006). While these
changes in energy balance are consistent with greater weight loss, changes in satiety and diet-induced
thermogenesis do not fully explain the differences in body composition or glycemic regulations observed
with higher-protein diets. Evaluating the role of protein in cardiometabolic health requires going beyond
the generic role of protein as a macronutrient to considering molecular roles of individual amino acids
and particularly roles in metabolic regulation within skeletal muscles.
The importance of skeletal muscle and physical activity to cardiometabolic health is well
accepted. Physical activity is a standard recommendation for all cardiometabolic disorders includ-
ing obesity, the metabolic syndrome, type 2 diabetes, and cardiovascular diseases (Klein et al., 2004;
Lee et al., 2005; American Diabetes Association, 2006). Routine exercise impacts energy expendi-
ture, glucose homeostasis, insulin sensitivity, lipid metabolism, mitochondrial function, and body
composition. Exercise alters skeletal muscle metabolism by changing total energy consumption and
the fuel balance of carbohydrates and fats and producing changes in both blood lipids and insulin
sensitivity. Further, age-related losses of muscle mass, strength, and metabolic functions are signifi-
cant risk factors for cardiometabolic disease and associated with increased morbidity and mortality
(Wolfe, 2006). Much less appreciated is that changing the dietary amount of protein and the ratio of
protein and carbohydrates produces similar metabolic effects on skeletal muscle as exercise. This
perspective might be considered a “protein-centric” view of metabolism recognizing specific roles
of dietary protein in skeletal muscle health (Layman et al., 2015).
The relationship of dietary protein to skeletal muscle mass and function is not a new concept.
Athletes and trainers routinely emphasize protein for muscle performance with long-held beliefs
that greater protein intake leads to greater muscle mass and performance. While many athletes
overconsume protein (Phillips, 2006), there is mounting evidence that the current Recommended
Dietary Allowance (RDA) of 0.8 g of protein/kg body weight/day is not adequate to optimize muscle
A Protein-Centric Perspective for Skeletal Muscle Metabolism and Cardiometabolic Health 335
development, repair, and remodeling (Burd et al., 2008). Likewise, there is a consensus develop-
ing that the current RDA is not adequate to maintain adult muscle health (Bauer et al., 2013). This
chapter focuses on the relationships of protein and skeletal muscle to cardiometabolic health. The
research will be reviewed in three areas: (1) weight management and obesity, (2) sarcopenia, and (3)
amino acid regulation of muscle metabolism. These diverse areas of research are providing new per-
spectives about optimum daily protein needs for adult health with the focus shifting from minimum
daily protein intake to the optimum amount and distribution of protein at individual meals.
PROTEIN, WEIGHT LOSS, AND MUSCLE MASS

The initial evidence linking higher-protein diets to body composition and muscle mass appeared in
the 1970s with acute weight loss studies (Flatt and Blackburn, 1974; Bistrain et al., 1977). During
extreme energy restrictions such as fasting or very-low-calorie diets (<900 kcal/day), the body is
forced to modify the balance of metabolic fuels. In a typical American diet, carbohydrates provide
~50% of energy and >1200 kcal/day. When total energy and dietary carbohydrates are restricted, the
body shifts toward the use of stored fats. In addition, lean tissues are degraded to maintain a constant
supply of amino acids for hepatic gluconeogenesis to maintain blood glucose and for the synthesis
of essential proteins for plasma and organs. This early research with very-low-calorie diets demon-
strated that providing a diet enriched in high-quality proteins produced a “protein-sparing” effect by
minimizing the degradation of lean tissues and reducing nitrogen loss. Researchers found that pro-
tein intakes of 1.5 g/kg/day could produce net protein gains even during extreme energy restriction.
During the 1990s, a carbohydrate-centric view of protein emerged with popular weight loss diets
including the Atkins diet (Atkins, 1992), the Zone diet (Sears, 1999), and the carbohydrate addict’s
diet (Heller and Heller, 1993). These diets all trumpeted the dangers of excess carbohydrates and
chronic hyperinsulinemia as major contributors to the onset of obesity, heart disease, and type 2 dia-
betes; and authors recommended replacing dietary carbohydrates with protein and/or fat. These diets
were characterized as high-protein diets, but protein was generally only high as a percentage of calo-
ries. Dietary protein expressed as percentage of energy increases by default as carbohydrate intake is
reduced. Most of these diets were moderate in the absolute amount of daily protein consumed, ranging
from about 1.0 to 1.5 g/kg body weight or about 80 to 130 g/day. These diets were typically effective
for short-term weight loss, but long-term efficacy was equivocal (Foster et al., 2003).
Since the late 1990s, there have been numerous well-controlled weight loss studies using higher-
protein diets. Diets with protein >1.4 g/kg/day produce greater weight loss and improve body com-
position by protecting lean body mass and partitioning energy loss to body fat. Many of these studies
have been summarized in two meta-analyses (Krieger et al., 2006; Wycherley et al., 2012). Krieger
et al. (2006) evaluated 87 weight loss studies examining the substitution of protein for carbohydrates
in reduced-energy diets. They found that diets with carbohydrates reduced below ~40% of energy
and protein above 1.05 g/kg/day produced greater reductions of body mass, fat mass, and fat as a
percentage of body weight and attenuated loss of fat-free mass. Wycherley et al. (2012) extended
these findings to weight loss studies that also included measurements of blood lipids, glucose, and
blood pressure. They reported that higher-protein diets increased the loss of body weight and body
fat and reduced serum triglycerides and blood pressure while mitigating reductions in fat-free mass
and resting energy expenditure.
While the majority of weight loss studies support the use of higher-protein diets, some ran-
domized controlled trials report no differences in weight loss or body composition comparing
diets with different macronutrient compositions (Dansigner et al., 2005; Sacks et al., 2009). These
investigators concluded that the critical factor in weight loss is the caloric deficit created by the
diet, and the macronutrient ratios are relatively unimportant. However, on further inspection of
these studies, a confounding factor was often poor diet compliance. In large clinical trials, pre-
cise diet monitoring is impossible and producing sustained lifestyle changes is difficult. Subjects
tend to relapse toward preexisting diet behaviors (Foster et al., 2003; Dansigner et al., 2005).
While diet acceptance is an important issue in evaluating diet merits, poor compliance obscures
the fundamental question about the efficacy of a diet.
In total, research studies provide evidence that higher-protein diets provide metabolic advantages
over high-carbohydrate diets for weight loss, but negative perceptions hinder the acceptance of these
diets (St. Jeor et al., 2001). As a macronutrient, protein provides 4 kcal/g, making it equal to carbo-
hydrates, but protein-rich foods are often energy dense, containing high amounts of cholesterol and
saturated fat. Further, protein produces ammonia and urea still considered by some to be a health risk.
From an energy perspective, the perceived risks of protein or protein-rich foods combined with survey
data showing that Americans typically consume protein in excess of the RDA temper enthusiasm for
higher-protein diets. In total, protein is often viewed as an expensive and inefficient form of calories.
Carbohydrate-centric and fat-phobic perspectives have been ingrained in the U.S. Dietary
Guidelines for over 50 years. Since the late 1960s, the major nutrition-related health concern for
Americans has been heart disease, and U.S. nutrition policy was formulated largely around using
carbohydrates to reduce the consumption of cholesterol and saturated fat (USDA, 2005). Protein was
not considered to be a nutrient at risk and was downplayed in dietary guidelines. The most visual
example of this policy was the iconic food guide pyramid emphasizing avoiding fat and specifically
animal products containing cholesterol and saturated fat and increasing consumption of grains. These
nutrition guidelines resulted in a reduced consumption of milk, eggs, and red meat by 31%, 30%, and
21%, respectively, from 1970 to 2000 (USDA, 2003). In place of these protein-rich foods, Americans
increased consumption of grains, plant oils, and potatoes by 40%, 56%, and 62%. Coincident with
these dietary changes were dramatic increases in obesity and cardiometabolic disorders. In 2010, the
Dietary Guidelines Advisory Committee established by the USDA and NIH attempted to define the
diet changes associated with the obesity epidemic and found that beginning in the 1980s Americans
increased daily energy consumption by over 350 kcal, and the top six food categories accounting for
the excess calories were grain-based desserts and snacks, yeast breads, pizza, pasta, breaded chicken
products, and sugar-sweetened soda and sports drinks (USDA-HHS, 2010). Based on these findings,
the USDA discontinued the use of the food guide pyramid and shifted nutrition policy to MyPlate
emphasizing vegetables and protein (including dairy) as the foundation of a healthy diet.
DEVELOPING AN AMINO ACID PERSPECTIVE

ABOUT CARDIOMETABOLIC HEALTH
The protein-centric perspective shifts the focus from a global view of protein as simply a macronutri-
ent similar to carbohydrates and fat to a molecular focus that recognizes the individual roles of each
of the 20 amino acids. Each amino acid has at least two important roles, one in protein synthesis as
a building block for new proteins and a second in the creation of new biomolecules and/or metabolic
signals (Wu, 2013). A few examples of secondary roles of amino acids include tryptophan for synthe-
sis of serotonin, arginine for synthesis of creatine, cysteine for synthesis of glutathione, and lysine for
synthesis of carnitine, and signal molecules include histidine for stimulation of GCN2 in the integrated
stress response, arginine for nitric oxide and vascular function, and leucine for the activation of the
mTORC1 signal cascade for stimulation of muscle protein synthesis (mPS). The leucine signal has
received extensive attention because of its role in stimulating mPS and has become a principal factor
in defining optimal dietary protein intake (Layman, 2003; Layman et al., 2015).
These diverse roles of amino acids are dependent upon intracellular concentrations and, as might
be expected, are optimized at different dietary intakes. In the hierarchy of metabolic roles, the fun-
damental role of amino acids as building blocks for new proteins appears to be satisfied first and
equates with the estimated average requirement. Additional roles of amino acids appear to be more
dependent on increases in intracellular concentrations of respective amino acids. The relationship
of the metabolic response to the intracellular concentration highlights the importance of the protein
content of individual meals. If the intracellular concentration of a specific amino acid does not
increase after a meal, the metabolic pathway or signal cascade is minimized. The protein-centric
perspective emphasizes the quantity, quality, and meal distribution of protein to optimize these met-
abolic signals and related health outcomes.
OPTIMIZING MEAL PATTERN OF PROTEIN FOR

BODY COMPOSITION AND SARCOPENIA
The fourth decade of life often marks a detectable age-related loss of muscle mass and strength
known as sarcopenia (Stenholm et al., 2008). The decline in muscle mass and strength leads to the
loss of mobility, increased likelihood of falls and injury, reduced glucose tolerance, and increased
risk for obesity (Rosenberg, 1997). Factors contributing to sarcopenia remain to be fully elucidated.
Declining physical activity certainly contributes, but there is also a progressive decline in the mPS
response to dietary protein. Skeletal muscle slowly becomes more refractory to anabolic stimulus,
a condition described as “anabolic resistance” (Cuthbertson et al., 2005; Rennie and Wilkes, 2005).
Maintaining muscle health requires continuous repair and remodeling of muscle structures and
enzymes dependent on continuous synthesis and breakdown of muscle proteins (Burd et al., 2008).
The balance between synthesis and breakdown is an oscillating pattern with anabolic periods occur-
ring after meals and catabolic periods during postabsorptive times. The anabolic periods after meals
are driven by a rapid stimulation of protein synthesis with small changes in protein breakdown.
The catabolic period between meals occurs with a decline in protein synthesis, while protein break-
down is maintained at higher rates. The net balance between the anabolic and catabolic periods deter-
mines changes in muscle mass; and there is general consensus that the postmeal anabolic periods are
the dominant factor. This perspective shifts the focus of dietary protein requirements from the current
recommendations based on total protein per day to a focus on the meal distribution of protein.
Historically, protein requirements have been viewed as the minimum amount of dietary protein per
day necessary to achieve efficient growth or prevent deficiencies. For adults, growth is not relevant
and acute deficiencies are rare. Instead, adult protein requirements are based on an Estimated Average
Requirement defined as the minimum amount of protein necessary to achieve nitrogen balance (Institute
of Medicine, 2002). Nitrogen balance studies to determine the Estimated Average Requirement have
been performed mostly in young healthy adults with short-term controlled feeding conditions. Protein
intakes above that required for nitrogen balance have been viewed as unnecessary and possibly unsafe.
While nitrogen balance is the conventional approach to define minimum protein requirements, it has
no clear relationship to any health outcomes. Further, using the Estimated Average Requirement as the
dietary guideline assumes that amino acid metabolism beyond minimum nitrogen balance provides no
metabolic advantage and should be avoided. Nitrogen balance methods are widely criticized for under-
estimating optimal amino acid needs (Elango et al., 2008; Millward et al., 2008; Wolfe and Miller,
2008). Emphasis on adult health is shifting attention from simple nitrogen balance to functional health
outcomes related to skeletal muscle mass, metabolic function, and physical performance.
Mechanisms to explain anabolic resistance and net loss of muscle mass during aging remain unclear,
but likely include changes in capillary blood flow, reduced membrane transport of essential nutrients,
and reduced anabolic signaling for the initiation of mPS (Cuthbertson et al., 2005; Fry et al., 2011;
Wall et al., 2014). Research from Volpi et al. (1999) demonstrates that anabolic resistance originates
from changes in protein synthesis (translation) versus defects in gene expression (transcription). In a
series of studies, these researchers found that mPS responds similarly in older (>60 years) and younger
(<25 years) adults given a large bolus of essential amino acids (EAA, 18 g). However, when subjects
were given only 7 g of EAA, mPS was stimulated in the young but not in older adults. These studies
demonstrated that older adults maintain similar mPS capacity if adequate amino acids are available.
Subsequent studies examined the effects of individual EAA. They found that the amino acid leucine
had a unique ability to overcome the anabolic resistance in older adults (Volpi et al., 2000, 2003). In a
seminal study, subjects consumed a drink containing 6.7 g of EAA with leucine accounting for 26%
(1.75 g) of the total EAA. The EAA and leucine contents of the test meal represent the EAA content of
a 15 g whey protein meal. This EAA drink produced a normal postmeal mPS response in young adults,
but no response in older adults. Then the investigators modified the drink by substituting additional
leucine for equal amounts of the other EAA to enrich the formula to 41% leucine (2.75 g). The leucine-
enriched formula stimulated mPS similarly in young and older subjects demonstrating that leucine was
the critical EAA limiting the anabolic response in older adults (Katsanos et al., 2006).
These investigators also examined supplementing the amino acid test meal with 40 g of glu-
cose. As expected, the glucose supplement increased plasma insulin. The glucose supplement also
increased mPS in young adults but inhibited mPS in the older adults (Volpi et al., 2000). This nega-
tive effect of glucose (or insulin) was confirmed in a subsequent study (Guillet et al., 2004). Insulin
is now thought to function in a permissive but not regulatory role in older adults. These findings
demonstrate that high-carbohydrate diets may have differential effects on adults depending on age
with insulin producing an anabolic response in young adults but contributing to anabolic resistance
and reduced mPS in older adults (Volpi et al., 2000). The differential effects of glucose and/or
insulin on mPS in young versus older adults have not been extensively studied. Preliminary studies
indicate that older adults exhibit reduced insulin-induced blood flow reducing amino acid delivery
to muscles (Rasmussen et al., 2006) and reduced signaling at mTORC1 (Cuthbertson et al., 2005)
consistent with the negative effects of hyperinsulinemia (Gual et al., 2005).
Consistent with studies using free EAA, Pennings et al. (2012) reported that the mPS response for
older adults was greater after a meal containing 40 g of whey protein compared with 20 or 10 g meals.
In most proteins, EAA account for ~50% of total amino acid content, such that the 40 g whey meal
provides ~20 g of EAA. These investigators found that the 10 g meal produced no response in mPS
compared with the fasted baseline, while 20 g produced an anabolic response equal to approximately
one-half of the 40 g meal. Whey protein was used in these studies because it is highly water soluble,
rapidly digested, and particularly high in leucine accounting for ~11% of the total amino acids. The
three whey protein meals provided approximately 4.4, 2.2, and 1.1 g of leucine, respectively.
Yang et al. (2012) extended these findings by examining the interaction of dietary protein with
exercise. In a sedentary control group, they found that 40 g of whey protein increased myofibrillar
protein synthesis more than 20 g, and 10 g was not different from the fasted baseline. Using the same
protein meals, they found that the rate of mPS was significantly higher after each of the protein meals if
subjects completed a bout of resistance exercise before the meal. The synergy of protein with exercise
appeared to lower the threshold of protein required to stimulate mPS such that adults consuming 20 g
of whey protein after resistance exercise produced a similar anabolic response in mPS as sedentary
adults consuming the 40 g meal. These studies help define the meal threshold for protein and demon-
strate that resistance exercise can reduce anabolic resistance and lower the threshold for older adults.
The meal perspective for muscle health is becoming well recognized (Paddon-Jones and
Rasmussen, 2009; Layman et al., 2015). A recent international study group concluded that older
adults require more protein than younger adults to maintain function and health and to offset catabolic
conditions and disease risk associated with aging (Bauer et al., 2013). They recommended that daily
protein should be increased at least to a range of 1.0–1.2 g/kg body weight with protein distributed in
meals containing at least 30 g of protein and providing at least 2.5 g of leucine. These recommenda-
tions received additional support from a study by Mamerow et al. (2014) demonstrating that subjects
consuming 90 g/day of protein had higher net daily mPS if the protein was distributed in three 30 g
meals versus an unbalanced distribution with meals containing 11, 16, and 63 g, respectively.
LEUCINE DEFINING A MEAL THRESHOLD FOR DIETARY PROTEIN

Metabolic pathways for the three branched-chain amino acids (BCAA), leucine, valine, and isoleu-
cine, have been studied extensively since the 1970s when researchers discovered that BCAA, unlike
other amino acids, were not degraded in the liver, but were predominately metabolized in skeletal
muscle, and that leucine had a unique role in stimulating mPS (Buse and Reid, 1975; Fulks et al.,
1975; Harper et al., 1984). These discoveries served to focus attention on leucine in the relationship
between dietary protein and muscle mass and led to the development of a meal threshold theory.
Equally important, the mPS response to leucine appears to contribute to the metabolic advantage of
dietary protein to increase thermogenesis, increase fat utilization, and improve body composition.
The unique roles of leucine are, at least in part, associated with the absence of the branched-
chain aminotransferase enzyme in liver, resulting in an enriched supply of the BCAA appearing in
blood (Harper et al., 1984). Dietary BCAA reach the blood virtually unaltered from the levels in the
diet, while all other dietary amino acids are extensively metabolized by the GI tract and liver with
the removal of 60%–98% before reaching the blood. Further, BCAA transport is in near equilib-
rium balance between extracellular and intracellular concentrations, allowing leucine concentration
within skeletal muscle to directly reflect dietary intake (Bergstrom et al., 1974). Muscle responds
to changes in leucine concentrations through multiple pathways including mTORC1, the branched-
chain ketoacid dehydrogenase (BCKAD) complex, Sirt-1, and adenosine monophosphate (AMP)
kinase (AMPK) (Suryawan et al., 1998; Vary and Lynch, 2007; Sun and Zemel, 2009; Wilson et al.,
2011). Collectively, these pathways connect the regulation of skeletal muscle protein turnover and
energy metabolism with dietary protein intake.
mPS is cyclical with stimulation after meals and inhibition during postabsorptive periods. After
a meal, the intracellular leucine concentration increases in proportion to the protein content of the
meal. If the meal contains adequate protein to produce approximately a threefold increase in intra-
cellular leucine (from fasted ~100 μmol/L to fed ~300 μmol/L), then mTORC1 is activated stimu-
lating downstream activation of translation factors eIF4E (initiation factor 4E) and rpS6 (ribosomal
protein S6). These translation factors serve to target specific mRNAs that increase the capacity for
the synthesis of structural myofibrillar proteins by 30%–40% (Crozier et al., 2005; Norton et al.,
2009). Meals with approximately 30 g of protein, and specifically providing more than 2.5 g of
leucine, produce an anabolic response that enhances muscle protein turnover (Layman et al., 2015).
Parallel with mTORC1 activation, increased intracellular leucine stimulates BCKAD, the rate-
limiting step in BCAA oxidation (Lynch et al., 2003) (Figure 18.1). BCAA (along with lysine)
[Leucine]
BCAT
mTORC1 Sirt-1
–NH2
αKIC
Protein synthesis PGC-1α
Glucose
BCKAD #
Fatty acid
CPT
PDH FA
Acetyl CoA
Pyruvate
–NH2 TCA cycle
Alanine
Mitochondria ATP
FIGURE 18.1 Roles of leucine in regulation of skeletal muscle metabolism. Increased intracellular leucine
concentration stimulates mTORC1 and BCKAD and shifts the production of acetyl CoA from glucose to fatty
acids. Solid lines represent metabolic pathways and dashed lines represent signaling pathways. Abbreviations:
BCAT, branched-chain aminotransferase; BCKAD, branched-chain ketoacid dehydrogenase; αKIC, alpha-keto
isocaproate; mTORC1, mechanistic target of rapamycin complex 1; Sirt-1, Sirtuin-1; PGC-1α, peroxisome
proliferator-activated receptor-γ coactivator-1α; CPT, carnitine palmitoyltransferase; PDH, pyruvate dehydro-
genase; #, mitochondrial biogenesis.
are the only ketogenic amino acids producing acetyl CoA similar to β-oxidation of fatty acids
and providing an important fuel source for skeletal muscle. Activation of the BCKAD complex
occurs through the phosphorylation of a kinase that also inhibits the pyruvate dehydrogenase
complex, regulating the conversion of pyruvate to acetyl CoA for entry into the mitochondria
(Chang and Goldberg, 1978). With inhibition of pyruvate dehydrogenase, pyruvate generated
through glycolysis from either blood glucose or muscle glycogen is converted to alanine with the
addition of a nitrogen group derived from BCAA degradation. Leucine concentration also appears
to stimulate signaling through Sirt-1 and PGC-1α (peroxisome proliferator-activated receptor-γ
coactivator-1α) to increase mitochondrial biogenesis and fatty acid oxidation (Sun and Zemel,
2009). The net effects of increased intracellular leucine are stimulation of mPS, increased energy
expenditure, a shift of mitochondrial fuel mixture from glucose to fatty acids, and changes in
glycemic regulations with increases in glucose recycling via the glucose–alanine cycle (Layman
and Baum, 2004).
The magnitude of the impact of the mPS response on energy status can be appreciated by moni-
toring ATP depletion. After a leucine-rich protein meal, we observed a significant depletion of mus-
cle ATP and activation of the cellular energy sensor AMPK (Wilson et al., 2011). These responses
are similar to the effects of exhaustive exercise. Activating AMPK serves to protect cellular ATP
levels by inhibiting mPS at the elongation stage of translation, reducing ATP expenditure, and stimu-
lating energy production by increasing mitochondrial fatty acid oxidation and glucose uptake for
glycolysis (Kahn et al., 2005). While equating a protein meal to exercise may be overextending the
data, the metabolic responses are similar to changes observed with endurance training (McArdle
et al., 1999) and estimates of energy costs of protein synthesis account for ~20% of total energy
expenditure (Waterlow, 1995) with the response proportional to the postmeal thermogenic effects
(Giordano and Castellino, 1997). Clearly, a meal pattern of dietary protein and mPS impacts daily
energy expenditure. These findings suggest that mPS is likely a major component of the increased
thermogenesis associated with dietary protein.
An additional piece of the meal-pattern puzzle is the duration of the anabolic response.
Multiple groups have observed that the duration of mPS after a meal is approximately 2–3 h and
muscle becomes refractory to further stimulation (Norton et al., 2009; Atherton et al., 2010).
The fact that a meal has a set duration may seem intuitively logical, but at 2 h, all of the anabolic
signals for mPS including mTORC1, eIF4E, rpS6, and plasma leucine concentration remain
elevated. This condition has been termed “muscle full.” The cause of the refractory, muscle full
response remains unknown but may be associated with the depletion of muscle ATP and inhibi-
tion of the elongation stage of translation (Wilson et al., 2011). The major energy cost of mPS
is associated with the elongation stage of peptide assembly. At approximately 2 h after a meal,
we observed an increase in the ratio of AMP/ATP, activation of the master energy sensor AMPK,
and inhibition of elongation factor 2 (Wilson et al., 2011). The net effects of these responses
are to slow mPS and protect muscle ATP concentrations from extreme depletion. After the post-
prandial anabolic response, muscle requires a period to recover or “reset” before responding to a
next meal. The pattern of meal response followed by a refractory period creates the anabolic to
catabolic cycling observed for skeletal muscle protein turnover. The need to reset the metabolic
machinery for mPS further highlights the important balance between the mTORC1 signal and
the BCKAD pathway allowing the leucine content of the meal to first create the anabolic signal
through mTORC1 and then BCKAD to restore the leucine concentration to baseline levels prior
to a next meal. These findings further support the importance of the meal distribution of dietary
protein and may ultimately determine potential metabolic advantages including diet-induced
thermogenesis.
Using a criterion of a leucine threshold to define the protein quantity and quality at each meal,
we designed a series of weight loss studies evaluating changes in body composition and cardio-
metabolic parameters. The studies were randomized controlled trials comparing a higher-protein,
reduced-carbohydrate (HP/LC) diet with a high-carbohydrate, low-protein (HC/LP) diet. The HC/LP
diet was designed to follow the guidelines of the Food Guide Pyramid. Both diets reduced energy
intake, while the HP/LC diet provided daily protein at 1.5 g/kg body weight including a minimum
of 2.5 g of leucine at each meal and carbohydrates <150 g/day; and the HC/LP diet set protein at
0.8 g/kg with carbohydrates at >200 g/day. The first study was a 10-week controlled feeding study
designed to minimize behavioral aspects of diet compliance (Layman et al., 2003a,b). Participants
were assigned diets with defined meal composition using a required 14-day menu rotation, and ~60%
of meals prepared at our Food Research Center. Participants also completed weekly 3-day weighed
food records. The HP/LC diet resulted in increased fat loss, greater improvement in body composition
(greater reduction in fat mass and attenuated loss of lean body mass), reduced triglycerides, increased
HDL-cholesterol, and stabilized postprandial glycemic responses including reducing postprandial
insulin peak and area-under-the-curve and eliminating the 2-h postmeal drop in blood glucose. The
second study was a 16-week evaluation of the same diets with or without exercise (Layman et al.,
2005). Participants were required to use the same meals tested in study 1 but were allowed free choice
for daily meal selection based on personal diet preferences. Participants completed weekly 3-day
weighed food records, and ~20% of meals were consumed at the Food Research Center. The primary
outcomes of increased fat loss, attenuated loss of lean mass, reduced triglycerides, and increased
HDL-cholesterol were consistent with study 1. Exercise further increased fat loss and attenuated loss
of lean mass with greater effects with the HP/LC diet. The third study was a multicenter, 12-month
randomized controlled trial with free-living subjects (Layman et al., 2009). Subjects were required
to attend a diet education meeting each week and complete 3-day weighed food records. The HP/LC
diet resulted in more subjects losing at least 10% of initial body weight with increased fat loss while
minimizing the loss of lean tissue mass compared with the HC/LP diet. In addition to improved body
composition, the HP diet improved cardiometabolic parameters including reducing triglycerides and
increasing HDL-cholesterol.
IMPACT OF PROTEIN VERSUS CARBOHYDRATES

ON CARDIOMETABOLIC RISK FACTORS
Glycemic Regulations
Diets with increased protein and reduced carbohydrates have been shown to improve glycemic
regulations and blood lipids in normal subjects, obese subjects, and people with type 2 diabetes.
Improvements include reduced postprandial glucose and insulin (i.e., area-under-the-curve) and
reduced triglycerides. However, the specific impact of dietary protein on cardiometabolic param-
eters beyond body composition and energy expenditure is difficult to differentiate from changes
associated with reduced carbohydrate or energy intake.
Interactions of protein and amino acids with carbohydrate metabolism are well known. Amino
acids directly contribute to de novo synthesis of glucose via gluconeogenesis and participate in the
recycling of glucose carbon from skeletal muscle via the glucose–alanine cycle. Further, dietary
protein and, specifically, the amino acids leucine and glycine stimulate insulin release from the pan-
creas, and leucine serves to modulate the intracellular insulin signal in skeletal muscle and adipose
tissue. Whereas potential interactions of amino acids with glucose metabolism have been experi-
mentally established, the physiological impact of amino acids on glucose homeostasis is dependent
on the experimental or dietary conditions.
Studies by Sweeney (1927) and Himsworth (1935) reported that normal subjects fed increas-
ing levels of carbohydrates increased their capacity for the disposal of an oral glucose load. These
studies are often cited as early evidence that high-carbohydrate diets increased insulin sensitiv-
ity, and the converse, reduced-carbohydrate diets reduce the capacity for glucose disposal. Other
studies reported that increasing plasma amino acid concentrations can decrease glucose disposal,
induce hyperinsulinemia and hyperglycemia, and potentially lead to insulin resistance (Schwenk
and Haymond, 1987; Ferrannini et al., 1988). These studies used the intravenous infusion of amino
acids with euglycemic clamp techniques to measure glucose uptake and insulin resistance. Acute
increases in plasma amino acids were found to increase plasma glucose concentrations, decrease
glucose uptake, and increase plasma insulin levels.
Contrary to these reports, studies examining metabolic responses in normal subjects consuming
meals in which protein was substituted for carbohydrates found that HP/LC meals reduced postpran-
dial insulin response and decreased 24-h area-under-the-curve for both glucose and insulin. Floyd et al.
(1966a,b) compared the intravenous infusion of amino acids with the oral consumption of a protein
meal and found acute hyperinsulinemia after the intravenous infusion but minimal postprandial insu-
lin response after oral consumption. Krezowski et al. (1986) reported similar effects on glycemic
regulations for normal, weight-stable subjects. Using isoenergetic meals, they found that substituting
dietary protein for carbohydrates reduced postmeal responses of both plasma glucose and insulin.
Treatment of type 2 diabetes with restriction of dietary carbohydrates produces similar improve-
ments for glycemic regulations. HP/LC diets decrease fasting plasma glucose and reduce HbA1c
when compared with responses to HC/LP diets with or without accompanying weight loss.
Gannon et al. compared HP/LC and HC/LP diets in weight-stable subjects with type 2 diabetes. They
found that HP/LC diets reduced fasting glucose, 24-h glucose area-under-the-curve, and HbA1c; the
reduction in HbA1c was proportional to the reduction in carbohydrate consumption (Krezowski
et al., 1986; Gannon et al., 1996; Gannon and Nuttall 2004). An important difference for people with
type 2 diabetes is that dietary protein appears to stimulate insulin release at levels similar to dietary
carbohydrates. Therefore, whereas the replacement of dietary carbohydrates with protein results
in reduced postprandial glucose response, protein maintains an equivalent insulin response that is
significantly higher than replacement with fat.
Blood Lipids
The most consistent effect of HP/LC diets on blood lipids is lowering serum triglycerides.
Normolipidemic subjects using HP/LC diets for weight loss generally experience 30%–55% reduc-
tions in triglyceride concentrations from baseline values (Krieger et al., 2006). Other changes in
blood lipids often associated with HP/LC diets include increased HDL-cholesterol concentration
and larger LDL particle size, but specific responses are influenced by baseline lipoprotein patterns,
fat content of the diet, total energy intake, weight loss, and insulin responsiveness of subjects
(Layman et al., 2008). Specific effects of protein are often difficult to untangle from changes in
energy and/or carbohydrate intakes; however, the OmniHeart Trial found the replacement of car-
bohydrates with protein had a greater effect on triglycerides than the replacement of carbohydrates
with unsaturated fat (Appel et al., 2005).
Controlled studies for energy intake that maintained stable body weight show reduced tri-
glyceride concentrations with HP/LC diets. Krauss et al. (2006) varied carbohydrate and protein
intakes with weight-stable subjects and found reduced triglycerides and increased LDL parti-
cle size with the primary effects associated with carbohydrate restriction. Likewise, Wolfe and
Giovannetti (1991) compared HP/LC and HC/LP diets that were isocaloric with weight-stable
subjects. They found that HP/LC reduced triglyceride and LDL-cholesterol concentrations and
increased HDL-cholesterol. Hence, both weight loss and weight-stable studies show that the iso-
caloric replacement of carbohydrates with protein is beneficial for blood lipoprotein patterns and
particularly for triglycerides.
Investigators have long recognized the associations of glucose intolerance, insulin resistance, and
increased triglycerides (Reaven, 1996). These relationships underlie the dyslipidemia of the metabolic
syndrome. Numerous studies with nondiabetic and diabetic subjects evaluated reduced-carbohydrate
diets for the treatment of dyslipidemia. Most of these studies reduced carbohydrates by replace-
ment with fat as an energy substitute (Reaven, 1996; Schwarz et al., 2003; Volk et al., 2014). These
studies consistently found that reducing dietary carbohydrates and increasing fat reduced triglycer-
ides and increased HDL. Similar findings are observed with substituting protein for carbohydrates
suggesting that the primary effects on lowering triglycerides arise from reducing dietary carbohy-
drates. McAuley et al. (2005) attempted to define the individual effects of protein, carbohydrates, and
fats on blood lipids. In a study with insulin-resistant, overweight women, they compared changes in
blood lipids using HP, high-carbohydrate (HC), or high-fat diets. After 24 weeks, the HP and high-fat
groups had greater reductions in body weight and triglycerides than the HC group. LDL-cholesterol
decreased for subjects in the HC and HP groups, and LDL was significantly lower in the HP com-
pared with the high-fat group. These findings suggest that lowering carbohydrates is most important
for reducing triglycerides and that replacing carbohydrates with protein may be more efficacious than
using fat for improving lipoprotein patterns. Further in support of benefits with high protein intake,
skeletal muscle mass appears to relate to triglyceride concentration by affecting VLDL secretion rates
(Sondergaard et al., 2015).
CONCLUSIONS
Dietary protein recommendations range from minimum requirements to prevent deficiencies defined
as the RDA to higher amounts established for optimal adult health. The RDA is 0.8 g/kg/day and
based on maintaining nitrogen balance, while optimal health is based on protecting skeletal muscle
mass and function with protein recommended at 1.2–1.5 g/kg/day (Bauer et al., 2013). Consistent
with the higher-protein targets, the 2015 Dietary Guidelines Advisory Committee recommended
healthy diets should provide protein at 155%–198% above the RDA (USDA, 2015). Further, opti-
mal muscle health requires daily protein to be consumed in meals containing a minimum of 30 g of
protein that also provide all of the EAA including at least 2.5 g of the EAA leucine.
Studies using energy-restricted diets for weight loss consistently find that diets with increased
protein and reduced carbohydrates (HP/LC) have beneficial effects on cardiometabolic outcomes.
These HP/LC diets produce a greater loss of weight and body fat while minimizing the loss of lean
tissue, and have been shown to improve dyslipidemia with reduced blood triglycerides and increased
HDL and to minimize postprandial swings in blood glucose and insulin. While higher-protein diets
appear to be beneficial for cardiometabolic health, interpreting the specific effects of protein is
difficult because of parallel changes in energy and carbohydrate intakes. Further, many long-term
clinical trials are confounded by the lack of subject compliance with diet protocols.
While metabolic advantages of higher-protein diets are often intertwined with reductions
in carbohydrates, evidence is accumulating that protein has unique effects on skeletal muscle
including increases in protein turnover, energy expenditure, and fat oxidation. These changes
are consistent with the increased fat loss and sparing of lean tissues during weight loss. Further,
research studies focused on healthy aging and exercise demonstrate that protein effects arise
from the meal distribution of protein and specifically the signaling effects of the EAA leucine
on initiation of mPS.
Meal-based effects of dietary protein on mPS appear to be associated with the initiation phase
of protein synthesis. In skeletal muscle, protein synthesis is a cyclical process stimulated during the
absorptive period after a meal providing adequate protein and energy and inhibited during posta-
bsorptive periods between meals. The signal for the postmeal stimulation of mPS is transmitted
through the mTORC1 signal complex to downstream initiation factors. In adults, mTORC1 is sensi-
tive to the amount of protein in the meal and specifically to the amount of the essential amino acid
leucine. Optimum stimulation of mPS requires meals providing at least 30 g of protein containing
at least 2.5 g of leucine. Studies evaluating the meal response to protein demonstrate the positive
effects of protein on muscle mass and strength. Further, changes in leucine concentrations influence
muscle metabolism and energy expenditure through multiple pathways including mTORC1, the
BCKAD complex, Sirt-1, and AMPK. mPS is estimated to account for up to 20% of total resting
energy expenditure. Together, these meal-based changes in mPS are consistent with the protection
of lean tissues and the increased energy expenditure (i.e., diet-induced thermogenesis) and fatty acid
oxidation observed with higher-protein diets.
The effects of HP/LC diets on glycemic regulations and blood lipoproteins appear to be largely
associated with reduced-carbohydrate intake. Multiple studies have shown that reducing carbohy-
drate intake by energy restriction or replacing carbohydrates with protein or fats will reduce blood
triglycerides, increase HDL, and reduce postmeal increases in glucose and insulin. While the pri-
mary effects appear to be associated with reducing carbohydrates, higher-protein diets may help
stabilize glycemic regulations by increasing the recycling of glucose via the glucose–alanine cycle
in skeletal muscle and increasing fatty acid oxidation.
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19 Protein Sources, CVD, Type 2
Diabetes, and Total Mortality
Peter Clifton
CONTENTS
Abbreviations................................................................................................................................. 350
Introduction.................................................................................................................................... 350
Scope of Review............................................................................................................................ 350
Red and Processed Meat................................................................................................................ 351
Epidemiological Studies........................................................................................................... 351
Total Mortality/CVD Mortality/CVD Events....................................................................... 351
Diabetes................................................................................................................................ 352
Characteristics of Red Meat Eaters...................................................................................... 353
Possible Mechanisms of a Harmful Effect of Red Meat...................................................... 353
Red Meat and CVD/Diabetes Risk Factors.......................................................................... 353
Dietary Interventions and Biomarkers of CVD and Diabetes Risk.......................................... 355
Dairy.............................................................................................................................................. 355
Epidemiological Studies........................................................................................................... 355
Total Mortality/CVD Mortality/CVD Events....................................................................... 355
Type 2 Diabetes.................................................................................................................... 357
Dairy and Risk Factors for Type 2 Diabetes........................................................................ 357
Dietary Interventions and Biomarkers of CVD and Diabetes................................................... 358
Longer-Term Feeding Studies.............................................................................................. 358
Acute Feeding Studies.......................................................................................................... 359
Poultry............................................................................................................................................ 360
Eggs................................................................................................................................................ 360
Fish................................................................................................................................................. 361
Epidemiological Studies of Fish Consumption and CVD and Diabetes................................... 361
Diabetes................................................................................................................................ 361
Dietary Interventions................................................................................................................. 362
Soy Intake, Heart Disease, and Type 2 Diabetes........................................................................... 362
Dietary Patterns Containing Soy............................................................................................... 363
Isoflavonoid Excretion.......................................................................................................... 363
Interventions with Soy on Cardiovascular and Diabetes Risk Markers............................... 363
Nontraditional Risk Factors.................................................................................................. 363
Acute Studies with Soy............................................................................................................. 364
Conclusion..................................................................................................................................... 364
References...................................................................................................................................... 364
349
ABSTRACT
Each of the main protein sources red and processed meat, dairy, poultry, chicken, fish, egg, and soy
will be discussed commencing with epidemiology relating to these foods and total mortality, CVD,
CHD, and stroke events and mortality and type 2 diabetes as well as risk markers of these diseases
such as weight, lipids and blood pressure. Acute and chronic interventions with these foods will then be
discussed. Although there is epidemiological evidence for red and processed meat to increase the risk
of CVD and type 2 diabetes, and for dairy to protect there is a complete absence of any evidence from
intervention studies to support these findings, except for a high dairy diet as part of the DASH diet low-
ering blood pressure. Red meat in place of carbohydrate also lowers blood pressure. The epidemiologi-
cal findings are very heterogeneous and only yogurt appears to be protective in the majority of studies.
ABBREVIATIONS
CARDIA Coronary Artery Risk Development in Young Adults
CCL5 Chemokine (C-C motif) ligand 5
EPIC European Prospective Investigation into Cancer and Nutrition
IHD Ischemic heart disease
MESA The Multi-Ethnic Study of Atherosclerosis
PREDIMED Prevención con Dieta Mediterránea
RANTES Regulated on activation, normal T cell expressed and secreted
INTRODUCTION
This is a very complex area in which it is almost impossible to isolate the protein source per se from
other macronutrients associated with that protein source in its usual food form at least in epidemio-
logical studies. Thus, red meat is always accompanied by a varied amount of saturated fat; dairy with
calcium, magnesium, and saturated fat; and fish with very widely varying amounts of long-chain
omega-3 (N3) fats, and poultry may be eaten with or without fatty skin. Vegetable sources of protein
except soy are always accompanied by carbohydrate and fiber and polyphenols. Only in acute stud-
ies are isolated sources of protein available, and interpretation of the physiological significance of
these studies is difficult. There are many differences in the way protein sources are consumed in the
United States, Europe, and Asia, so combining data from many regions may obscure any relation-
ships. In addition, as in all epidemiological studies, the results may be confounded by unmeasured
variables, for example, red meat eaters have higher mortality and fish eaters lower mortality because
of other behavioral attributes inadequately captured by the data and not due to meat or fish itself.
SCOPE OF REVIEW
Sources of protein in the U.S. diet (O’Neil et al. 2012) are shown in Table 19.1. Although wheat- and
corn flour–based products rank first in contribution to protein in this table, they will not be discussed
in detail except for acute studies using gluten. Eggs are an important source of protein, but the accom-
panying cholesterol makes interpretation of the effect of protein difficult except in acute studies using
egg albumin. Although soy and other legumes contribute only 1%–2% protein, they will be discussed
as they are important in non-U.S. diets. Nuts and seeds will not be discussed as they are not major con-
tributors to protein intake for most people and contribute mostly fat. This is similar for legumes, which
contribute fat, carbohydrate, and fiber. Red meat epidemiology usually includes unprocessed pork as
red meat, whereas in this analysis it is mixed with pork-based processed goods. Red and processed
meats together are the major source of protein at 24.1%, while animal protein accounts for about
75%–78%. Meat, poultry, and fish account for about 25% of the saturated fat intake as does dairy.
Protein Sources, CVD, Type 2 Diabetes, and Total Mortality 351
TABLE 19.1
Data from NHANES 2003–2006 Was Used to Look at Protein
Sources in the U.S. Diet in 9490 Individuals
Food Group Adults 19 Years and Over (% Total)
Bread, cakes, biscuits, etc. (wheat and corn) At least 20
Poultry 14.4
Beef 14.0
Cheese 8.5
Milk 6.9
Pork, ham, and bacon 5.7
Fish and shellfish 5.0
Frankfurters, sausages, luncheon meats 4.4
Eggs 3.2
Nuts, seeds 2.1
Source: O’Neil, C.E. et al., Nutrients, 4(12), 2097, 2012.
RED AND PROCESSED MEAT

Epidemiological Studies
Total Mortality/CVD Mortality/CVD Events
Total mortality is shown in Table 19.2 and CVD mortality and events in Table 19.3. The most recent meta-
analysis done by Wang et al. (2015) found a 15% increase in total mortality and CVD mortality per 100 g
of processed meats/day (both p < 0.001; five and six studies, respectively) with similar findings for total
meat. There was evidence of a nonlinear relationship for CVD mortality (steeper for the first 20 g and
flatter thereafter), and the effect was seen only in U.S. populations and not in European and Asian popula-
tions. An ecological study of eight Asian prospective studies found no association between red meat and
all-cause or CVD mortality (Lee et al. 2013). However, in Shanghai, red meat intake (mostly pork) was
associated with an 18% difference in total and IHD mortality between top and bottom quintiles in men
TABLE 19.2
Total Mortality: Increase in Risk from Highest to Lowest Intake Group or per 50–100 g
Total Meat Processed Processed Fresh Meat
(High vs. Total Meat Meat (High Meat (per (High vs. Low) Fresh Meat
Study Low) (per 100 g) vs. Low) 50 g) (per 100 g)
Wang et al. (2015) 15% (n = 6) 15% (per
Meta-analysis 100 g) (n = 5)
Larsson and Orsini 29% (n = 5) 23% (n = 6) 10% (NS)
(2014) Significant only
Meta-analysis in men, USA
Abete et al. (2014) 22%
Meta-analysis
Takata et al. (2013) 18% (men
Shanghai study only)
Notes: Lowest intake varied from <1 day/week, <6 times/month, or 0.25 servings/day, 0.51 servings/day, 0.28 servings per
1000 kcal, 9.1 g/1000 kcal, 15 g/day, or 21.4 g/day (all for total meat). Highest intake was 4–7 times/week, >45 times/month,
1.2 servings/1000 kcal, 65.8–68 g/1000 kcal, 2.07–2.17 servings/day, 160 g/day. Overall 4–11 times greater than the lowest.
TABLE 19.3
CVD Mortality: Increase in Risk from Highest to Lowest Intake Group or per 50–100 g/day
Processed Processed
Total Meat Total Meat Meat Meat Fresh Meat Fresh Meat
Study (High vs. Low) (per 100 g) (High vs. Low) (per 50 g) (High vs. Low) (per 100 g)
Wang et al. (2015) 15% 15% (per 100 g)
Meta-analysis (n = 6)
Micha et al. 42%
(2010, 2012) (incidence)
Meta-analysis (n = 5)
Abete et al. 16% 18%
(2014)
Meta-analysis
Takata et al. 18% (men
(2013) only)
Shanghai study
Kaluza et al. Stroke 13% Stroke 11% per Stroke 11%
(2012) per serving serving per
Meta-analysis serving
6 cohorts
only (Takata et al. 2013). In Japan no effect of meat was seen on IHD, stroke, or total CVD deaths (Nagao
et al. 2012). Larsson and Orsini (2014) separated out fresh red meat from total meat and found that the risk
for this component for total mortality was not significant (risk ratio [RR] 1.10, six studies) but also found a
higher risk for processed meat (highest vs. lowest consumption of about 50 g/day, RR 1.23, six studies) and
total meat (1.29 or 100 g/day, five studies). They also found that the relationship for unprocessed meat was
significant only in men and in U.S. populations, whereas processed meat and total meat were significant
in both men and women and in both the United States and Europe. Some of the same authors examined
six cohorts (Kaluza et al. 2012) and found a significant relationship between red meat and stroke. For each
serving per day increase in fresh red meat, processed meat, and total red meat consumption, the RRs of
total stroke were 1.11, 1.13, and 1.11, respectively (all p < 0.05), and unlike the studies on total and CVD
mortality, there was no heterogeneity in the stroke studies. Only thrombotic stroke was related to meat
intake (four cohorts). Abete et al. (2014) examined CVD mortality in 13 cohorts and found similar results
to Wang et al. (2015) with a 22% and 18% increase in all-cause and CVD mortality in the highest category
of consumption of processed meat and 16% for total red meat consumption and CVD mortality. Micha
et al. (2010) found that red meat intake was not related to CHD risk (four studies) while processed meat
was related to a 42% increase per 50 g (five studies, p = 0.04). Similar results were found in an updated
analysis, and it was concluded that the 400% difference in sodium content between fresh and processed
meat could explain two-thirds of the difference in risk (Micha et al. 2012). In the ARIC study, protein
sources were unrelated to CHD risk (Haring et al. 2014).
Diabetes
The most recent meta-analysis was performed by Feskens et al. (2013). Per 100 g of total meat, the
relative risk for type 2 diabetes was 1.15 and for unprocessed red meat 1.13, and for processed meat,
the relative risk per 50 g was 1.32, and all were significant. Poultry intake was unrelated to risk. Pan
et al. (2011) estimated that substitutions of one serving of nuts, low-fat dairy, and whole grains per day
for one serving of red meat per day were associated with a 16%–35% lower risk of type 2 diabetes.
Pan et al. (2013) also examined the effects of changes in red meat intake. Increasing red meat intake
of more than 0.50 servings/day (1 serving = 13 g for bacon; 28–45 g for various processed meat items;
85 g for unprocessed red meat) was associated with a 48% (p < 0.001) elevated risk in the subsequent
4-year period, and the association was modestly attenuated to 30% after further adjustment for initial
body mass index (BMI) and concurrent weight gain. Reducing red meat consumption by more than
0.5 servings/day from baseline to the first 4 years of follow-up was associated with a significant 14%
lower risk during the subsequent entire follow-up through 2006 or 2007. In the Malmo study, there was
a 16% increased risk with processed meat intake in the highest quintile (Ericson et al. 2013). In the
MESA study, a higher intake of meat saturated fat was associated with greater CVD risk with an increase
of 5 g/day or 5% of energy from meat increasing risk significantly by 26% and 48%, respectively, with a
clear dose–response relationship. The substitution of 2% of energy from meat saturated fat with energy
from dairy saturated fat was associated with a 25% lower CVD risk (de Oliveira Otto et al. 2012). A meta-
analysis of seven vegetarian studies showed a 29% lower mortality from IHD and a 12% lower mortality
from cerebrovascular disease (Huang et al. 2012). This result is a summation of the absence of meat plus
the addition of more fruit and vegetables. In meat eaters, increasing fruit and vegetable intake from less
than 3 servings/day to more than 5 servings/day reduces CHD risk by 17% suggesting that half of the
protective effect seen in vegetarians may be due to the absence of meat (He et al. 2007).
Characteristics of Red Meat Eaters

In the EPIC study (N = 449,000), men and women in the top categories of processed meat intake in gen-
eral consumed fewer fruits and vegetables than those with low intake. Red and processed meat consumers
were more likely to be current smokers and less likely to have a university degree. Men with high red meat
intake consumed more alcohol than men with a low intake, which was not seen in women. Individuals
consuming more than 80 g poultry/day had a higher consumption of fruits and vegetables than those with
an intake of less than 5 g/day; there was no difference in smoking habits at baseline. After adjustment for
these factors, processed meat was associated with 44% higher mortality. Mutual adjustment for other meat
sources did not change the processed meat association. The authors estimated that 3.3% of all deaths could
be avoided if processed meat consumption was less than 20 g/day (Rohrmann et al. 2013).
In the Health Professional Follow-Up Study (HPFS), men in the top quintile of meat consumption
were heavier and less active and a greater proportion of them were smokers compared with men in the
bottom quintile. They also had 50% greater energy intake, consumed more alcohol, eggs, coffee, soft
drinks, dairy, and trans fat and slightly less fish, poultry, and fruit and vegetables, and consumed 60%
less fiber (Pan et al. 2013). Their polyunsaturated-fat-to-saturated-fat ratio was about 60% of that in the
bottom quintile. Similar but less dramatic findings were seen in both Nurses’ Health Study (NHS) cohorts
(Pan et al. 2013), although in NHSII the high red meat consumers also ate more fish and more poultry. A
diabetes score was calculated from trans fat and glycemic load, cereal fiber, and the ratio of polyunsatu-
rated to saturated fat, and this score and BMI, along with alcohol, energy intake, and physical activity,
were used to adjust the RRs. Using dietary variables rather than the diet score did not change the results.
Possible Mechanisms of a Harmful Effect of Red Meat

Red meat has variable amounts of saturated fat and is rich in iron, phosphatidylcholine, and carni-
tine, the latter two of which are sources of trimethylamine, which, as discussed in later text, has been
linked to CVD and type 2 diabetes (Koeth et al. 2013, Tang et al. 2013). Processed meat is rich in
fat, salt, and nitrates. Although iron intake and iron stores have been linked to CVD (Hunnicutt et al.
2014), many cuts of chicken have more myoglobin than does pork (http://ndb.nal.usda.gov/ndb/
search), so the relationship of CVD with food sources of iron is not clear. Advanced glycation end
products in meat is another possibility. Overall, although there are many mechanisms for effects of
red meat on both CVD and diabetes (Kim et al. 2015), none stand out as obvious candidates.
Red Meat and CVD/Diabetes Risk Factors

Weight
A recent meta-analysis showed that those in the highest intake group of red and processed meat had
a 37% and a 32% higher BMI, respectively. Waist circumference was increased by 2.8 cm in this
group. There was, however, considerable heterogeneity in the studies (Rouhani et al. 2014). A high
saturated fat intake enhances the association between obesity gene risk scores and BMI, and meat
contributes about 25% of saturated fat to the diet (Casas-Agustench et al. 2014).
Saturated Fat/Cholesterol
Surprisingly, there are few epidemiological studies of meat intake and serum cholesterol. The
Harbin study (Na et al. 2015) showed a relationship between a high meat dietary pattern (1 of 5
patterns) and hypertriglyceridemia (86% greater in the highest tertile), but there was no relationship
with hypercholesterolemia. In South Korea, a high meat and takeaway food diet was associated with
more hypercholesterolemia (Shin et al. 2014).
Blood Pressure
In the Oxford cohort of the EPIC study, male meat eaters have an incidence of hypertension of 15%
compared with a rate of 5.8% in male vegans. Fish eaters and vegetarians have intermediate and similar
values suggesting the replacement of meat with fish will beneficially influence blood pressure (Appleby
et al. 2002). In the CARDIA study (Steffen et al. 2005), positive dose–response relations for elevated
blood pressure incidence were observed across increasing quintiles of meat intake (p trend = 0.004).
Telomeres
Processed meat was associated with shorter telomeres in the MESA study (Nettleton et al. 2008),
and it is known that telomeres are shorter in those with CVD (Aviv 2012).
Inflammatory Markers
Although C-reactive protein (CRP) was related to meat intake in the Nurses’ Health Study, the rela-
tionship was no longer significant after adjustment for BMI (Ley et al. 2014).
Serum/Urine Trimethylamine Oxide (TMAO)

Both phosphatidylcholine and carnitine can be converted by the microbiome to trimethylamine, which
is absorbed and oxidized in the liver. Omnivores have fourfold higher urinary TMAO excretion and
25%–30% higher plasma TMAO compared to vegans (Koeth et al. 2013). In addition, feeding carnitine
or steak increased plasma TMAO up to 6 μM in omnivores with no change in vegans. Individuals with
an enterotype characterized by enriched proportions of the genus Prevotella (n = 4) demonstrated higher
(p < 0.05) plasma TMAO levels than subjects with an enterotype notable for enrichment of Bacteroides
(n = 49) genus. There were significant differences in microbiota composition between vegans/vegetarians
and omnivores. Plasma carnitine levels predicted increased risks for both prevalent CVD and incident
cardiac events, but only among subjects with high TMAO levels. Three-year follow-up in 4007 patients
showed that plasma TMAO levels predicted increased CVD events with a 2.5-fold increase in the highest
vs. the lowest quartile (Tang et al. 2013). People with type 2 diabetes also have higher levels of TMAO (Li
et al. 2015). Fish, however, has high levels of TMAO that can be converted to trimethylamine by bacteria,
yet as described in the following text, fish intake is associated with a reduced risk of CVD.
Metabolomics
The EPIC study was a nested case–control study of 688 cases and 1993 controls. Total red
meat intake was associated with a 26% increase in risk of type 2 diabetes per 11 g/MJ. Six
biomarkers (ferritin, glycine, diacylphosphatidylcholines 36:4 and 38:4, lysophosphatidylcho-
line 17:0, and hydroxysphingomyelin 14:1) were associated with red meat consumption and
diabetes risk and, when added to the regression model, eliminated the association of red meat
to risk. Because the biomarkers accounted for 69% of the variance in outcomes, it was assumed
that they were mediators of risk. However, it is plausible that ferritin, for example, may only be
a marker of red meat intake (Wittenbecher et al. 2015) and would thus be expected to eliminate
the association of red meat with diabetes risk. As noted previously, the relationship between
iron and CVD risk is conflicted and controversial.
Dietary Interventions and Biomarkers of CVD and Diabetes Risk

Feeding hypercholesterolemic volunteers lean beef, poultry, or fish for 26 days lowered LDL
cholesterol by 5%–9% compared with the usual average American high saturated fat diet, with no
differences between the diets (Beauchesne-Rondeau et al. 2003). This is not a surprise given the low
saturated fat of all the diets, but it does not represent usual fatty American beef and so provides no
insight into the effect of this kind of palmitic acid–rich beef on CVD risk factors. A meta-analysis
of eight studies confirmed that there were no lipid differences comparing beef to chicken/fish (Maki
et al. 2012). Nevertheless, it is clear that in experimental studies, the major saturated fat in red meat,
palmitic acid, elevates LDL cholesterol (Mensink et al. 2003).
The DASH diet emphasizes a low intake of red meat in addition to high intake of fruit, vegetables, and
dairy, but in a recent DASH-like study, substitution of lean pork for fish and chicken did not affect the blood
pressure–lowering ability of the diet (Sayer et al. 2015). Lean red meat lowers blood pressure by 5 mmHg
systolic when 5% energy from carbohydrate is replaced with protein (Hodgson et al. 2006). Notably, mark-
ers of oxidation and inflammation were not found to be increased by red meat (Hodgson et al. 2007).
One of the few studies using red meat as normally consumed showed that substituting an oily
fish for red meat four times per week for 8 weeks in young women with low iron stores lowered
fasting insulin and increased HDL. Adhesion molecules, lipid peroxides, and LDL cholesterol were
not different across diets (Navas-Carretero et al. 2009). Insulin increased by 20% from baseline on
the red meat diet and did not change with the fish diet, although saturated fat intake was the same in
both diet groups and iron stores were no different. Saturated fat intake does not appear to influence
insulin sensitivity in humans (Lovejoy et al. 2002), suggesting it is not the saturated fat content of
meat that accounts for the apparent increased risk of type 2 diabetes. Chiu et al. (2014) showed that
increased saturated fat and protein from dairy sources had no effect on insulin sensitivity.
DAIRY
Epidemiological Studies
Although dairy fat contains about 50% saturated fat and is very similar in composition to beef fat, its
association with disease is quite different from red meat with no clear association with CVD and appar-
ent protection from type 2 diabetes, although the data are very heterogeneous and often of poor quality.
Total Mortality/CVD Mortality/CVD Events

In the most recent meta-analysis in 2015, an inverse association was found between total dairy consump-
tion and overall risk of CVD (9 studies; RR 0.88, p < 0.05) and stroke (12 studies; RR 0.87, p < 0.05).
However, no association was established between dairy consumption and CHD risk (RR 0.94; 95% CI
0.82, 1.07; 12 studies). Stroke risk was significantly reduced by consumption of low-fat dairy (six studies;
RR 0.93, p < 0.05) and cheese (four studies; RR 0.91, p < 0.05), and CHD risk was significantly lowered
by 16% by cheese consumption (seven studies; p = 0.05). Heterogeneity across studies was found for
stroke and CHD analyses, and publication bias was found for stroke analysis (Qin et al. 2015). A previous
meta-analysis of eight prospective studies focused particularly on milk (Soedamah-Muthu et al. 2011)
also reported reduced total CVD but found no effect of dairy on stroke; of note, the number of studies was
limited. Milk intake was not associated with total mortality (Soedamah-Muthu et al. 2011).
Rice (2014) reviewed papers published between 2009 and 2013 and found 18 prospective stud-
ies in this time period but did not perform a meta-analysis or add them to the meta-analysis of
Soedamah-Muthu et al. (2011); rather, they only discussed the findings. Six of the cohorts had at
least one positive finding of protection from dairy, three had negative findings, and nine were neu-
tral. Full-fat cheese and fermented milk are most often associated with apparent protection. Several
of these studies are not included in the 2015 meta-analysis (Qin et al. 2015).
Studies not included in the 2015 meta-analysis include the Hoorn study (van Aerde et al.
2013), which showed that overall total dairy intake was not associated with CVD mortality or
all-cause mortality. Each standard deviation increase in high-fat dairy; intake was associated with a
significant 32% higher risk of CVD mortality. In Costa Rican adults, dairy product intake as assessed
by adipose tissue fatty acids 15:0 and 17:0 and by food frequency questionnaire (FFQ) was not asso-
ciated with change in the risk of myocardial infarction (MI) (Aslibekyan et al. 2012). In a nested
case–control study from Sweden, milk fat biomarkers 15:0 and 17:0 were inversely associated with
the risk of first MI with a significant 26% reduction in women only. Quartiles of reported intake of
cheese (men and women) and fermented milk products (men) were inversely related to a first MI
(p trend < 0.05 for all) (Warensjo et al. 2010).
In elderly women, yogurt intake was inversely related to carotid intima-media thickness (IMT)
baseline risk factor–adjusted standard β = −0.075, p = 0.015. Participants who consumed >100 g
yogurt/day had a significantly lower common carotid artery IMT than did participants with lower
consumption (multivariable adjustment = −0.023 mm, p = 0.003) (Ivey et al. 2011).
In the Dutch Epic cohort (34,409 men and women), cheese was modestly inversely associated
with CVD mortality, particularly stroke mortality, with a 41% reduction in the latter (Praagman et al.
2015). In the Norwegian counties study, ruminant trans fat (from the two major protein sources—beef
and dairy) was associated with a 30% increase in CVD deaths, 30% increase in heart disease deaths,
and a 27% increase in sudden death in women only from highest compared to lowest intake category
(Laake et al. 2012). In the Swedish mammography study, the highest vs. lowest quintile of total dairy
food intake was associated with a 23% decreased incidence of MI (P < 0.05) and the highest vs. lowest
quintile of total cheese associated with a 26% decreased incidence of MI (p = 0.006). No significant
association between milk, cultured milk, or cream and MI was seen. In the highest vs. lowest quartile
of full-fat cheese, there was a 17% decreased incidence of MI (p = 0.035) (Patterson et al. 2013).
In an effort to determine whether calcium was a protective nutrient linking dairy to CVD, an
updated analysis of mineral intake in the Nurses’ Health Study showed no association between
calcium intake and stroke, although potassium and magnesium were protective. In an updated meta-
analysis of all cohort studies, calcium was not protective (Adebamowo et al. 2015).
Elwood et al. (2010) performed a meta-analysis of dairy intake and total mortality in eight cohorts but
omitted data from two cohorts. In the six cohorts included, there was a small but significant reduction
in total mortality in the subjects with the highest dairy consumption, relative to the risk in the subjects
with the lowest consumption (RR) 0.87, 95% confidence limits (0.77, 0.98). However, the two omitted
cohorts could have been validly included, and their omission biases the results. One, the Adventist cohort
compared 3+ glasses of milk per week to <1 glass per week and found a nonsignificant 0.98 RR (Kahn
et al. 1984). The Dutch Civil Servants cohort (Vijver et al. 1992) compared the top third of calcium intake
to the bottom and found no significant reduction with wide risk estimates, so the addition of these two
studies to the meta- analysis would likely make the relationship nonsignificant. It is reasonable to say
that dairy probably does not alter total mortality, but as noted earlier, there are some studies suggesting it
may increase total mortality. Kelemen et al. (2005) found a 41% increase in CHD mortality (and a 44%
increase with red meat) in the highest vs. the lowest quintile of intake substituting animal protein for an
equal number of servings of carbohydrate using the Willett technique (Willett 1998). Dairy was not sig-
nificantly associated with total mortality, but red meat increased mortality by 16%, which fits with other
meta-analytic results as discussed earlier. The Elwood meta-analysis on total mortality was dominated by
van der Pols’ paper on the Boyd Orr cohort (van der Pols et al. 2009), which examined childhood family
dairy intake and mortality 65 years later.
Similarly, in the meta-analysis of CHD deaths and dairy (mostly milk), Elwood used a derived
figure from the Hu report of the Nurses’ Health Study and ignored the significant risk estimate
of 1.67 for two glasses of whole milk per day vs. less than 1 glass/week and essentially used the
significant skimmed milk estimate of 0.78. In the Hu paper itself (Hu et al. 1999), the ratio of high-
fat to low-fat dairy was associated with CHD risk with an increase of 27% in the highest quintile
(p < 0.0004) after full adjustment. Given this data treatment, all estimates in the paper are suspect.
Overall, it is not clear if total dairy or any dairy form is related to the risk of CVD or CHD or
mortality from these diseases.
Type 2 Diabetes
The epidemiology of dairy intake and protection from type 2 diabetes is contradictory. Many stud-
ies show protection with low-fat dairy but at the same time also show protection with a fermented
food—cheese (Aune et al. 2013). Perhaps the most convincing data comes from the Harvard group as
their studies have dietary assessments every 4 years and a long follow-up period of 18–20 years. They
showed no link with total dairy or low-fat or high-fat dairy in a combined analysis of the NHS and
NHSII and the HPFS, but 1 serving/day of yogurt (226 g) was associated with a 17% reduction in the
incidence of type 2 diabetes (Chen et al. 2014). They also performed a meta-analysis with the addi-
tion of one new study and the updated Harvard information to the studies in the original Aune meta-
analysis (Aune et al. 2013). No protection was shown for total dairy (11 studies), whereas 1 serving
of yogurt/day was associated with an 18% reduction in risk (only 6 studies separated out yogurt).
The EPIC interact study showed that cheese intake tended to have an inverse association with dia-
betes (p-trend = 0.01), and a higher combined intake of fermented dairy products (cheese, yogurt, and
thick fermented milk) was inversely associated with diabetes (12% reduction in highest quintile, p-trend
= 0.02) in adjusted analyses (Sluijs et al. 2012). In the EPIC-Norfolk study (O’Connor et al. 2014),
an inverse association was found between diabetes and low-fat fermented dairy product intake (24%
reduction in highest tertile p trend = 0.049) and specifically with yogurt intake (28% reduction p trend =
0.017) in multivariable adjusted analyses. In the Malmo Diet and Cancer Study, there was a 23% reduc-
tion in risk in the highest quintile of high-fat dairy, particularly cream, high-fat fermented milk, and
cheese in women. Intakes of saturated fat with 4–14 carbons were associated with a decreased risk
(Ericson et al. 2015). The Malmo study stands out as providing opposite results to virtually all other
studies except for the findings for cheese and fermented milk. In the PREDIMED study (Díaz-López
et al. 2015), total dairy and low-fat dairy were associated with a 32%–35% reduced incidence of type 2
diabetes in the top tertile. Total yogurt reduced the incidence by 40% and increasing the consumption of
low-fat dairy and total yogurt reduced the risk even further to 45%–56%. However, the total number of
cases (270) was relatively small, so the estimates have wide confidence intervals.
In conclusion, yogurt seems to be convincingly associated with protection, while cheese is in
doubt. Astrup (2014) discusses the findings relating to yogurt in a recent review.
Dairy and Risk Factors for Type 2 Diabetes

Weight and Metabolic Syndrome
Kratz et al. (2013) examined 16 cohorts and found that high-fat dairy was associated with decreased
adiposity in 11 of them. Similarly, dairy intake was inversely associated with an incidence or prevalence
of the metabolic syndrome in 7 out of 13 studies. Three studies found no association and three had
mixed findings (Crichton et al. 2011). The Hoorn study (Snijder et al. 2007) found that dairy intake had
no relation to current weight or metabolic syndrome components (except lower diastolic blood pressure
and higher glucose) and no relation to the development of metabolic syndrome over 6.4 years. In sub-
jects with a BMI < 25, high dairy intake was associated with weight gain (Snijder et al. 2008). A review
in 2011 concludes that the data linking dairy food with protection from obesity was suggestive but not
consistent with 3 out of 10 studies in children and 5 of 9 studies in adults showing protection (Louie et al.
2011). In the Stanislas cohort in men only, a higher consumption of dairy products was associated with
beneficial changes in the metabolic profile (lower glucose and higher HDL cholesterol) over a 5-year
period; a higher calcium consumption was associated with a lower 5-year increase of the BMI and waist
circumference (Samara et al. 2013). The DESIR study showed a beneficial association between dietary
calcium and arterial blood pressure, insulin, and HDL cholesterol levels in women, whereas in men there
was only a beneficial association with diastolic blood pressure (Drouillet et al. 2007).
Blood pressure is the CVD risk marker most strongly associated with dairy. In a meta-analysis,
Soedamah-Muthu et al. (2012) showed that total dairy, low-fat dairy, and milk were associated with pro-
tection from hypertension (6–9 cohorts). The risk reduction was small at 3%–4% per 200 g/day. High-fat
dairy (six studies), total fermented dairy (four studies), yogurt (five studies), and cheese (eight studies)
were not significantly associated with hypertension incidence. A second meta-analysis with five cohorts
showed that the highest intake of low-fat dairy reduced hypertension by 16% (Ralston et al. 2012).
In the Luxembourg study (Crichton and Alkerwi 2014a,b), higher intakes of whole-fat milk,
yogurt, and cheese were associated with better cardiovascular health as defined by the American Heart
Association (smoking, BMI, physical activity, total cholesterol, blood pressure, and fasting plasma
glucose). Even when controlling for demographic and dietary variables, those who consumed at least
5 servings/week of these dairy products had a significantly higher cardiovascular health score than
those who consumed these products less frequently. Obesity was reduced by 55% in the highest tertile
of dairy intake. Clearly, a high dairy intake is a marker of those with better lifestyle choices such as
less smoking and more physical activity. In Portugal, adolescents with high milk intake were 47% less
likely to have a high cardiometabolic risk score than those with low milk intake (Abreu et al. 2014). No
association was found between this score and total dairy, yogurt, and cheese intake. The PREDIMED
study found that a higher intake of low-fat dairy, low-fat yogurt and milk, and full-fat yogurt were
associated with a significant 20%–28% reduction in the incidence of metabolic syndrome in the upper
tertile (Babio et al. 2015). Interestingly, cheese was associated with a 31% increase in risk. A higher
prevalence of Inuit participants with metabolic syndrome was observed in the highest tertile compared
with the lowest tertile of total dairy (10.3% vs. 1.6%; p < 0.001) (Ferland et al. 2011).
Dietary Interventions and Biomarkers of CVD and Diabetes

Longer-Term Feeding Studies
Chen et al. (2012) analyzed 29 randomized controlled trials with 2010 participants and found that dairy
had no effect on weight overall with a positive effect in studies less than 1 year in duration and a negative
effect in longer-term studies. Interventions focusing on changing dairy intake without changing weight or
exercise patterns are sparse. We recently reviewed the dairy intervention studies and found a very mixed
picture with no clear outcomes on glucose and (Turner et al. 2015a). We performed a three-way 4-week
crossover diet study in 47 overweight and obese people contrasting high-dairy diets with a high-meat
diet and a control diet containing neither dairy or red meat. The high-dairy diet caused increased insulin
resistance relative to both red meat and control diets (Turner et al. 2015b).
A large parallel design 4-week intervention (n = 158) that increased fat and protein derived
mostly from dairy found no effect on insulin sensitivity as assessed by intravenous glucose toler-
ance tests. However, changes in plasma branched-chain amino acids (BCAAs) across all diets were
negatively correlated with changes in the metabolic clearance rate of insulin (ρ = −0.18, p = 0.03)
and positively correlated with changes in the acute insulin response to glucose (ρ = 0.15, p = 0.05)
(Chiu et al. 2014). BCCAs are related to increases in the risk of insulin resistance and in fasting and
2 h glucose levels (Würtz et al. 2012, 2013).
In the Dairy Health study (Bohl et al. 2015), 63 healthy adults with abdominal obesity were
randomly allocated to 1 of 4 diets: 60 g casein or whey plus 63 g of milk fat with either high or low
medium-chain saturated fatty acids for 12 weeks. No effects on fasting lipids or glucose were seen,
but whey reduced postprandial apoB48 and decreased glucagon-like peptide 1 (GLP1) compared
with casein. There was no control diet. It was hypothesized that CVD risk would be lowered by
whey because of its effects on apoB48.
Benatar et al. (2013) examined 20 studies with 1677 participants over a mean of 26 weeks. Dairy
was increased by 3.6 servings, and there was a weight increase with low- and high-fat dairy of
0.4–0.8 kg. No other variable changed significantly.
Dairy fat increases both HDL and LDL cholesterol as expected based on their fatty acid com-
position compared with control diets, but no meta-analysis has been performed to look at different
components of dairy or to contrast saturated fat from dairy vs. saturated fat from other food sources
(Huth and Park 2012). Tholstrup et al. (2004) originally showed that cheese elevated LDL cholesterol
less than expected based on its composition and less than butter for the same amount of saturated
fat, and a meta-analysis of 12 studies showed a 0.22 mmol/L difference in LDL cholesterol between
cheese and butter for the same amount of saturated fat (de Goede et al. 2015). Rosqvist et al. (2015)
examined 40 g/day of dairy fat with and without a milk fat globule membrane (whipped cream or
butter) for 8 weeks in a parallel study. LDL cholesterol increased by 0.36 mmol/L with butter and by
0.04 mmol/L with whipped cream (p = 0.024 for difference). Sphingolipids are important components
of the milk globule membrane and have been shown to reduce cholesterol absorption (Conway et al.
2013). Butter milk also lowers blood pressure compared with a macro- and micronutrient-matched
placebo (Conway et al. 2014). Metabolomic studies have been performed on dairy interventions exam-
ining either urine or postprandial plasma and have found distinctive changes, but these have not so far
been related to CVD and dietary risk markers (Piccolo et al. 2015, Zheng et al. 2015)
A meta-analysis of 14 randomized placebo-controlled trials involving 702 participants showed
that probiotic fermented milk produced a significant reduction of 3.10 mmHg in systolic BP and
1.09 mmHg in diastolic BP (Dong et al. 2013). A second meta-analysis of nine studies showed
that probiotic consumption significantly changed systolic BP by −3.56 mmHg and diastolic BP by
−2.38 mmHg compared with control groups. A greater reduction was found with multiple as com-
pared with single species of probiotics, and interventions of <8 weeks did not result in a significant
reduction in systolic or diastolic BP. A daily dose of probiotics >1011 colony-forming units was
required for a significant effect (Khalesi et al. 2014).
Probiotics compared with placebo significantly reduced fasting glucose (−0.31 mmol/L;
p = 0.02) and fasting plasma insulin (−1.29 μU/mL; p = 0.004) in 17 randomized trials with 1105
participants (Ruan et al. 2015).
Acute Feeding Studies

Clemente et al. (2003) examined postprandial lipemia after milk, mozzarella cheese, and butter
and found the same triglyceride peak height, but the peak occurred earlier for milk than cheese or
butter. This may reflect differences between solid and liquid foods rather than anything specific to
the individual whole dairy foods.
Most of the acute studies have examined isolated dairy proteins rather than intact dairy products.
Four liquid test meals containing whey, tuna, turkey, and egg albumin and eaten on four separate
occasions were followed 4 h later by a standard ad libitum test meal in 22 lean healthy men. The
blood glucose response was significantly lower with the whey meal than with the turkey (p < 0.023)
and egg (p < 0.001) meals, but it was similar to the tuna meal. The area under the curve (AUC) for
blood insulin was significantly higher with the whey meal than with the tuna, turkey, and egg meals
(all p < 0.001). The AUC for the rating of hunger was significantly lower with the whey meal than
with the tuna (p < 0.033), turkey (p < 0.001), and egg (p < 0.001) meals. Mean energy intake for an
ad libitum meal consumed 4 h after the test meal was significantly lower (p < 0.001) with the whey
meal than with the tuna, egg, and turkey meals. There was a strong relationship between self-rated
appetite, postprandial insulin response, and energy intake at lunch (Pal and Ellis 2010).
Twenty-five healthy subjects consumed 10% or 25% of meal energy either as casein, soy, or
whey protein. At both levels of the protein, whey triggered the strongest responses in concentrations
of active GLP1 (p < 0.05) and insulin (p < 0.05) compared with casein and/or soy. There were no
differences in energy intake at a test meal 3 h later (Veldhorst et al. 2009).
Cod protein was compared with cottage cheese or soy in 17 healthy women (von Post-Skagegård
et al. 2006). The blood glucose response after the cod protein meal differed from that of the soy pro-
tein meal, with a larger AUC calculated up to 120 min. The serum insulin response after the cottage
cheese differed from that of the cod protein meal with a larger insulin AUC calculated up to 240 min.
The insulin/C-peptide was higher after the cottage cheese meal (suggesting delayed insulin clear-
ance) compared to the cod and soy protein meals at 120 min. The insulin/glucose ratio was lower
after the cod protein meal compared to the cottage cheese and soy protein meals at 120 min. Based
on these observations, it was suggested that cottage cheese may be metabolically harmful.
Protein-rich meals containing 45 g casein, whey, cod, or gluten as part of a 100 g butter meal were
fed to 12 people with type 2 diabetes. The incremental area under the curve (iAUC) for triglyceride
was significantly lower after the whey meal than after the other meals. Free fatty acids were most
pronouncedly suppressed after the whey meal. The glucose response was lower after the whey meal
than after the other meals, whereas no significant differences were found in insulin, glucagon, GLP1,
and glucose-dependent insulinotropic peptide responses (Mortensen et al. 2009).
Eleven obese nondiabetic subjects were fed a fat-rich meal with either whey, casein, or gluten. Whey
protein caused lower postprandial lipemia (p < 0.05) compared to supplementation with cod protein
and gluten. This was primarily due to lower triglyceride concentrations in the chylomicron-rich fraction
(p < 0.05) (Holmer-Jensen et al. 2013). The iAUC for CCL5/RANTES (pro-inflammatory molecules)
was significantly lower after the whey meal compared with the cod and casein meals (p = 0.0053). The
iAUC for monocyte chemoattractant protein 1 was significantly higher after the whey meal compared to
the cod and gluten meals (p = 0.04), which would be interpreted as adverse for atherosclerosis develop-
ment despite the lower postprandial lipemia. The overall effect on the risk of atherosclerosis is not clear.
An acute intervention with 45 g casein, whey, or glucose supplemented to a breakfast test meal had
no differential effects over 6 h on blood pressure, vascular function, or IL-6, TNF-α, and CRP (Pal and
Ellis 2011). However, there was a significant decrease in the triglyceride AUC by 21% and 27% after
consuming the whey meal compared to control and casein meals, respectively (Pal et al. 2010).
Overall, whey appears to stimulate insulin and lower triglyceride and glucose responses compared
with other proteins. The increased insulin may stimulate lipoprotein lipase and enhance triglyceride
clearance. Although this would be perceived as beneficial, it is possible that enhanced stimulation of
insulin may not be metabolically benign in those individuals predisposed to pancreatic failure. The
metabolic benefit of whey has been reviewed by Sousa et al. (2012), while the use of dairy foods
for the management of type 2 diabetes has also been reviewed (Astrup 2014). Whey-containing pre-
meal drinks can dampen meal glycemic responses (Clifton et al. 2014).
POULTRY
Poultry in moderate amounts is a component of the Mediterranean diet, and it and other dietary pat-
terns that replace red meat with white meat are associated with lower rates of CVD and type 2 diabetes
(Esposito et al. 2010), but whether poultry per se has a protective as opposed to a neutral effect is not
clear. The healthy dietary patterns reduced type 2 diabetes by 15%–83% in all 10 prospective cohorts
examined. Poultry is recommended as an important component of a healthy diet (Marangoni et al. 2015).
EGGS
Because dietary cholesterol elevates LDL cholesterol to a small degree (Weggemans et al. 2001),
there has been controversy about the role of whole eggs as a source of dietary protein. Two meta-
analyses have been performed examining the relationship between eggs and CVD. Li et al. (2013a)
examined 14 studies with 320,778 subjects. Comparing the highest to the lowest egg intake groups,
CVD risk was increased significantly in the whole population by 19%. In those with diabetes, the
risk was greater, with a significant 83% increase. For each 4 eggs/week increase in intake, the risk
for CVD increased by 6% and the development of type 2 diabetes increased by 40% in the whole
population, and both were significant. For unknown reasons, eggs appeared to be twice as harmful
in non-U.S. Western countries compared with the United States (Li et al. 2013a). A second meta-
analysis (Shin et al. 2013) examined 22 separate cohorts and did not find any increase in risk for
CVD or its separate components for a comparison of the highest (1 egg/day or more ) to the lowest
(<1 egg/week) intake. However in people with type 2 diabetes, there was a significant increase in
overall CVD risk of 69%. There was also a 42% increase in the risk of developing type 2 diabe-
tes. Despite this and Weggemans meta-analysis from egg intervention studies, the 2015 Dietary
Guidelines Advisory Committee removed dietary cholesterol from its nutrients of concern. Notably,
eggs are rich in choline, a dietary precursor of trimethylamine, which has been associated with CVD
risk (Miller et al. 2014), but the trimethylamine area is controversial.
FISH
Epidemiological Studies of Fish Consumption and CVD and Diabetes
Epidemiology relating fish to CVD has been mixed. The Physicians Health Study found no asso-
ciation of fish to CVD risk (Morris et al. 1995), whereas a meta-analysis of 14 cohort and 5 case–
control studies found that any vs. no fish consumption was associated with 14% less total CHD and
17% less fatal CHD (Whelton et al. 2004). A second meta-analysis (He et al. 2004a) found that
weekly fish intake reduced fatal CHD by 15%, with a 38% reduction when fish was consumed five
or more times per week. Fish type was not assessed. A very small amount (~20 g/day) of fish could
reduce fatal CHD by 7% (He et al. 2004a). Strokes were also reduced by this low level of intake
(He et al. 2004b). This level of fish intake would be unlikely to change levels of N3 fatty acids in
RBC membranes, and indeed lean fish is also protective (Kromhout et al. 1985), so N3 fats may not
be required for protection. In PREDIMED, the Mediterranean diet, which reduced CVD rates by
13% for a two-point change in diet score, is a fish-rich diet (Martinez-Gonzalez and Bes-Rastrollo,
2014). Although measurement of N3 fats in plasma phospholipids is related to total and CHD mor-
tality (27% reduction in the highest quintile) in older adults (Mozaffarian et al. 2013), this does not
necessarily mean that the N3 fats are the cause of the reduction as they may be a marker of a healthy
diet abundant in fish and with little red meat. A higher N3 fat level may also reflect individuals with
superior incorporation of N3 fats into phospholipids as well as a higher intake of fish. A primary
prevention intervention using 1 g/day of fish oil fatty acids in high-risk individuals had no effect on
cardiovascular disease endpoints (Risk and Prevention Study Collaborative Group 2013).
In Japan a high fish intake of 180 g/day (8 times/week) was associated with reduced definite myocar-
dial infarction by 56% compared with a low intake of 23 g/day of fish. The effect was predominantly on
nonfatal events as opposed to the U.S. epidemiology. The effect appeared to be related to N3 fatty acids
rather than fish protein as the highest intake of N3 fats reduced definite MI by 65% (Iso et al. 2006).
A recent meta-analysis examined 11 prospective studies and found that the highest intake of
fish (>4 times/week) was associated with a 21% reduction in acute coronary syndromes. Each
100 g serving of fish per week lowered the risk by 5% (Leung Yinko et al. 2014). Heart failure
was also reduced by fish consumption in five prospective studies with a 14% reduction for
consumption five or more times per week. An increase of 20 g/day of fish reduced the risk by a
further 6% (Li et al. 2013b).
In some studies a separation of the effect of fish per se and fish oil fatty acids could be seen.
For instance, in the Nurses’ Health Study in diabetic women, there was a 74% reduction in CHD
mortality in the highest fish consumption group (5 or more times/week), while higher consumption
of long-chain N3 fatty acids was associated with an insignificant 31% reduction in CHD mortality
(Hu et al. 2003). This suggests that there was a benefit from fish protein (or some other compo-
nents in fish) replacing other forms of protein, in particular red meat, for reducing the risk of CHD
mortality. In addition, in Western populations, red meat intake may also be a significant source of
long-chain N3 fatty acids (Welch et al. 2010), which will confound the relationships. This disparity
between fish and N3 fats was not seen in the whole Nurses’ Health Study cohort (Hu et al. 2002)
for CHD, but total strokes were reduced by 52% in the highest fish intake group and by 33% in the
highest quintile of fish oil intake (Iso et al. 2001).
Diabetes
Fish/seafood or DHA and EPA consumption had no overall effect on the risk of type 2 diabetes in
18 separate cohorts (Wu et al. 2012). However, in Asian cohorts, there was an 11% reduced risk
in the highest intake, while there was a significant 20% increase (p < 0.02 for interaction) risk in
diabetes in Western populations (North America and Europe). Asian subjects with type 2 diabetes
also had significantly lower tissue levels of 22:6 N3 compared with those without diabetes (Zheng
et al. 2012). Another meta-analysis found a significant protective effect of oily fish only, with a
20% reduced risk of type 2 diabetes per 80 g of fish/day. Ethnicity was not assessed in this study
(Zhang et al. 2013). In Japan, fish intake was associated with a significant 27%–32% reduced risk
of type 2 diabetes in men, but there was no significant effect in women. The fat content of fish was
unrelated to risk (Nanri et al. 2011). In the Women’s Health Study, both fish and N3 intake were
associated with increased risk of type 2 diabetes, but adjustment of fish for DHA content removed
the risk, suggesting it was not fish per se but fish oil fatty acids that increased the risk (Djoussé
et al. 2011). This may be due to N3 fats modulating the insulin receptor lipid microdomain.
Dietary Interventions
Most fish interventions have aimed to increase the intake of long-chain N3 fats, and there are few
studies using lean fish. The WISH-CARE study examined 273 people with metabolic syndrome after
8 weeks of intervention with 100 g/day of hake, a lean fish. There was no control nonfish protein used,
but participants had an 8-week fish-and-seafood-free period in a randomized crossover study. The inves-
tigators found a significant effect of the intervention with white fish on reducing waist circumference
(p < 0.001) and diastolic blood pressure (p = 0.014). A significant lowering of serum LDL cholesterol
concentrations (p = 0.048) was also seen. A significant rise in serum EPA and DHA following white
fish consumption suggested that the effects may not be due only to fish protein (Vázquez et al. 2014).
Four weeks of lean fish per week (100–150 g per meal) decreased systolic and diastolic blood
pressure (p < 0.01 and p < 0.02, respectively, group by time interaction) in volunteers with cardiac
disease, with no effects in the fatty fish or lean meat fed control groups (Erkkilä et al. 2008).
Eight weeks of four mixed fish meals per week compared to a control group had no effect
on CRP (n = 80), IL-1β (n = 33), or IL-6 (n = 21) concentrations, blood pressure, or lipids
(Grieger et al. 2014). Daily fish diets in people with type 2 diabetes increased HbA1c by 0.5%
and fasting glucose by 0.57 mmol/L (Dunstan et al. 1997).
The effects of lean white fish on plasma lipoproteins also have been investigated in pre- and
postmenopausal women fed a low-fat, high-polyunsaturated/saturated-fat ratio diet (Jacques et al.
1992). In postmenopausal women, lean white fish compared with other animal protein products
induced higher concentrations of plasma cholesterol, LDL-apoB and HDL cholesterol, mainly in the
HDL3 fraction. In premenopausal women, lean white fish induced lower concentrations of VLDL
triglycerides and higher concentrations of LDL-apoB in plasma (Gascon et al. 1996).
SOY INTAKE, HEART DISEASE, AND TYPE 2 DIABETES

The Singapore Chinese Health Study was a population-based study that recruited 63,257 Chinese
adults aged 45–74 years old from 1993 to 1998. The median intake was 5.2 g/day for soy protein,
15.8 mg/day for soy isoflavones, and 87.4 g/day for soy expressed as tofu equivalents. Cardiovascular
deaths (n = 4780) occurred until 2011. After adjustment for sociodemographic, lifestyle, and other
dietary factors, soy protein intake was not significantly associated with cardiovascular disease.
Similarly, no significant association was observed for soy isoflavones and total tofu equivalents
when deaths from CHD (n = 2697) and stroke (n = 1298) were considered separately (Talaei et al.
2014). In the same cohort (Mueller et al. 2012), the highest quintile of unsweetened soy intake was
associated with a 28% reduced risk of type 2 diabetes (p trend = 0.015), while sweetened soybean
was associated with 13% more diabetes in the highest quintile of intake (p trend = 0.013).
In a case–control study in women from Fukuoka City, Japan, with 660 cases and 1277 controls,
tofu consumption was inversely related to the risk of acute myocardial infarction; relative risks for
eating tofu <2, 2–3, and 4 or more times per week were 1.0, 0.8, and 0.5, respectively, after adjust-
ment for nondietary factors (p trend = 0.01). Further adjustment for the consumption of fruit and fish
did not alter the findings (Sasazuki et al. 2001). In the Japan Public Health Center–based cohort of
40,462 men and women, consuming soy more than five times per week was associated with a 69%
reduction in CVD mortality. Similar relationships were seen with isoflavone intake. The observation
was mostly in postmenopausal women and was not seen in men (Kokubo et al. 2007).
Dietary Patterns Containing Soy

A dietary pattern rich in soy, fruits, and vegetables (Odegaard et al. 2014) was associated with a
25% reduction in all-cause mortality in the highest quintile (p trend < 0.0001), while a diet rich
in dim sum and meat was associated with a 27% increase in CVD mortality in the highest quintile
(p trend = 0.001). In never smokers only, type 2 diabetes was reduced by 25% (p = 0.0005) with the
healthy diet and increased by 47% (p < 0.0001) with the meat diet (Odegaard et al. 2011).
In the INTERHEART China case–control study (Guo et al. 2013), the fruit and vegetables and tofu
groups had a 30% reduction in risk of type 2 diabetes in the highest vs. lowest intake quartile (p = 0.0001).
Isoflavonoid Excretion
In a nested case–control study (377 cases and 753 controls) with the Shanghai cohort, urinary equol
excretion (but not total isoflavonoids) showed a significant inverse association with CHD in women
with a 54% reduction in the highest vs. lowest quartile (Hall et al. 2005).
Interventions with Soy on Cardiovascular and Diabetes Risk Markers

LDL Cholesterol
Soy protein has been shown in many studies to lower LDL cholesterol, and there have been
several meta-analyses confirming these findings. The most recent one was performed by
Anderson and Bush (2011), and 20 parallel studies and 23 crossover studies were examined.
LDL cholesterol was lowered by 5.5% in parallel studies and 4.2% in crossover studies with a
median dose of 30 g/day of soy protein in comparison with non-soy protein. Only in parallel
studies was HDL cholesterol elevated by 3.2% and triglyceride lowered by 10.2%. Studies of
soy protein with intact isoflavones (n = 23) showed very similar effects to the earlier meta-
analysis (Zhan and Ho 2005) with a dose–response relationship found between isoflavone
daily intake and LDL cholesterol lowering, but this was not adjusted for soy protein intake.
Soy may be effective because it is displacing other protein sources that may be associated with
LDL-elevating components such as saturated fat.
A meta-analysis showed no overall effect of isoflavone extracts on lipids (Taku et al. 2008), and
a comparison of soy enriched or depleted in isoflavones (11 studies) showed no reduction in total
cholesterol but isoflavone enrichment resulting in a small significant reduction in LDL cholesterol of
3.5% (Taku et al. 2007). Thus, it would appear that isolated soy protein lowers LDL cholesterol with
some additional benefit from intrinsic isoflavones. Isolated isoflavones appear to have no effect, and
there is no dose–response relationship between the amount of isoflavones and LDL cholesterol low-
ering (Weggemans and Trautwein 2003). A Cochrane meta-analysis found no effect of isoflavones
on plasma lipids (Qin et al. 2013).
Nontraditional Risk Factors

There have been few studies on nontraditional risk markers, but 40 g of soy protein (89.3 mg of
isoflavones) for 8 weeks in comparison with 40 g milk protein supplement or 40 g complex car-
bohydrate in a randomized crossover study lowered E-selectin (p = 0.014) and leptin (p = 0.011).
However, given that 20 comparisons were made, these changes could well have occurred by chance
(Rebholz et al. 2013). No effect was found on adhesion molecules in postmenopausal women (Blum
et al. 2003), on adhesion molecules or other inflammatory markers in 117 postmenopausal women
(Hall et al. 2005), or on CRP in a meta-analysis of 17 studies, but there was some evidence of benefit
of soy in women with a high baseline CRP (Dong et al. 2011). In the Hall study (Hall et al. 2005),
there was no effect of soy on lipids, glucose, and insulin.
Thirty men were fed 25 g/day of protein from soy with or without isoflavones, or milk. A high-
fat test meal resulted in an increase in postprandial triglycerides after the soy alone diet and not the
other two diets (Santo et al. 2010). Feeding natto (viscous fermented soybeans) and viscous vegeta-
bles as a breakfast with rice for 2 weeks compared with nonviscous soybeans, potatoes, and broccoli
and rice breakfast improved glucose and insulin responses to the breakfast, while the comparator
showed no improvement with time. Furthermore, the test meal of natto and viscous vegetables also
lowered glucose and insulin response compared with the control meal prior to the 2-week feeding
period (Taniguchi-Fukatsu et al. 2012).
Acute Studies with Soy

These have been mostly confined to muscle protein synthesis studies that find that soy is inferior
to whey but is better than (Tang et al. 2009), equivalent (Luiking et al. 2011), or worse than casein
(Luiking et al. 2005) at acutely stimulating protein synthesis. Soy is similar to whey and gluten at
reducing appetite over 3 h and reducing food intake at a buffet meal by 10% and stimulating GLP1
and CCK (Bowen et al. 2006).
CONCLUSION
Meat is associated with worse outcomes, while dairy is probably neutral, although yogurt may
protect against type 2 diabetes. Fish is mostly associated with protection from CVD but the exact
mechanism is not clear. Poultry is neutral and soy may be beneficial. Most data are based on epi-
demiology, while the few interventions that have been performed have failed to show clear cardio-
metabolic benefit.
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Section V
Dietary Food Groups, Patterns,
and Cardiometabolic Health
20 Consumption of Foods,
Food Groups, and
Cardiometabolic Risk
Edward Yu and Frank B. Hu
CONTENTS
Introduction.....................................................................................................................................373
Beverages........................................................................................................................................374
Sugar-Sweetened Beverages......................................................................................................374
Coffee��377
Alcohol.......................................................................................................................................378
Foods.............................................................................................................................................. 379
Fruits and Vegetables..................................................................................................................379
Dairy (Cheese, Milk, Yogurt).....................................................................................................381
Eggs��382
Meats��383
Fish��384
Grains and Fiber.........................................................................................................................385
Nuts and Legumes......................................................................................................................386
Conclusion......................................................................................................................................387
References.......................................................................................................................................388
ABSTRACT
Knowledge about the effect of regular intake of specific food groups on cardiometabolic health is
primarily derived from large prospective cohort studies. With careful study design and analysis, results
from observational data offer opportunities for causal inference. Sugar-sweetened beverage intake
is consistently associated with increased risk of obesity, cardiovascular disease (CVD), and type 2 dia-
betes owing to the excess amounts of added sugar, as well as mediation by increased weight gain.
Coffee consumption is associated with lower incidence of CVD, with the largest benefit observed
around 3–5 cups/day. Higher consumption of fruits and vegetables is associated with lower risk of heart
disease, although data on type 2 diabetes is conflicted. Dairy consumption has mixed associations with
cardiometabolic diseases, depending on the types of dairy products. Red meat, especially processed red
meat, is associated with increased risk of diabetes and CVD. Fish intake is inversely associated with
CVD incidence, although high-dose fish oil supplementation does not appear to be beneficial. Regular
consumption of whole grains and nuts is associated with lower risk of both diabetes and CVD. Future
research should focus on studying food subtypes, as well as adopting a holistic view of nutrition.
INTRODUCTION
For decades, scientists have recommended the intake of specific foods in order to achieve a balanced
diet. As a result, food groups have become a popular area of study in nutritional science, as they
allow for easier understanding, reporting, and public health recommendations.
373
At present, the evidence for the effects of intake of specific foods is dominated by epidemiologic
studies. Although randomized clinical trials are regarded as the gold standard for causal inference,
such studies are especially difficult to carry out for foods, owing to the high attrition rate, lack of
compliance, short follow-up time, and small sample sizes. Narrowness of exposure is also a prob-
lem; an investigator of a randomized trial must specify a quantity of food or drink to administer
before the study commences, whereas epidemiologic studies may draw from a range of intakes pro-
vided by the participants. Finally, dietary trials are difficult to blind, and so the interventional nature
of trials may affect participant expectations and results.
On the contrary, epidemiologic studies collect information from free-living participants and
consist primarily of case–control and cohort studies. Case–control studies are most often con-
ducted retrospectively, when the investigation begins after the outcome has occurred. As a result,
they are prone to both recall bias and reverse causation, which prevents strong arguments for causa-
tion. Consequently, large prospective cohort studies provide the best current knowledge for the car-
diometabolic effects of food groups. Cohorts such as the European Prospective Investigation into
Cancer and Nutrition (EPIC), the Health Professionals’ Follow-Up Study (HPFS), and the Nurses’
Health Study (NHS) consist of tens of thousands of participants that periodically report their eat-
ing habits and incidence of disease. The prospective nature of cohort studies obviates the problems
of reverse causation and recall bias, although residual confounding remains a major obstacle to
causal inference. However, with prudent study design and statistical analysis, such studies can
provide a robust case for causation. Major well-known covariates such as smoking, body mass
index, and common demographic variables will almost always be accounted for in the analyses of
published studies. Furthermore, once data regarding food groups from several populations have
been released, a meta-analysis can be performed to quantitatively combine results in order to report
a pooled effect of exposure. Meta-analyses provide much more statistical power than a single study
alone, and sources of heterogeneity can also be explored. Though they are still imperfect, such
results have provided the basis for many public health recommendations and policies.
Assessment of food intake has long been a source of both criticism and misunderstanding. While
diet records provide the reference method for quantifying food and drink, the expense and burden
of this technique has kept its widespread use in all but small randomized trials and validation stud-
ies (Bingham et al. 1994, Newby et al. 2003). Random dietary recalls, where a trained investigator
interviews participants on their eating habits, have also been shown to be highly reliable and accurate
(Briefel 1994, Hu 2008, Wright et al. 2007). For large prospective cohort studies, semiquantitative
food frequency questionnaires (FFQs) remain the method of choice for dietary assessment. Different
FFQs, such as the Block or Willett FFQ, have been validated against reference methods for both accu-
racy and reproducibility (Kroke et al. 1999, Subar et al. 2001). Although precise measurement of food
quantity is difficult if not impossible with FFQs, investigators can gain great insight by examining the
relative ranking of food intakes among participants.
The present chapter summarizes the current knowledge of nine major groups of food and drink
that have been linked to cardiometabolic risk (Tables 20.1 and 20.2). Evidence originates primar-
ily from the systematic review and meta-analyses of large prospective cohort studies. Potential
mechanisms based on results from animal studies, as well as small randomized trials, will also
be discussed.
BEVERAGES
Sugar-Sweetened Beverages
Sugar-sweetened beverages (SSBs) are a class of drinks that include soft drinks, sports drinks, energy
drinks, and fruit juices with added sugar. This class of beverage represents the primary source of
added sugar in the U.S. diet and is composed of sweeteners such as sucrose, high-fructose corn
syrup, and fruit juice concentrate (100% fruit juice with no added sugar is not considered a SSB)
Consumption of Foods, Food Groups, and Cardiometabolic Risk 375
TABLE 20.1
Summary of Food Group Constituents and Nutrients
Food/Beverage Constituents of Interest Major Nutrients/Compounds
Sugar-sweetened Soft drinks, sports drinks, energy drinks, fruit juice Added sugar
beverages
Coffee Caffeinated and decaffeinated, filtered and unfiltered Caffeine, chlorogenic acid, lignans, flavonoids
Alcohol Beer, wine, spirits Ethanol, resveratrol (red wine)
Fruits Tree fruits, berries, melons, citrus Phytochemicals, flavonoids, folate, fiber
Vegetables Green leafy, cruciferous, root
Dairy Milk, cheese, yogurt Saturated fat, ruminant fat, protein, lactose,
calcium, probiotics
Eggs Cholesterol, protein, sodium
Red meats Beef, pork, lamb, game meats Sodium, preservatives, L-carnitine, protein,
fatty acids
White meats Chicken, turkey
Fish Oily (sardines, herring, anchovies, salmon, trout, tuna), Polyunsaturated fatty acids, protein
whitefish (cod, kutum, whiting, haddock, hake, pollock)
Grains Rice, wheat, bread, corn Starch, fiber
Nuts and legumes Tree nuts, peanuts, peas, beans Unsaturated fats, plant protein, minerals,
vitamins, polyphenols
(Malik et al. 2010a). Data from the National Health and Nutrition Examination Survey (NHANES)
from 2005 to 2008 have shown that roughly half the U.S. population consumes SSBs on any given
day, that at least a quarter of the population consumes at least 200 calories from SSBs, and that 5%
consume at least 567 calories (Ogden et al. 2011). Although consumption has seen a modest decline
over the past decade, soft drink sales continue to rise outside the United States (Kleiman et al. 2012,
Welsh et al. 2011), and such a trend poses a major public health challenge.
As also discussed in Chapters 13 and 14 of this text, ample observational evidence has strongly
suggested a detrimental effect of SSB consumption in relation to obesity, type 2 diabetes mel-
litus (T2DM), and CVD. Most (Hu and Malik 2010, Malik et al. 2006, 2009, 2010a, Vartanian
et al. 2007), but not all (Forshee et al. 2008), systematic reviews of epidemiologic studies have
concluded that there is a positive relationship between SSB intake and risk of overweight/obesity
in both children and adults. This association is strongest among large prospective cohort studies
with long duration of follow-up and without adjustment for total energy intake (Malik et al. 2009).
Findings from experimental studies also support this hypothesis. A meta-analysis of six random-
ized trials performed by Mattes et al. (2011) demonstrates that the addition of SSBs to the diets of
participants significantly increased body weight, although in the same publication, no weight loss
benefit was seen in a meta-analysis of six different trials aimed at reducing SSB intake. However,
Mattes et al. (2011) note methodological limitations of these trials, including short duration, small
sample sizes, poor compliance, and lack of blinding. Genetic factors may also affect the strength
of association between SSB intake and weight gain. Based on a genetic predisposition score of
32 obesity genes, those who consumed >1 serving/day of SSBs showed more than twice the
genetic effect on obesity risk compared to those who consumed <1 serving/month, suggesting
that the benefit of healthy beverage choice is greater among individuals genetically predisposed
to weight gain (Qi et al. 2012).
Systematic reviews and meta-analyses of prospective cohort studies have also clearly linked SSB
consumption with risk of type 2 diabetes and metabolic syndrome (Hu and Malik 2010, Malik et al.
2010a,b, Vartanian et al. 2007). Among 310,819 individuals, those in the highest category of SSB
intake (most often 1–2 servings/day) had a 26% greater incidence of T2DM compared to those in
TABLE 20.2
Summary of Relative Risks of Food Groups in Relation to CVD, T2DM, and
Strength of Evidence
Pooled Risk Ratio Level of Pooled Risk Ratio for Level of
Food or Beverage for CVD Overall Evidence T2DM Overall Evidence
SSBs 1.16 (1.10–1.23)a + Strong 1.26 (1.12–1.41)b + Strong
(Huang et al. 2014) (Malik et al. 2010a)
Coffee 0.85 (0.80–0.90)c U Strong 0.91 (0.89–0.94)a − Moderate
(Ding et al. 2014b) (Ding et al. 2014a)
Alcohol 0.80 (0.78–0.83)b U Strong 0.87 (0.76–1.00)d U Weak
(Corrao et al. 2000) (Baliunas et al. 2009)
Fruits and Vegetables 0.96 (0.92–0.99)a − Strong 0.90 (0.80–1.01)c 0 Moderate
(Wang et al. 2014) (Cooper et al. 2012)
Dairy 0.92 (0.80–0.99)c − Moderate 0.99 (0.98, 1.01)a − Moderate
(Elwood et al. 2010) (Chen et al. 2014)
Eggs 0.96 (0.88–1.05)c 0 Moderate 1.06 (0.86–1.30)c + Weak
(Shin et al. 2013) (Djoussé et al. 2016)
Red Meats 1.09 (1.01–1.18)c + Weak 1.19 (1.04–1.37)a + Moderate
(Chen et al. 2013) (Pan et al. 2011)
Processed Meats 1.42 (1.07–1.89)a + Strong 1.51 (1.25–1.83)a + Strong
(Micha et al. 2010) (Pan et al. 2011)
White Meats 1.00 (0.87–1.15)a 0 Weak 1.04 (0.82–1.32)a 0 Weak
(Abete et al. 2014) (Feskens et al. 2013)
Fish 0.62 (0.46–0.82)c − Strong 0.99 (0.85–1.16)c 0 Moderate
(He et al. 2004) (Xun and He 2012)
Whole Grains 0.79 (0.74–0.85)c − Strong 0.74 (0.69–0.80)b − Strong
(Ye et al. 2012) (Ye et al. 2012)
Refined Grains 1.07 (0.94–1.22)c 0 Moderate 0.95 (0.88–1.04)b 0 Moderate
(Mellen et al. 2008) (Aune et al. 2013b)
Nuts/Legumes 0.71 (0.59–0.85)a − Strong 0.72 (0.64–0.81)a − Strong
(Luo et al. 2014) (Luo et al. 2014)
a For 1 serving/day increase.

b For 3–5 serving/day increase.
c Comparing those in highest to lowest categories of intake.
d U-shaped relationship where relative risk is at optimal intake.
the lowest category of intake (none or <1 serving/month) (95% CI = 1.12–1.41). For metabolic
syndrome, the relative risk was 1.20 (95% CI = 1.02–1.42) (Malik et al. 2010b). SSB intake has also
been shown to have a detrimental effect on cardiovascular health, leading to an increased risk of
total cardiovascular disease, hypertension, and coronary heart disease (CHD), but not stroke (Huang
et al. 2014, Xi et al. 2015). In particular, a pooled analysis of 173,753 participants indicated that a
one serving per day increase in SSB consumption was associated with a 16% increased risk in CHD
(95% CI = 1.10–1.23) and that the effect was especially prominent among men and for individuals
living in the United States (Huang et al. 2014).
It is thought that SSBs lead to weight gain through their high levels of added sugar, which pro-
vide ample energy but low satiety due to the liquid calorie content, leading to additional food intake
and net positive energy balance (Malik et al. 2006, 2010a). Widespread consumption of SSBs con-
tributes to high glycemic load, which has been shown to lead to increased concentrations of inflam-
matory biomarkers, such as C-reactive protein (Liu et al. 2002), and insulin resistance, especially
among overweight individuals (Schulze et al. 2004). The effect of SSB intake on type 2 diabetes and
metabolic syndrome is thought to act in part through weight gain. However, the added sugar in SSBs
may also have an independent effect in the development of these diseases. Schulze et al. (2004) esti-
mate that roughly half the effect of SSBs on type diabetes was through obesity. SSBs have also been
shown to dramatically increase postprandial blood glucose and insulin concentrations (Janssens et al.
1999). Recent experimental studies have indicated an important role of fructose, a constituent of both
sucrose and high-fructose corn syrup, in inducing adverse metabolic effects. Fructose has been shown
to increase hepatic lipogenesis, lower high density lipoprotein (HDL) cholesterol, and promote insu-
lin resistance (Bray 2007). Compared to glucose-containing beverages, fructose-containing beverages
may promote additional visceral adiposity (Stanhope et al. 2009).
Although the case against SSB consumption is robust, observational data continue to be criticized
on the grounds that they cannot infer causality. Both Hu (2013) and Huffman (2012) have appealed to
the Hill’s criteria, a set of conditions to establish causality, to argue for the true effect of SSBs consump-
tion on negative cardiometabolic outcomes. While the health consequences of SSBs have been well
established, additional research into appropriate and cost–effect measures to curb SSB use is needed.
Coffee
Coffee is one of the most widely consumed beverages in the world. Historically, the link
between coffee and disease had remained unclear due to strong confounding by smoking, but
research on coffee consumption has seen a recent flurry of interest following the initial publication
of a strong decrease in risk of type 2 diabetes with increasing coffee consumption among 17,111
Dutch adults (van Dam and Feskens 2002). In this study, those who drank at least seven cups of
coffee a day were half as likely to develop type 2 diabetes compared to those who drank two cups
or fewer a day. This finding was subsequently confirmed in several other large cohorts, including
the NHS/HPFS (Salazar-Martinez et al. 2004), NHANES (Greenberg et al. 2005), and the Women’s
Health Study (Song et al. 2005). A meta-analysis of prospective cohort studies including 50,595
cases of T2DM among 1,096,647 participants reported a relative risk of 0.71 (95% CI = 0.67–0.76)
for the highest level of coffee intake compared to lowest (Jiang et al. 2014).
Interestingly, caffeine content did not appear to alter the magnitude of the results, with decaf-
feinated coffee having a similar relative risk of 0.79 (95% CI = 0.69–0.91), as well as total caffeine
content alone with a relative risk of 0.70 (95% CI = 0.65–0.75) (Jiang et al. 2014). In a dose–
response analysis, Ding et al. (2014a) reported that a 1 cup/day increase was associated with a 0.91
(95% CI = 0.89–0.94) reduction in T2DM risk for regular coffee and 0.94 (95% CI = 0.91–0.98)
for decaffeinated coffee. This trend appeared to be linear, with more coffee leading to a monotoni-
cally increasing benefit.
Pooled analyses by Sofi et al. (2006) and Malerba et al. (2013) show that risk of both CHD and
CVD mortality are reduced when comparing heavy to light coffee drinkers. Two additional meta-
analyses that are modeling nonlinear associations point to drinking 3–5 cups/day to have the optimal
reduction in risk of approximately 15% (95% CI = 0.80–0.90), and heavy coffee intake having a null
effect (Ding et al. 2014b, Mostofsky et al. 2012). A meta-analysis performed by Mesas et al. (2011)
concluded that although administration of caffeine produces an acute increase in blood pressure,
there was no evidence of an increased risk of CVD among hypertensive subjects.
The primary component responsible for the cardiometabolic benefit of coffee remains unclear, as
brewed coffee contains over a thousand identified chemicals. Caffeine, once thought to be detrimen-
tal to insulin sensitivity and glucose tolerance (Cheraskin et al. 1967, Keijzers et al. 2002, Pizziol
et al. 1998), appears to have little effect, since both caffeinated and decaffeinated coffee are indepen-
dently associated with benefit. One hypothesis is that long-term intake of coffee causes a tolerance to
develop against caffeine (van Dam et al. 2004). Another hypothesis is that other components found in
coffee antagonize the adverse effects of caffeine (Natella and Scaccini 2012).
Several clinical studies have suggested important roles for polyphenols and antioxidants,
similar to those found in fruits and vegetables. Findings from randomized trials indicate that
improved adipocyte and hepatocyte function induced by changes in adiponectin and fetuin-A
concentrations may play a role in the benefit of coffee (Wedick et al. 2011). Coffee also appears
to improve markers of inflammation and endothelial dysfunction, such as TNF-α, E-selectin,
C-reactive protein, and VCAM-1 (Kempf et al. 2010, Natella and Scaccini 2012). The pres-
ence of phytochemicals in coffee such as flavonoids, lignans, and chlorogenic acid has also
been observed to independently increase glucose tolerance and insulin sensitivity (Natella and
Scaccini 2012, van Dijk et al. 2009). Chlorogenic acid in particular has recently caught the atten-
tion of researchers, as it appears to reduce fasting glucose (Rodriguez de Sotillo et al. 2006),
increase insulin sensitivity (Shearer et al. 2003), and attenuate the appearance of glucose in
blood after challenging with glucose (Bassoli et al. 2008).
Method of preparation is an area deserving of future research. Although Ranheim and Halvorsen
(2005) speculate that boiled coffee is particularly beneficial due to its antioxidant content, van Dam
et al. (2006) reported that there was no difference in the benefit of filtered coffee (RR = 0.86, 95%
CI = 0.82–0.90) or instant coffee (RR = 0.83, 95% CI = 0.74–0.93) on T2DM.
Alcohol
Alcohol intake has been studied extensively in the past several decades. Among food groups exam-
ined in epidemiologic studies of diet, modest alcohol has presented the most consistent association
with lower cardiometabolic risk (Rimm et al. 1996). This is also discussed in Chapter 31 of this text.
Alcohol can be classified into beers, wines, and distilled beverages (spirits), although there appears
to be no difference in the type of drink on health benefit (Rimm et al. 1996), offering enticing evi-
dence that the causal component of risk reduction is due to the ethanol itself.
Pooled analyses of alcohol intake have also repeatedly shown a U-shaped relationship with both
CVD risk (Holmes et al. 2014, Ronksley et al. 2011) and T2DM risk (Baliunas et al. 2009, Carlsson
et al. 2005, Schrieks et al. 2015). Reviewing 84 eligible studies, Ronksley et al. (2011) reported that
the relative risks for drinkers vs. nondrinkers were 0.75 (95% CI = 0.70–0.80) for total CVD mor-
tality, 0.71 (95% CI = 0.66–0.77) for incident CHD, 0.75 (95% CI = 0.68–0.81) for CHD-specific
mortality, 0.98 (95% CI = 0.91–1.06) for incident stroke, and 1.06 (95% CI = 0.91–1.23) for stroke-
specific mortality. The strongest reduction in CVD risk was seen at one to two drinks per day. Corrao
et al. (2000) report similar results, with optimal intake of one to two drinks per day but a deleterious
effect above six drinks per day. Evidence from experimental studies is also encouraging, as a meta-
analysis of 42 trials demonstrated that moderate alcohol intake was associated with increased HDL,
apolipoprotein A1, and lower fibrinogen (Rimm et al. 1999).
For T2DM, a recent meta-analysis found a similar U-shaped relationship (Baliunas et al. 2009).
Incidence of T2DM was lowest among men drinking 22 g alcohol/day (about 2 drinks) with a
relative risk of 0.87 (95% CI = 0.76–1.00), and increased significantly compared to nondrinkers at
over 60 g/day (about 5 drinks). The effects among women are even stronger, with consumption of
24 g/day alcohol having a relative risk of 0.60 (95% CI = 0.52–0.69), and becoming deleterious
at 50 g/day (about 4 drinks). A meta-analysis of 14 clinical trials indicated that moderate alcohol
consumption reduced fasting insulin and improved insulin sensitivity among women but not men
(Schrieks et al. 2015).
Alcohol has been shown to increase circulating HDL cholesterol, which is thought to be the
most important mechanism for its cardioprotective effect (Gaziano et al. 1993). It is estimated that
approximately two drinks per day increases HDL levels by 4.0 mg/dL (Rimm et al. 1999), which is
greater than that produced by gemfibrozil, a drug used to raise HDL levels (Stampfer et al. 1991).
However, a risk reduction is observed for CHD even when controlling for HDL cholesterol, impli-
cating additional pathways (Criqui et al. 1987). Moreover, as discussed elsewhere in this volume
(Chapter 31), there is little evidence to date that an increase in HDL cholesterol causes a reduction
in CVD risk. Ethanol also modulates several biochemical pathways, including platelet aggregation
and clotting, omega-3 fatty acid processing, and vascular integrity (Di Castelnuovo et al. 2009).
Moderate alcohol intake may also affect inflammation, a process associated with atherosclerosis,
and lower plasma concentration of C-reactive protein, a molecule correlated with CVD risk (Albert
et al. 2003). On the other hand, alcohol consumption appears to increase levels of triglyceride,
which is associated with an increased incidence of CVD (Stampfer et al. 1996). However, the bal-
ance appears to be in favor of reduced risk.
While epidemiologic studies have indicated the benefits of moderate alcohol intake on health
outcomes, public health experts have not endorsed alcohol consumption as a prophylactic for
cardiometabolic disease, owing to the dangers of overconsumption. At higher intake, risk of death
attributed to CVD has been found to be comparable or worse in relation to nondrinkers, which
may due to direct myocardial toxicity or tendency of alcohol to induce arrhythmias (Moore and
Pearson 1986). Researchers have pointed out the strong potential for confounding in observa-
tional studies of alcohol (Mukamal and Rimm 2001). Abstainers may not drink due to illness
or have quit due to former alcohol abuse. Moderate drinkers also tend be younger, leaner, more
physically active, and of higher socioeconomic status and are likelier to be married compared
to nondrinkers (Mukamal and Rimm 2001). Furthermore, although randomized trials of alcohol
address the question of causality, they tended to be of small sample size, short duration, and mea-
sure only intermediate outcomes (Rimm et al. 1999, Schrieks et al. 2015).
A novel way of addressing the causal role of alcohol has originated from genetic studies. Because
polymorphisms in alcohol dehydrogenase and aldehyde dehydrogenase are associated with aversion
to alcohol, a genetic association study of single nucleotide polymorphisms in these genes may act
as a Mendelian randomized trial of alcohol consumption. Studies of alcohol dehydrogenase (Hines
et al. 2001) and cholesteryl ester transfer protein (Jensen et al. 2008) indicate a benefit of moderate
alcohol consumption that was modified by genotype. However, in a large pooled analysis of 261,991
individuals, Holmes et al. (2014) reported that carriers of a genetic variant in alcohol dehydrogenase
1B, which is associated with nondrinking, presented lower systolic blood pressure, interleukin-6 lev-
els, waist circumference, body mass index, odds of CHD, and odds of ischemic stroke. The authors
concluded that alcohol consumption increases cardiovascular risk among all drinkers, including
those who drink moderately, contrasting with the body of epidemiologic evidence. However, the use
of genetic variants as instrumental variables in dietary association analyses remains controversial,
and thus additional studies are needed.
FOODS
Fruits and Vegetables
Fruits are the seed-bearing structures that develop from ovaries of flowering plants, whereas
vegetables are all the other plant parts, such as leaves, roots, and stems. However, both fruits and
vegetables are similar in that they are rich in folate, fiber, vitamins, minerals, and phytochemicals,
including polyphenols (Dauchet et al. 2006). They have been historically recommended as part of
a healthy diet since the earliest iteration of the USDA guidelines in 1980 and for good reason—
evidence for fruits and vegetables overwhelmingly support their positive effects on cardiometabolic
and overall health.
Among the earliest systematic reviews, examining fruits and vegetables was conducted by Ness
and Powles (1997), who found a strong protective effect against stroke and weaker benefit for CHD.
The earliest quantitative meta-analysis of fruits and vegetables for CHD risk was performed by
Dauchet et al. (2006), who reported a relative risk of 0.96 (95% CI = 0.93–0.99) for each additional
serving of fruits and vegetables. In the same year, He et al. (2006) reported a reduction of 26% in
stroke incidence for those consuming 3–5 servings/d of fruits and vegetables. A later analysis per-
formed by He et al. (2007) reached a similar conclusion for CHD: compared with those who had less
than 3 servings/d of fruits and vegetables, those who consumed more than 5 servings/d had a relative
risk of 0.83 (95% CI = 0.77–0.89) for CHD. The most recent meta-analysis of fruits and vegetables
conducted by Wang et al. (2014) consisted of 833,234 subjects—more than triple to that of the
analysis conducted by He et al. (2007). It found that a 1 serving/day increase in fruits or vegetables
was associated with a risk ratio of 0.96 (95% CI = 0.92–0.99) for CVD mortality.
The findings for fruit and vegetable consumption on the risk of T2DM are less clear. A meta-
analysis of four cohort studies consisting of 223,512 subjects conducted by Carter et al. (2010)
reported that there was no association between vegetable, fruit, or total fruit and vegetables and
T2DM incidence, although green leafy vegetables specifically were associated with a relative risk
of 0.86 (95% CI = 0.77–0.97). Cooper et al. (2012) reported no association between total fruits
and vegetables and T2DM, although root and green leafy vegetables specifically may reduce
T2DM incidence. An updated meta-analysis of 10 cohort studies consisting of 434,342 subjects
performed by Li et al. (2014) concluded that a 1 serving/day increase in total fruit intake was asso-
ciated with a 7% decrease in T2DM risk (RR = 0.93, 95% CI = 0.88–0.99). No association was
found for total vegetable intake (RR = 0.90, 95% CI = 0.80–1.01). Again, similar to the previous
study by Carter et al. (2010) and Cooper et al. (2012), green leafy vegetables were found to be
protective against T2DM, with a 0.2 serving/day increase conferring a relative risk of 0.87 (95%
CI = 0.81–0.93).
A major component of the cardioprotective effect of fruits and vegetables is thought to arise
from plant-derived phytochemicals, such as sulfides, carotenoids, flavonoids, phenols, lignans, and
resveratrol (Van Duyn and Pivonka 2000). These biomolecules are not found in other nonplant
foods and have been shown to modulate the detoxification of enzymes, stimulate the immune
system, reduce inflammation, regulate cholesterol synthesis and hormone metabolism, and also
demonstrate antioxidant, antibacterial, and antiviral effects (Lampe 1999). Such effects have been
reported not only in animal and cell-culture models but also in numerous clinical trials (Dillard and
Bruce German 2000).
One developing frontier of research has been into flavonoids, a class of polyphenols found
exclusively in plant and plant products such as tea and wine. Interest in flavonoids originated from
examination of the French paradox, the observation that Mediterranean populations consuming
diets high in red wine and saturated fat (SFA) present low CVD mortality rate (Nijveldt et al.
2001). As isolated molecules, flavonoids have been easier to study in a clinical context than whole
foods and have been shown to protect cells against oxidative damage, interfere with nitric-oxide
synthase activity, scavenge free radicals, mobilize leukocytes, and interact with other enzymes
to produce beneficial effects (Nijveldt et al. 2001). Among observational data, regular flavonoid
intake has been shown to be associated with reduced blood pressure (Cassidy et al. 2011) and
T2DM incidence (Liu et al. 2014). In a meta-analysis of 133 randomized trials of flavonoids
among 6557 participants, Hooper et al. (2008) concluded that soy protein isolate, cocoa, and
green tea confer the strongest effects of cardiovascular risk factors via antihypertensive and LDL-
lowering actions. Additional research is required to elucidate the different mechanisms through
which flavonoid subclasses operate.
There is also a growing need to study the effects of specific fruit and vegetable subtypes because
the composition of micronutrients and phytochemicals differ between food classes. For example,
the specific benefit of green leafy vegetables on T2DM may be attributed to its high magnesium
and α-linolenic acid content compared to other types of vegetables (Carter et al. 2010). Regarding
flavonoids, citrus fruits contain virtually all the flavanones consumed in the American diet, whereas
berries are the richest sources of anthocyanins (Cassidy et al. 2011). Furthermore, different nutri-
tional constituents, such as flavonoid subclasses, may have different physiological effects (Hooper
et al. 2008). Muraki et al. (2013) recently published results from the NHS and HPFS and found sig-
nificant heterogeneity between individual fruits. For instance, 3 servings/week of blueberries were
strongly protective (RR = 0.74, 95% CI = 0.66–0.83) while 3 servings/week of cantaloupe appeared
to be detrimental (RR = 1.10, 95% CI = 1.02–1.18). These results signify that future studies should
not classify all fruits and vegetables into one exposure category but should differentiate between
individual foods wherever possible.
Dairy (Cheese, Milk, Yogurt)

Dairy consists of milk or products derived from milk, such as cheese, butter, and yogurt. They
are a major source of SFAs in the United States, accounting for about 21% of total SFA con-
sumption (U.S. Department of Agriculture 2015). As a result, research into dairy has been
closely linked to the controversy regarding SFA. Current recommendations from the World
Health Organization and from federal dietary guidelines in the United States suggest consum-
ing less than 10% of total energy from SFA in order to decrease the risk of CVD, with the
American Heart Association setting a target at less than 7% of total energy. SFAs have been
known to increase circulatory low-density lipoprotein (LDL) cholesterol (Mensink et al. 2003),
a major risk factor of CVD risk. Furthermore, it is known that following a diet high in poly-
unsaturated fat compared to SFA leads to a lower risk of total CHD events (Mozaffarian et al.
2010, Skeaff and Miller 2009).
Despite the high SFA content in dairy, current findings from prospective cohort studies indicate
no clear evidence of an increased risk in CVD with milk or dairy consumption. Qin et al. (2015)
reported that among 22 studies, dairy intake was found to be associated with a modest reduction
in overall CVD risk (RR = 0.88, 95% CI = 0.81–0.96), stroke (RR = 0.87, 95% CI = 0.77–0.99),
but not CHD (RR = 0.94, 95% CI = 0.82–1.07). No link was found comparing high-fat dairy and
CHD (RR = 1.04, 95% CI = 0.89–1.21) or low-fat dairy and CHD (RR = 0.93, 95% CI = 0.74–1.17)
(Soedamah-Muthu et al. 2011). However, low-fat dairy may reduce the risk of stroke (RR = 0.93,
95% CI = 0.88–0.99).
Results regarding T2DM are conflicting. Two meta-analyses of 14 and 17 cohort studies,
each consisting of over 400,000 subjects, have concluded that a significant inverse association
exists between dairy consumption and T2DM risk (Aune et al. 2013a, Gao et al. 2013), but a
more recent analysis concluded a null association (Chen et al. 2014). However, yogurt intake
specifically has been shown to be predictive of reduced T2DM risk (Chen et al. 2014, Gao
et al. 2013).
Several hypotheses have been proposed to explain the null or modest benefit of dairy despite
its high SFA content. One explanation is that milk and dairy products are rich mixtures of
macro- and micronutrients, many of which confer cardioprotective effects. For example, epide-
miologic evidence has suggested an inverse association between vitamin D and calcium intake
and the development of metabolic syndrome and T2DM (Tremblay and Gilbert 2009). Another
explanation is that while SFAs may elevate LDL cholesterol, SFAs may also affect other lipid
biomarkers in different ways depending on the macronutrient composition of the food (Micha
and Mozaffarian 2010, Mozaffarian and Clarke 2009). For example, substituting carbohy-
drates with SFAs increase total cholesterol and LDL cholesterol, but also lowers triglycer-
ides (TG) and increases HDL cholesterol. The overall effect is that the TG/HDL cholesterol
ratio is not altered, which is arguably the best predictor of CVD risk (Micha and Mozaffarian
2010). Additionally, the possibilities of residual confounding and measurement error cannot be
excluded when considering observational evidence. Finally, the type of dairy product is seldom
considered in these studies. Fermented dairy like yogurt and cheese may have different effects
from unfermented varieties such as milk (St-Onge et al. 2000).
Despite the need for experimental studies, well-conducted trials of dairy are extremely rare.
Steinmetz et al. (1994) assessed the lipid profile of eight males consuming 2–3 cups/day of whole
or skimmed milk and found that compared to whole milk, skimmed milk significantly lowered
total cholesterol and LDL cholesterol, but did not alter other lipids, lipoproteins, or apolipopro-
teins. In a study of 49 males, Hjerpsted et al. (2011) reported that a 6-week intervention of cheese
lowered serum total, LDL, and HDL cholesterol concentrations and raised glucose concentra-
tions compared to a butter diet. There has also been interest in yogurt, owing to its probiotic
potential, but evidence from randomized trials has been vitiated by various design issues such
as adequate control for energy and macronutrient intake, adjustment for bacterial concentration,
study duration, and prebiotic confounding (Huth and Park 2012). Nonetheless, results from a few
randomized trials support the conclusion that yogurts fermented by probiotic strains may affect
the lipid profile more favorably compared to conventional yogurts (Cho and Kim 2015, Ejtahed
et al. 2011, Mohamadshahi et al. 2014). Future studies should incorporate dairy into long-term
interventions and continue to track lipids, inflammatory markers, and gut hormones as predictors
of cardiometabolic risk.
Eggs
Historically, the health benefits of regular egg consumption have been viewed with skepti-
cism from consumers and scientists alike due to its high cholesterol content. The 1980 USDA
Dietary Guidelines admonished that “[eggs] can be eaten in moderation, as long as your over-
all cholesterol intake is not excessive” (USDA 1980). Indeed, a single large 50 g egg contains
about 186 mg of total cholesterol (USDA 2015). However, most randomized trials conclude
that high doses of dietary cholesterol are not significantly associated with serum cholesterol
(Bowman et al. 1988, Buzzard et al. 1982, Chenoweth et al. 1981, Kummerow et al. 1977, Vorster
et al. 1992). A meta-analysis of 395 metabolic ward studies indicated that consuming 1 egg/d
increased both LDL cholesterol by 4.1 mg/dL and HDL cholesterol by 0.9 mg/dL, resulting in a
negligible effect on LDL/HDL ratio, a significant predictor of CHD (Clarke et al. 1997). Research
by Miettinen and Kesäniemi (1989) demonstrated that endogenous cholesterol levels are highly
regulated and that greater cholesterol intake is offset by higher rates of biliary secretion and fecal
elimination, and lower rates of cholesterol synthesis.
With respect to cardiovascular disease, a meta-analysis of 17 total studies concluded a null
relationship for each additional egg consumed per day and risk of CHD (RR = 0.99, 95%
CI = 0.85–1.15) (Rong et al. 2013). Consumption of 1 egg/d was also not associated with total
stroke (RR = 0.91, 95% CI = 0.81–1.02), although the authors noted a significant decrease in
risk of hemorrhagic stroke (RR = 0.75, 95% CI = 0.57–0.99). Among diabetic patients, how-
ever, those in the highest category of egg consumption were found to have a 1.54-fold (95%
CI = 1.14–2.09) increase in CHD risk compared to those in the lowest category, signifying
possible effect modification by pre-existing illness. For total CVD risk, a later meta-analysis
of 14 studies and 320,778 subjects indicated that an additional 4 eggs/week was associated
with a significant increase in total CVD (RR = 1.06, 95% CI = 1.03–1.10) and that the effect
is strengthened among diabetics (RR = 1.40, 95% CI = 1.25–1.57) (Li et al. 2013). However, a
contemporary meta-analysis of 16 studies found that compared to an intake of <1 egg/d, intake
of ≥1 egg/d was not associated with total CVD (RR = 0.96, 95% CI = 0.88–1.05), ischemic heart
disease (IHD) (RR = 0.97, 95% CI = 0.86–1.09), stroke (RR = 0.93, 95% CI = 0.81–1.07), IHD
mortality (RR = 0.98, 95% CI = 0.77–1.24), or stroke mortality (RR = 0.92, 95% CI = 0.56–1.50)
(Shin et al. 2013). The disparity in results for total CVD may be due to differences in methodolo-
gies and included studies, although the observation that egg consumption is detrimental for heart
disease in populations with T2DM is consistent. However, these findings contrast with a random-
ized trial of 140 T2DM patients assigned to 12 eggs/week vs. <2 eggs/week, which indicated no
between-group differences for HDL, LDL, total cholesterol, triglycerides, or glycemic control
after 3 months (Fuller et al. 2015).
In contrast to CVD, those consuming high amounts of eggs appear to be at heightened risk for
T2DM. Every increase in consumption by 4 eggs/week was associated with a 1.29-fold (95% CI
= 1.21–1.37) greater risk of T2DM (Li et al. 2013), and an alternate categorical comparison of
≥1 egg/d vs. <1 egg/d yielded similar results (RR = 1.42, 95% CI = 1.09–1.86) (Shin et al. 2013).
However, a more recent meta-analysis of egg intake and T2DM considered 12 cohorts totaling
219,979 individuals and concluded a null relationship when comparing highest to lowest category of
intake (RR = 1.06, 95% CI = 0.86–1.30). The authors discovered heterogeneity by country of origin,
reporting that among studies conducted in the United States only, those in the highest category of
consumption had a 1.39-fold (95% CI = 1.21–1.60) risk of T2DM compared to those in the lowest
category (Djoussé et al. 2016). An association for T2DM comparing highest vs. lowest intake in
non-U.S. countries was not observed (RR = 0.89, 95% CI = 0.79–1.02). Although several publica-
tions have suggested that high cholesterol intake alone increases chronic inflammation (Tannock
et al. 2005) and risk of T2DM (Feskens and Kromhout 1990, Meyer et al. 2001, Salmerón et al.
2001), a 12-week randomized trial that assigned 31 subjects to 3 eggs/d vs. placebo (egg substitute
with no cholesterol) reported no changes in fasting blood glucose or insulin sensitivity (Mutungi
et al. 2008, Ratliff et al. 2009).
The relationship between eggs and cardiometabolic risk remains unsettled. The conflicting con-
clusions involving T2DM outcomes demonstrate the unique challenges of the study of eggs in an
epidemiologic context. Likely explanations for these conflicting results include unmeasured con-
founding in the observational data, particularly strong measurement error, and short follow-up dura-
tion in RCTs. Future research should focus on reconciling these differences and exploring plausible
biological mechanisms that may mediate an increase in T2DM incidence. Another unexplored area
of interest is the method of preparation (e.g., raw, fried, scrambled, hard-boiled), since different
methods can alter the nutrient content of the eggs, especially with the addition of cooking oils and
salt or when used as an ingredient in baked goods.
Meats
Meats consist of a heterogeneous range of foods from white meat (e.g., turkey, chicken) to red meat
(e.g., pork, beef) to seafood. The most often studied meats in observational studies are red meat and
white meat. Fish is also commonly consumed, but is covered in a separate section. Meats present
several nutritional benefits, as they are rich in protein, iron, zinc, and B vitamins, but they also con-
tain high amounts of SFAs and cholesterol (Rohrmann et al. 2013).
Red meats consist of beef, lamb, pork, and game meat and exclude poultry and fish. They may
come in processed or unprocessed varieties. Processed meats are defined as meats that have been
preserved by smoking, curing, salting, or addition of chemical preservatives. Examples of pro-
cessed meats include hamburgers, bacon, salami, sausages, hot dogs, and deli and luncheon meats.
A meta-analysis of 20 prospective and case–control studies consisting of 1,218,380 individuals
(Micha et al. 2010) indicated that a 100 g/day increase in red meat consumption was not associated
with CHD (RR = 1.00, 95% CI = 0.81–1.23). However, the authors note the critical gaps in the
literature when it comes to differentiating between processed and unprocessed meats. In the same
meta-analysis, Micha et al. (2010) note that a 50 g/day increased in processed meat consumption
was associated with a relative risk of 1.42 for CHD (95% CI = 1.07–1.89) and 1.19 for T2DM (95%
CI = 1.11–1.27). However, it should be noted that this meta-analysis included only a small number
of studies, and additional research is warranted. A later analysis of six cohort studies revealed that
unprocessed red meat, processed meat, and total red meat consumption were all associated with
elevated risks of total stroke (Kaluza et al. 2012). Finally, data from the EPIC cohort consisting
of 448,568 individuals who were not included in the previous analyses indicated no association
between red meat intake and mortality but a moderate positive association between processed meat
consumption (RR = 1.30, 95% CI = 1.17–1.45 for a 50 g/day increase) and mortality (Rohrmann
et al. 2013). A pooled analysis by Pan et al. (2011) addressed the issue of T2DM; they conclude
that a 1 serving/day intake of unprocessed, processed, or total red meat were all associated with an
increased risk of T2DM.
On the other hand, regular intake of white meats, which include chicken, turkey, and rabbit, was
not found to be associated with CVD risk based on a meta-analysis of 13 cohort studies of 1,674,272
participants (Abete et al. 2014). A 100 g/day increase in white meats was associated with a relative
risk for CVD mortality of 1.00 (95% CI = 0.87–1.15), and 1.10 (95% CI = 0.63–1.89) for IHD mor-
tality. Similarly, the consumption of unprocessed poultry was not associated with T2DM risk (RR =
1.04, 95% CI = 0.82–1.32) (Feskens et al. 2013).
The subject of unprocessed red meat and CVD risk warrants further investigation. Traditionally,
the high SFA content found in red meat (but not in white meat) is thought to heighten cardiovas-
cular risk by increasing LDL cholesterol levels. However, a randomized trial of lean red meats and
lean white meats showed no significant difference in serum lipid levels among hypercholesterol-
emic patients (Davidson et al. 1999). Heme iron has been implicated as a mediator in the relation-
ship between red meat intake and CVD, as high concentrations have been shown to be linked with
chronic inflammation and increased oxidative stress (Wagener et al. 2001). Studies in mice have also
indicated that l-carnitine, a nutrient found in high levels in red meat, may be processed by intesti-
nal microbiota to produce trimethylamine-N-oxide, which may reduce reverse cholesterol transport
and precipitate atherosclerosis (Koeth et al. 2013). Yet, processed meats contain much lower levels
of l-carnitine levels than red meats and are also associated with higher CVD risk. This may be
explained by the addition of preservatives to processed meats. In the United States, processed meats
contain on average four times the amount of sodium and 50% more nitrates than unprocessed variet-
ies (Micha et al. 2010).
Future investigations should aim to clarify the link between fresh meats and CVD risk. Using
data from the Nurses’ Health Study, Bernstein et al. (2010) concluded that replacing red meats with
alternative protein sources such as poultry, fish, low-fat dairy, nuts, and beans is associated with a
significant reduction in CHD incidence. Cutting out meats altogether may also be a potential method
of reducing cardiometabolic risk; compared to non-vegetarians, vegetarians present lower risk of
IHD, CVD mortality, stroke, all-cause mortality, and cancer (Huang et al. 2012), although there
is strong potential for confounding by lifestyle factors among these individuals. However, clinical
trials of vegetarian diets have shown their potential for lowering blood pressure as well as improv-
ing glycemic control (Yokoyama et al. 2014a,b). Such a dietary pattern may prove meaningful as a
nonpharmacologic method of improving health.
Fish
Epidemiologists have long suspected fish intake to be protective against CHD based on early studies of
Alaskan and Greenland Eskimo (Bang et al. 1980, Kromann and Green 1980). This is also discussed in
Chapter 19. Fish has also historically been an integral component of a Mediterranean dietary pattern,
which has been shown to drastically reduce the incidence of CVD (Estruch et al. 2013). Indeed, the
current body of evidence suggests that whole fish intake is inversely associated with heart disease. Two
meta-analyses published concurrently in 2004 both concluded that fish intake is an important modifi-
able lifestyle factor for reducing CHD risk. He et al. (2004) reported that among 11 prospective cohort
studies and 222,364 individuals, a higher consumption of fish was associated with a dose-dependent
reduction in CHD mortality, with the highest category of intake of 2–4 times per week resulting in a
risk ratio of 0.62 (95% CI = 0.46–0.82). In a meta-analysis of 228,864 subjects, comparing all fish
consumers to nonconsumers, Whelton et al. (2004) reported a risk ratio of 0.83 (95% CI = 0.76–0.90)
for fatal CHD and 0.86 (95% CI = 0.81–0.92) for total CHD incidence.
While fish appears to be salutary for heart health, epidemiologic data do not support the same
benefit for T2DM. In a recent meta-analysis of nine cohort studies and 438,214 individuals, Xun and
He (2012) reported that comparing those eating fish five or more times per week to those who did
not eat or ate fish less than once per week, the risk of developing T2DM over follow-up was 0.99
(95% CI = 0.85–1.16). The authors note that significant heterogeneity existed when comparing stud-
ies in Eastern vs. Western populations, with fish intake being significantly beneficial in populations
based in the East (Japan and China).
Traditionally, the cardioprotective effect of fish has been explained by the rich amounts of poly-
unsaturated fatty acids (PUFA) found in fish, especially omega-3 fatty acids such as eicosapen-
taenoic and docosahexaenoic acid. As discussed further in Chapter 10, the physiological benefits
of these fatty acids are numerous and act through various molecular pathways to lower inflamma-
tion and maintain vascular function (Mozaffarian and Wu 2011). Furthermore, various studies have
indicated that omega-3 fatty acids decrease the risk of cardiac arrest, reduce blood pressure, improve
the lipid profile, and decrease platelet aggregation (Dyerberg et al. 1978, Kris-Etherton et al. 2002,
Nestel 1990, Siscovick et al. 1995). Nonetheless, Rizos et al. (2012) concluded in a meta-analysis
of 20 clinical trials of 68,680 patients that fish oil supplementation conferred no significant benefit
for total CVD, CVD subtypes, or all-cause mortality. Similarly, Chowdhury et al. (2012) found a
significant protective effect of fish in epidemiologic studies, but no benefit of omega-3 supplementa-
tion in randomized trials.
The discordance between the epidemiologic and experimental evidence has led scientists to
believe that other nutrients in fish, such as vitamins, trace elements, and essential amino acids, may
drive its effect on CVD. Others have argued that insufficient attention has been paid to confound-
ers such as red meat intake, baseline drug use, or method of preparation (Chowdhury et al. 2012).
Furthermore, FFQs typically do not specify type of fish consumed (e.g., oily fish vs. whitefish),
only total intake of fish. Since different fish species contain varying amounts of omega-3 fatty acids,
pooling fish intake into one exposure type may introduce measurement error. Lastly, although fish
intake correlates with circulating omega-3 fatty acid concentration, only 20%–25% of this variation
can be explained by fish intake alone (Welch et al. 2006).
Despite the effectiveness of high-dose fish oil supplementation being called into question, consump-
tion of whole fish remains a wise choice for maintaining cardiovascular health. However, public health
officials must also note complications arising from mercury and polychlorinated biphenyl and consider
the risks vs. benefits of regularly consuming seafood (Storelli 2008). Future research should focus on
elucidating the mechanism by which fish helps to lower CHD risk, as well as whether whole fish intake
confers any additional benefit for T2DM and glycemic control and insulin resistance.
Grains and Fiber

Grains provide about two-thirds of the total energy and protein intake in the world, even more in devel-
oping countries, and about a quarter of total energy in the United States (Pedersen et al. 1989). Cereal
grains include wheat, rice, corn, barley, sorghum, millet, oat, and rye. These grains are usually pro-
cessed in order to modify flavor, color, texture, and appearance. Whereas whole grains are left with
the bran, germ, and endosperm intact, refined grains undergo additional processing and retain only the
endosperm in order to produce a finer texture and prolong shelf life. This refining process decreases the
nutritional value of the grain because while the bran and germ are rich in dietary fiber, phytochemicals,
iron, and B vitamins (such as folate), the endosperm contains mostly starch (Slavin 2000).
Regular intake of whole grains is inversely associated with all CVD events (RR = 0.79, 95%
CI = 0.73–0.85) according to a meta-analysis of seven cohort studies (Mellen et al. 2008). Similar
estimates were indicated for CVD subtypes, such as CHD, stroke, and CVD death. An expanded
meta-analysis conducted in 2012 consisting of 66 studies arrived at a very similar estimate (RR =
0.79, 95% CI = 0.74–0.85) (Ye et al. 2012). In contrast, refined grain intake was found to have no
association with all CVD events (RR = 1.07, 95% CI = 0.94–1.22) (Mellen et al. 2008). Investigators
have also analyzed epidemiologic data from the perspective of dietary fiber, which is thought to be a
major contributor to the cardioprotective effect of whole grains. A meta-analysis of 22 cohort studies
by Threapleton et al. (2013) found that greater total dietary fiber intake was significantly associated
with a lower risk of first stroke, with an additional 7 g/day conferring a relative risk of 0.93 (95%
CI = 0.88–0.98). Total fiber intake was also found to be protective for CHD incidence (RR = 0.93,
95% CI = 0.91–0.96), and for CHD mortality (RR = 0.83, 95% CI = 0.76–0.91) in a meta-analysis
of 18 studies involving 672,408 participants (Wu et al. 2015).
Whole grain intake also appears to be associated with a decreased risk of T2DM. A meta-analysis
of six studies among 286,125 participants conducted by de Munter et al. (2007) indicates that a
2 serving/d (20 g/day) increase in whole grain intake was associated with a relative risk of 0.79 (95%
CI = 0.72–0.87). Similarly, a 3–5 servings/d increase was associated with a relative risk of 0.74 (95%
CI = 0.69–0.80) (Ye et al. 2012). For refined grains, Aune et al. (2013b) conclude from a pooled
analysis of 16 cohort studies that a 3 serving/day increase of refined grains was not associated with
T2DM (RR = 0.95, 95% CI = 0.88–1.04). There has also been recent interest in white rice, as it is one
of the most commonly consumed food items in the world, especially in Asian countries. Combining
data from four cohort studies consisting of 352,384 subjects, Hu et al. (2012) found that a 1 serving/
day increment in white rice intake was associated with a relative risk of 1.11 (95% CI = 1.08–1.14) of
T2DM. Finally, a meta-analysis of total dietary fiber and T2DM reported an inverse correlation, with
a 2 g/day increment resulting in a risk ratio of 0.94 (95% CI = 0.93–0.96) (Yao et al. 2014).
Although micronutrients and phytochemicals may play a role in the differences between whole
and refined grains, dietary fiber has been the most studied component. Regarding its cardioprotec-
tive effects, dietary fibers in the form of guar gum, glucan, and psyllium have been shown to lower
blood pressure (Anderson et al. 2009) and decrease LDL cholesterol (Anderson et al. 2000, Brown
et al. 1999, Whitehead et al. 2014). It is thought that fiber binds to bile acids in the small intestine
and increases their excretion in the feces; the fermentation of fibers in the colon with production of
propionate may also contribute to the hypocholesterolemic effects of fiber (Anderson et al. 2009).
Dietary fiber also improves glycemic control and insulin sensitivity, although additional studies will
be needed to elucidate this mechanism (Weickert and Pfeiffer 2008).
Currently, few studies have differentiated between soluble and insoluble fiber, and from what
sources these fibers originate. Threapleton et al. (2013) found a null relationship between soluble
fiber intake (RR = 0.94, 95% CI = 0.88–1.01) and risk of first stroke, but also remarked that more
studies of fiber types needed to be performed. The differences in physiological response between
soluble and insoluble fiber are well known. Insoluble fiber slows gastric emptying, reduces nutrient
intake, and can blunt the rise in plasma glucose after a glucose challenge. Soluble fiber, due to its
high water-retaining capacity, can be fermented by intestinal bacteria, and may increase utilization
of fatty acids, thereby increasing energy absorption. Although both types appear to lead to weight
loss, how insoluble and soluble fiber interacts with diet is not well understood, and more research is
needed in this area (Lattimer and Haub 2010).
Nuts and Legumes

Although nuts were once considered unhealthy foods due to their high caloric and fat content, their
cardioprotective effects (further discussed in Chapter 24) have been demonstrated by recent epi-
demiologic and clinical data. In a review of 25 observational studies and 501,791 subjects, Afshin
et al. (2014) report that nut consumption was inversely associated with fatal IHD (RR = 0.76, 95%
CI = 0.69–0.84), nonfatal IHD (RR = 0.78, 95% CI = 0.67–0.92), but not stroke. Similarly, legume
consumption was associated with a lower risk of total IHD (RR = 0.86, 95% CI = 0.78–0.94), but
not stroke. Two other concurrent meta-analyses corroborate these findings (Luo et al. 2014, Zhou
et al. 2014). Luo et al. (2014) additionally reported that nut consumption also reduced the risk of
all-cause mortality (RR = 0.85, 95% CI = 0.79–0.91). Zhou et al. (2014) meta-analyzed 40,102
subjects in studies of blood pressure as well and found that nut consumption was protective against
hypertension (RR = 0.66, 95% CI = 0.44–1.00). In a recent cohort study of 120,852 subjects not
included in previous pooled analyses, van den Brandt and Schouten (2015) indicate that total nut but
not peanut butter intake was related to lower incidence of cardiovascular disease. The PREDIMED
trial, an exceptional intervention study of 7,447 individuals, demonstrated that compared to a tradi-
tional low-fat diet, a Mediterranean diet supplemented with 30 g/day of nuts had a relative risk for
all cardiovascular events of 0.72 (95% CI = 0.54–0.96) (Estruch et al. 2013).
All three of the previous meta-analyses concluded a null effect of nut consumption on the
risk of T2DM after adjustment for BMI (Afshin et al. 2014, Luo et al. 2014, Zhou et al. 2014),
although the point estimate for risk reduction remained below 1, signaling that the pooled
analyses may still be underpowered. Furthermore, van den Brandt and Schouten (2015) reported
an inverse association between nut intake and diabetes in the Netherlands Cohort Study. Despite
a historic aversion to nuts owing to their high-energy density, nuts have not been linked to
obesity (Rajaram and Sabate 2006). This may be due to both decreased fat absorption and the
satiating effect of nut consumption (Ros 2009). Furthermore, nuts and legumes typically have
low glycemic indices (i.e., they do not drastically increase postprandial blood glucose levels),
and consumption of low glycemic index foods in place of high glycemic index foods have been
linked to lower risk of T2DM (Willett et al. 2002).
One of the main explanations for the health benefits of nuts is their high unsaturated fat content.
Indeed, numerous clinical studies have demonstrated that short-term nut supplementation decreases
LDL cholesterol, total cholesterol, apoB, and triglycerides (Del Gobbo et al. 2015, Sabate et al. 2010).
However, nuts and legumes additionally contain ample amounts of plant protein, fiber, folate, vita-
mins, minerals, and phytochemicals such as flavonoids. Key constituents also include L-arginine, the
precursor of nitric oxide, α-linolenic acid and phenolic antioxidants (Ros 2010). All of these nutrients
have been shown to boost cardiovascular health beyond their effects on blood cholesterol modulation
(Mozaffarian et al. 2011). Various short-term clinical trials have shown that nut and legume consump-
tion may improve insulin sensitivity and produce beneficial endothelial effects (Nash and Nash 2008).
Nuts also decrease LDL oxidation, and favorably modulate biomarkers such as adiponectin (Ros 2009).
Future studies should focus on T2DM risk, as well as provide data on specific nut and legume sub-
types. It is known that different nuts offer different nutrient profiles, for example, walnuts contain the
highest concentration of PUFAs among any nut subtype (Ros 2010). Moreover, evidence from the NHS
and NHSII confirm that consumption of walnuts was linearly associated with decreased risk of T2DM,
with those consuming at least 2 servings/week having a relative risk of 0.67 (95% CI = 0.54–0.82) com-
pared to nonconsumers (Pan et al. 2013). Furthermore, a meta-analysis of clinical trials revealed that
walnuts significantly decrease total and LDL cholesterol (Banel and Hu 2009). A higher consumption
of peanuts has also been associated with lower risk of CHD (Hu et al. 1998) and T2DM (Jiang et al.
2002). In the latter study, even peanut butter was found to lower the risk of T2DM when comparing
those consuming ≥5 times a week to nonconsumers (RR = 0.79, 95% CI = 0.68–0.91).
CONCLUSION
This chapter aimed to summarize the epidemiologic and clinical evidence regarding food groups
and their relationships with cardiometabolic disease. Different foods and beverages confer differ-
ent health consequences depending on their macro- and micronutrient composition. However, the
effects of foods appear similar for both CVD and T2DM. In other words, there does not appear to
be a food item that is clearly beneficial for one condition and detrimental for the other, perhaps due
to similar metabolic mechanisms underlying both conditions. Public health officials should recom-
mend foods such as nuts, whole grains, fruits, and vegetables and discourage excessive consump-
tion of red meats and sugar-sweetened beverages. Furthermore, processed varieties are generally
unhealthier than unprocessed varieties of foods, as is the case with whole grains vs. refined grains.
Coffee appears to be beneficial with moderate intake but neutral with heavy intake. Moderate alcohol
consumption is also beneficial, but heavy consumption is associated with deleterious effects on both
diabetes and CVD. One should exercise caution when prescribing these beverages for improving health.
These recommendations are described in more detail in the 2015 Dietary Guidelines for Americans
(U.S. Department of Health and Human Services and U.S. Department of Agriculture 2015).
Our present knowledge of food groups originates from data from large prospective cohort studies
in conjunction with results from randomized trials. Yet, despite our gains in knowledge, questions of
causation still linger for various types of foods and beverages. For these concerns, future investigators
may require large randomized trials to settle issues once and for all. However, such trials, which are
often infeasible due to practical and ethical considerations, present their own set of disadvantages, such
as expense, high attrition rates, nonadherence, and inadequate blinding (Satija et al. 2015).
Nutrition research has also begun to see a shift in thinking from a reductionist to a holistic perspective.
The traditional reductionist perspective holds that parts of the diet, such as single nutrients, are respon-
sible for the health benefits or detriments observed in the literature (Hoffmann 2003). Following the
pharmacological paradigm, classical nutritionists and clinicians have isolated hundreds of active ingre-
dients in hopes of finding a magic bullet, but very few have been successful. Among many randomized
trials, for example, there has been no significant effect of the use of multivitamins/multiminerals on
death due to vascular causes or cancer (Macpherson et al. 2013), fish oil supplements on heart disease
(Rizos et al. 2012), or antioxidant vitamins on heart disease (Ye et al. 2013). Furthermore, dosages
administered in trials are often much greater than what one would normally consume from food sources.
In one case, concentrated doses of vitamin A, as well as beta-carotene in combination with vitamin E,
appeared to increase incidence of all-cause mortality (Bjelakovic et al. 2013). On the contrary, the holis-
tic view of nutrition incorporates complex relationships between foods, as well as multicausal nonlinear
associations (Fardet and Rock 2014). Such a view can facilitate the translation of scientific evidence into
healthful dietary patterns for the prevention of cardiometabolic diseases.
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21 Dietary Patterns and
Cardiometabolic Disease
Elizabeth M. Cespedes Feliciano and Frank B. Hu
CONTENTS
Introduction.................................................................................................................................... 398
Dietary Patterns Methodology........................................................................................................398
Major Study Designs.......................................................................................................................402
Mediterranean Diet.........................................................................................................................402
Other Commonly Used A Priori Dietary Patterns..........................................................................406
Dash Diet........................................................................................................................................407
Healthy Eating Index......................................................................................................................408
Alternate Healthy Eating Index......................................................................................................409
Cardiovascular Disease..............................................................................................................409
CVD Risk Factors......................................................................................................................410
Type 2 Diabetes.....................................................................................................................410
Body Fatness and Body Mass Index.....................................................................................410
Blood Pressure, Blood Lipids, and Inflammation.................................................................410
Other Popular Diets.........................................................................................................................410
A Posteriori Patterns.......................................................................................................................411
Cardiovascular Disease..............................................................................................................411
CVD Risk Factors......................................................................................................................412
Type 2 Diabetes.....................................................................................................................412
Body Fatness and Body Mass Index.....................................................................................413
Blood Pressure, Blood Lipids, and Inflammation.................................................................413
Implications and Directions for Future Research...........................................................................413
References.......................................................................................................................................415
ABSTRACT
Appropriate dietary recommendations are essential to the prevention and management of cardiovascular
disease. Dietary patterns attempt to encompass the synergistic and cumulative effects of the whole diet
and, as such, may be more easily translated into guidelines for healthful nutrition than knowledge of
the disease associations of single foods or nutrients. In this chapter, we describe the methodology used
to study dietary patterns, synthesize the evidence for the most commonly studied dietary patterns and
cardiovascular health, and describe the emerging role of dietary patterns in national nutrition policy.
The benefits of a Mediterranean dietary pattern characterized by the abundant consumption of olive oil,
fruits, and vegetables and moderate consumption of fish, dairy, and alcohol and low intake of red and
processed meats are among the best studied, strongest, and the most consistent. While the Mediterranean
diet is the only dietary pattern to have been tested in a large, randomized controlled trial for the primary
prevention of cardiovascular endpoints, there are other patterns, such as the Dietary Approaches to Stop
Hypertension (DASH), whose effects on cardiovascular disease risk factors such as blood pressure,
blood lipids, and glycemic control have been rigorously tested and show compelling benefits. Across
healthful dietary patterns, there are common elements that likely drive their protective associations with
397
cardiovascular disease, including an emphasis on plant foods including fruits, vegetables, and whole
grains. Often, fish is a signature component, while red and processed meat and added sugar are limited
and nuts, legumes, and moderate alcohol consumption are considered beneficial. In sum, the study of
dietary patterns provides a complementary evidence base to the study of individual foods and nutrients
and has great potential for informing nutrition policy to reduce cardiovascular disease risk.
INTRODUCTION
Identifying the optimal diet (or diets) for the prevention of cardiometabolic diseases is a public health pri-
ority. While traditional nutrition research in cardiovascular health has focused on single nutrients or spe-
cific foods as exposures, there are limitations to this classical approach to nutritional epidemiology: foods
and nutrients are often highly correlated and may have cumulative, synergistic, or antagonistic effects.
Dietary pattern analysis offers a complementary approach by describing the overall diet: the foods, food
groups, and nutrients included in the pattern; their combination and variety; and the frequency and quan-
tity with which they are habitually consumed (Hu 2002). Further, the overall quality of the diet may have
a stronger relationship with cardiovascular outcomes than do individual foods or nutrients. This makes
dietary pattern analysis a potentially useful tool to detect effects in the context of measurement error and
misclassification, which reduce a study’s statistical power (Willett 2012). Dietary pattern analysis can
also provide a summary measure of diet quality with which to control for potential confounding by diet
when studying the relationship of other exposures (e.g., physical activity or individual dietary compo-
nents) to cardiovascular risk. From the perspective of national guidelines, recommendations based on a
composite measure of diet quality may be most applicable: individuals consume foods in combination,
and when intake is manipulated, there are important substitution effects best captured by examining
the entirety of the diet. As methods have been refined and standardized, recent dietary guidelines in the
United States (United States Department of Agriculture 2015a) and other countries (United Kingdom
Department of Health 1994) have shifted toward advice based on dietary patterns.
This chapter describes prominent methods for assessing dietary patterns, current evidence on the role
of major dietary patterns for the primary prevention and management of cardiometabolic diseases and
major cardiovascular risk factors in adult populations, implications, and future directions for research.
DIETARY PATTERNS METHODOLOGY

The most prominent statistical methods to assess dietary patterns can be divided into a posteriori
and a priori approaches, as shown in Figure 21.1.
A posteriori methods take nutritional data (typically semiquantitative food frequency question-
naires or diet records) and apply multivariable statistical techniques to empirically identify correlated
groups of foods. Principal components analysis identifies nonexclusive factors to explain variation
among food items based on intercorrelations, whereas cluster analysis groups individuals into mutually
Methods to derive patterns
Use of prior information Exploratory methods
Indices Confirmatory Reduced Principal Cluster

and scores factor rank component analysis
analysis regression analysis
FIGURE 21.1 Major approaches to dietary patterns analysis (Jackson and Hu 2014, Horton 2005). (Adapted
from Schulze, M.B. and Hoffmann, K., Br. J. Nutr., 95(5), 860, 2006; Hu, F., Obesity Epidemiology, New York,
Oxford University Press, 2008.)
Dietary Patterns and Cardiometabolic Disease 399
exclusive clusters based on differences in mean intakes (Newby and Tucker 2004). In both of these
outcome-independent techniques, foods and nutrients are typically grouped into a smaller number of
input variables (e.g., food groups, quantified by frequency of consumption, weight, or contribution to
daily energy) before patterns are derived. Reduced rank regression is another type of a posteriori tech-
nique but is outcome dependent: it incorporates scientific knowledge by deriving patterns that explain
the largest variation in intermediate outcomes such as biomarkers or nutrients previously known to be
associated with disease (Hoffmann et al. 2004). Often, patterns derived via reduced rank regression
are more strongly associated with cardiovascular disease (CVD) endpoints than those obtained from
outcome-independent techniques. Regardless of the method of derivation, a posteriori patterns are
often grouped into two broad categories: healthy/prudent and unhealthy/Western. The most common
characteristics of these two categories of empirical dietary patterns are described in Table 21.1.
Importantly, dietary patterns defined using a posteriori techniques are empirically derived and thus may
not represent optimal diets based on scientific evidence for chronic disease prevention. One alternative to
describe a dietary pattern consistent with the current knowledge on dietary risk factors and/or on current
dietary recommendations is to use an a priori approach such as numerical indices, which measure adher-
ence to predefined diets. As is the case for a posteriori methods, the dietary input variables for a priori
methods are typically assessed through self-reported intake on a food frequency questionnaire, diet record,
or 24-hour recall. Food and nutrient intakes recorded through these instruments are then used to character-
ize the overall diet. One approach to quantifying dietary patterns is to define the pattern based on individual
preference or behavior or on excluded foods; lacto-ovo-vegetarianism is a prominent example. However,
the most common a priori approach is to create numerical indices that describe the degree to which an
individual’s habitual intake represents the predefined dietary pattern in question. Table 21.2 lists the most
TABLE 21.1
Major A Posteriori Dietary Patterns: Constituents and Scoring Criteria
Dietary Pattern Constituents Emphasized Scoring Criteriaa
A posteriori A posteriori patterns describe For factor analysis, input variables are typically entered into
clusters of foods and nutrients principal components analysis using varimax (orthogonal) rotation
commonly eaten together identified and eigenvalues (which indicate that the factor explains more of
through statistical methods (factor the variance in the correlations than is explained by a single
analysis, utilizing intercorrelations variable). Often, factor scores are categorized for further analysis.
between dietary items, or cluster The number of factors that are derived ranges substantially
analysis that is based on individual depending on the analysis from just a few to dozens. The
differences in mean intakes). percent variance explained by the factors may also range.
Prudent • Fruit A pattern with high-factor loadings for several healthy foods,
• Vegetables including fruit, vegetables, legumes, and fish, is typically
• Legumes called prudent. Individuals receive a score that defines the
• Fish position of each individual along a gradient for each derived
• Whole grains pattern.
• Poultry
Western • Red and processed meats Pattern with high-factor loadings for red and processed meats,
• Eggs eggs, refined grains, and sugar is typically called Western.
• Refined grains
• Sweets/sweetened beverages
• Fried foods
a For both a posteriori and a priori methods, the primary dietary assessment method may be a 24-hour recall or diet record,
though food frequency questionnaires are used most often in prospective cohort studies. In both cases, different dietary
input variables may be quantified differently, for example, by frequency (servings), weight (grams), or daily percent energy
contribution and are at times adjusted for total energy intake.
400
TABLE 21.2
Scoring Criteria for Major Diet Quality Indices with Sample Population Intakesa
HEI-2010 (Guenther et al. 2013) AHEI-2010 (Chiuve et al. 2012) DASH (Fung et al. 2008) aMED (Fung et al. 2009)
0–100 Points 0–110 Points 8–40 Points 0–9 Points
12 Components, Each 5–20 Points 11 Components, Each 10 Points 8 Components, Each 5 Points 9 Components, Each 1 Point
Criteria and
Score Min Max Min Max Min Max Min Max
Fruit 0 cups 0 ≥0.8 cup total 5 0 cups 0 ≥4 servings/ 10 Low quintile 1 High 5 <Median 0 >Median 1
fruit; ≥0.4 cup day excluding 0.46 cups quintile
whole juices 3 cups
fruit/1000 kcal
Vegetables 0 cups 0 ≥1.1 cup total; 5 0 cups 0 ≥5 servings/ 10 Low quintile 1 High 5 <Median 0 >Median 1
≥0.2 cup day 0.44 cups quintile 1 cup/day
greens/beans 2 cups
Dairy 0 cups 0 ≥1.3 cup/1000 10 0 10 Low quintile 1 High 5
kcal, includes 0.09 cups quintile
high fat 3 cups
Nuts and Allocated to 0 serving/day 0 ≥1 servings/ 10 High quintile 1 Low 5 <Median 0 >Median 1
legumes total or plant day 0.09 oz quintile 0.25 cups/day
proteins or 2 oz legumes, 0.15
vegetables oz/day nuts
Fish 0 oz 0 ≥0.8 oz/1000 <Median 0 >Median 1
kcal (seafood/ 0.45 oz/day
plant proteins)
Oils/fats Ratio (PUFA + >4% trans; 0 mg 0 ≤0.5% trans; 10 <Median 0 >Median ratio 1
MUFA):SFA EPA + DHA; 250 mg EPA + MUFA:SFA
<2% PUFA DHA; ≥10% 1.14
PUFA
Total protein 0 oz 0 >2.5 oz/1000 10
foods kcal
Whole grains 0 oz 0 >1.5 oz/1000 10 0 g/day 0 75 (women) 10 Low quintile 1 High 5 <Median 0 >Median 1
kcal or 90 (men) 0.22 oz quintile 1 oz/day
g/day 3 oz
(Continued)
Scoring Criteria for Major Diet Quality Indices with Sample Population Intakesa
HEI-2010 (Guenther et al. 2013) AHEI-2010 (Chiuve et al. 2012) DASH (Fung et al. 2008) aMED (Fung et al. 2009)
0–100 Points 0–110 Points 8–40 Points 0–9 Points
12 Components, Each 5–20 Points 11 Components, Each 10 Points 8 Components, Each 5 Points 9 Components, Each 1 Point
Criteria and
Score Min Max Min Max Min Max Min Max
Refined ≥4.3 oz 0 ≤1.8 oz/1000 10
grains kcal
Sugar- >1 servings 0 0 serving 10 High quintile 1 Low 5
sweetened including juice 1 serving/day quintile
beverages 0 serving/
day
Dietary Patterns and Cardiometabolic Disease
Red and ≥1.5 serving/ 0 0 oz 10 High quintile 1 Low 5 >Median 0 <Median 1

processed day 0.34 oz quintile 1 oz/day
meats 4 oz
Sodium ≥2.0 g 0 ≤1.1 g per 1000 10 High decile 0 Low decile 10 High quintile 1 Low 5
kcal mg/day mg/day 1382 mg quintile
4314 mg
Empty ≥50% 0 ≤19% kcal from
calories kcal solid fat, added
sugars, alcohol
Alcohol >2 drinks/day ≥2.5 (women) 0 0.5–1.5 10 <5 or 0 5–15 g 1
toward empty or ≥3.5 (men) (women) or 2 >15 g
kcal drinks/day; (men) drinks/
nondrinker day
2.5 pts
a Adapted from Cespedes et al. (2016) and George et al. (2014), with sample intakes from Cespedes et al. (2016) analysis of multiple dietary patterns and diabetes risk in the Women’s
Health Initiative.
401
common components and scoring criteria for the major numerical indices. Adherence to the dietary pat-
tern is represented in a summary score based on assigning points for the consumption of component foods
and nutrients in predefined relative or absolute quantities. Often, multiple indices describe variations of the
same dietary pattern, for example, multiple indices describe adherence to a Mediterranean-style dietary
pattern, with two of the most prominent being the Mediterranean Diet Score (MDS) (Trichopoulou et al.
2003) and the Alternate Mediterranean Diet Score (aMED) (Fung et al. 2009). Different numerical indi-
ces also use different scoring and weighting schemes. For example, some use population-specific intakes
(e.g., Dietary Approaches to Stop Hypertension [DASH] (Fung et al. 2008) scores individuals based on
quintiles of intake for the population studied) versus fixed cutoffs for recommended intakes (e.g., Alternate
Healthy Eating Index [AHEI] (Chiuve et al. 2012) scores individuals primarily on the servings/day of par-
ticular foods or nutrient groups). These differences in scoring criteria make synthesizing evidence across
studies a challenge. Dietary patterns methodology continues to evolve to address these limitations, with
recent efforts to standardize numerical dietary quality indices across various population-based cohorts
(Liese et al. 2015).
MAJOR STUDY DESIGNS

In addition to multiple approaches to quantifying dietary patterns, there are multiple study designs,
from the ecologic to the prospective cohort to the randomized controlled trial, for testing their asso-
ciations with disease. While observational studies of dietary patterns and health outcomes are often
cost effective and allow for wider exposure ranges and longer follow-up in which clinical endpoints
can be observed, testing the efficacy of dietary patterns through randomized controlled trials of
intermediate endpoints provides a complementary evidence base. One such approach is controlled
feeding trials, in which all food is provided to participants over the course of weeks or months.
These studies allow the researcher to precisely define the dietary exposures tested. Prominent exam-
ples include the evaluation of the effects of the DASH diet with and without sodium reduction on
blood pressure lowering (Appel et al. 1997, Sacks et al. 2001); the benefits of DASH for CVD
outcomes were later affirmed in large cohort studies showing that adherence to the DASH-style diet
decreased the risk of CVD, coronary heart disease, stroke, and heart failure by 20%, 21%, 19%, and
29%, respectively (Salehi-Abargouei et al. 2013). Long-term randomized trials of dietary pattern
interventions on hard endpoints are rare due to prohibitive cost and lack of dietary compliance in
the long run, though there are important exceptions (e.g., the Prevención con Dieta Mediterránea
[PREDIMED] trial) (Estruch et al. 2013).
Table 21.3 presents pooled relative risk (RR) estimates drawn from meta-analyses comparing
the highest to lowest categories of adherence to major a priori and a posteriori dietary patterns.
In the case of Mediterranean-style diets, the evidence base is particularly strong: meta-analyses of
prospective cohort studies are accompanied with published results of a randomized controlled trial
of clinical endpoints. For the a posteriori patterns, not only are there no interventions testing the
influence of these diets on clinical outcomes, findings from prospective cohort studies have yielded
inconsistent or null results, and, as indicated by the blanks in the table, there are some clinical out-
comes for which no existing meta-analysis of the dietary pattern could be found. The subsequent
section of the chapter describes in greater detail the current state of knowledge for each of the
dietary patterns.
MEDITERRANEAN DIET
Research on the role of the Mediterranean diet for the primary prevention of CVD began to take off
after the Seven Countries Study in the 1960s (Keys 1970), which observed exceptional longevity
among populations living along the shores of the Mediterranean. The lower incidence of CVD was
hypothesized to be due in part to the traditional dietary pattern characterized by olive oil as a primary
culinary fat; abundant plant-based foods; moderate consumption of fish, dairy, and alcohol with
TABLE 21.3
Relationship of Major Dietary Patterns to Cardiovascular Disease and
Cardiovascular Risk Factors
Relative Risk (95% Confidence Interval)
Dietary Pattern CVD CHD Stroke T2D
A posteriori
Prudent 0.69 (0.60–0.78)a 0.83 (0.75–0.92)a 0.86 (0.74–1.01)a 0.85 (0.80–0.91)b
Western 1.14 (0.92–1.42)a 1.03 (0.90–1.17)a 1.05 (0.91–2.22)a 1.41 (1.32–1.52)b
A priori
Mediterranean 0.90 (0.87–0.92)c 0.63 (0.53–0.72)e 0.71 (0.57–0.89)f 0.77 (0.66–0.89)g
0.87 (0.87–0.90)d
DASH 0.80 (0.74–0.86)h 0.79 (0.71–0.88)h 0.81 (0.72–0.92)h 0.79 (0.66–0.95)i
0.80 (0.76–0.85)h
AHEI-2010 0.74 (0.72–0.77)i 0.77 (0.68–0.86)i
HEI-2010 0.82 (0.79–0.85)i 0.82 (0.76–0.88)i
a Rodriguez-Monforte, Flores-Mateo, and Sanchez (2015).

b McEvoy et al. (2014).
c Includes CHD and stroke in composite “CVD” endpoint and reports result per 2-point increase in a
Mediterranean diet adherence scores Sofi et al. (2014).
d Martinez-Gonzalez and Bes-Rastrollo (2014).
e Mente et al. (2009).
f Psaltopoulou et al. (2013).
g Koloverou et al. (2014).
h Salehi-Abargouei et al. (2013).
i Schwingshackl and Hoffmann (2015).
meals; and a comparatively low intake of red meat. Contemporary prospective cohort studies pro-
vide consistent evidence regarding the benefits of adherence to a Mediterranean-style diet for CVD
prevention (notably, the findings for CVD prevention are more consistent than the results observed
for CHD or for cerebrovascular disease individually) (D’Alessandro and De Pergola 2015). Of all
of the dietary patterns described in this chapter, the Mediterranean diet has arguably the strongest
evidence of causality since this dietary pattern has been tested in a large, randomized controlled trial
for the primary prevention of CVD. Testing a dietary pattern through a randomized controlled trial
is very rare due to the expense, difficulty, and length of follow-up required; thus, the PREDIMED
trial (Estruch et al. 2013) represents a key advance that warrants description here.
Conducted in Spain among 7447 participants with CVD risk factors, PREDIMED compared
advice to follow a low-fat diet (control) with advice to follow a Mediterranean-style diet (high in
vegetables, fruits, legumes, fish, and poultry but low in red meats, sweets, and whole-fat dairy)
along with the provision of either extra virgin olive oil (~1 L per week) or nuts (30 g/day; half
walnuts and one-quarter each hazelnuts and almonds). It bears mention that a major criticism of the
trial was the lack of a comparably intense intervention in the low-fat group, which did not achieve
radical reductions in fat intake. Indeed, one reason that low-fat diets have not been effective for
long-term CVD risk reduction may be the substitution of fat by refined carbohydrate, as well as
the difficulty of adhering to such a diet in the long term without continued reinforcement (Howard
et al. 2006). Despite these limitations, compared to the low-fat control group, both PREDIMED
intervention groups experienced ~30% reductions in CVD events after a median 4.8 years of fol-
low-up, and the data safety monitoring board stopped the trial early. While subgroup analyses of
randomized trials must be interpreted with caution, those within the PREDIMED trial suggested a
beneficial effect of the Mediterranean diet on multiple CVD endpoints and intermediate markers
of risk. For example, there was a clear protective effect of a Mediterranean diet supplemented with
olive oil on stroke compared to the low-fat control, but the protective effects for myocardial infarc-
tion (MI) and CVD death did not achieve statistical significance (Estruch et al. 2013). Building
on this success, the PREDIMED research group has launched the “PREDIMED-Plus” trial, which
will test the efficacy for the primary prevention of adding calorie restriction, an intensive lifestyle
program with physical activity and behavioral therapy to achieve weight loss goals to the original
PREDIMED diet (Corella et al. 2013). The results from the landmark PREDIMED trial, together
with consistent observational evidence from prospective cohort studies (Martinez-Gonzalez and
Bes-Rastrollo 2014) indicating that greater adherence to a Mediterranean-style diet (see Figure 21.2)
is strongly associated with lower incidence of CVD, provide convincing evidence to support the
health benefits of the Mediterranean diet for the primary prevention of CVD.
As mentioned earlier, in addition to robust evidence from randomized controlled trials, a large
body of observational evidence has examined adherence to a Mediterranean diet with results that are
generally consistent with findings from short- and long-term randomized trials (Estruch et al. 2013;
Martinez-Gonzalez and Bes-Rastrollo 2014). One challenge in summarizing this evidence, however,
is that more than a dozen scores describe adherence to a “Mediterranean-style diet” (D’Alessandro
and De Pergola 2015). One of the most popular is an adaptation of the MDS (Trichopoulou et al.
2003) known as the aMED (Fung et al. 2009), which ranges from 0 (minimal adherence to the
Mediterranean diet) to 9 (maximal adherence). Table 21.2 shows the components and scoring cri-
teria for aMED in comparison with other major dietary pattern indices. For each of nine foods or
Study Weight (%) RR (95% Cl)

Trichopoulou et al. (1995) 0.7 0.67 (0.47, 0.95)
Knoops et al. (2004) 5.3 0.84 (0.76, 0.94)
Mitrou et al. (2007) (M) 13.2 0.92 (0.89, 0.96)

Mitrou et al. (2007) (F) 10.0 0.93 (0.88, 0.99)
Fung et al. (2009) (CHD) 10.5 0.87 (0.82, 0.92)
Fung et al. (2009) (stroke) 9.0 0.95 (0.88, 1.01)
Buckland et al. (2010) 6.4 0.89 (0.81, 0.97)
Sjorgen et al. (2010) 0.3 0.86 (0.49, 1.53)
Martinez-Gonzalez et al. (2011) 1.2 0.80 (0.62, 1.03)
Agnoli et al. (2011) (stroke) 0.9 0.60 (0.45, 0.82)
Buckland et al. (2011) 7.1 0.88 (0.81, 0.96)
Gardener et al. (2011) 5.0 0.90 (0.81, 1.01)
Tognon et al. (2012) (M) 4.1 0.98 (0.86, 1.11)
Tognon et al. (2012) (F) 2.1 0.81 (0.67, 0.98)
Dilis et al. (2012) (M) 1.8 0.81 (0.66, 0.99)
Dilis et al. (2012) (F) 1.0 0.75 (0.57, 0.99)
Hoevenaar-Blom et al. (2012) (CVD) 12.6 0.95 (0.91, 0.99)
Hoevenaar-Blom et al. (2012) (stroke) 4.3 0.88 (0.78, 0.99)
Misirli et al. (2012) (M) 2.4 0.88 (0.74, 1.05)
Misirli et al. (2012) (F) 2.0 0.81 (0.67, 0.98)
Total (95% Cl) 100.0 0.90 (0.87, 0.92)
0.4 0.6 0.8 1.0 1.2 1.4
Reduced risk Increased risk
RR (95% Cl)
FIGURE 21.2 Meta-analysis forest plot of greater Mediterranean diet adherence scores and cardiovascular
incidence and/or mortality risk. (From Sofi, F. et al., Public Health Nutr., 17(12), 2769, December 2014.)
nutrients, the score assigns a value of 1 to beneficial components for which the individual stud-
ied consumes above the sex-specific median intake (vegetables, legumes, fruits and nuts, cereal,
fish, ratio of monounsaturated to saturated fat) as well as detrimental components for which the
individual consumption is below the sex-specific median (meat, poultry, and dairy products). The
aMED is scored identically to the original MDS but includes dietary patterns and eating behaviors
consistently associated with lower risks of chronic disease, for example, excluding potato products
from the vegetable group, including only whole-grain products in the cereal category, and assigning
a lower (15 g instead of 25 g) value to moderate alcohol intake. In the following summary of the
Mediterranean diet’s influence on major cardiometabolic risk factors, we summarize evidence from
randomized controlled trials where available (primarily subgroup analyses of the PREDIMED trial)
and rely on observational research utilizing a priori scores such as the aMED otherwise.
In addition to CVD endpoints, adherence to a Mediterranean-style dietary pattern has also been
associated with a lower risk of metabolic syndrome and beneficial effects on components including
waist circumference, high-density lipoprotein (HDL) cholesterol, triglycerides (TG), systolic and
diastolic blood pressure, and blood glucose (Kastorini et al. 2011), as well as lower annual weight
gain and lower risk of obesity, which have been reported in some but not all studies (Beunza et al.
2010, Romaguera et al. 2010). Importantly, though trials show benefits of a Mediterranean dietary
pattern on both blood pressure and lipids as summarized earlier, there is very limited observational
research examining benefits of Mediterranean-style diets on blood pressure (Nunez-Cordoba et al.
2009) and results for lipids have been inconsistent (Tortosa et al. 2007, Rumawas et al. 2009) sug-
gesting that the level of adherence achieved through an intensive dietary intervention may be critical
to achieving maximum CVD protection.
With regard to risk of type 2 diabetes (T2D), the trials leading up to PREDIMED as well as
post hoc analyses of PREDIMED suggest a protective effect of Mediterranean diets supplemented
with olive oil on the incidence of T2D (Salas-Salvadó et al. 2011, 2014) and glycemic control:
compared to control (advice to follow a low-fat diet), the Mediterranean diet supplemented with
extra virgin olive oil (RR 0.60, 95% confidence interval [CI] 0.43–0.85) and the Mediterranean
diet supplemented with nuts (RR 0.82, 95% CI 0.61–1.10) reduced the risk for T2D (Salas-
Salvadó et al. 2014). In observational research, most scores measuring a greater adherence to
a Mediterranean-style diet have a protective association with T2D overall (Schwingshackl et al.
2015). However, in research examining multiethnic populations in the United States, results have
been mixed, with some studies finding no association or disparate associations according to race/
ethnicity (Abiemo et al. 2013, Jacobs et al. 2014, Cespedes et al. 2016). One potential reason for
the weaker and at times null associations in diverse U.S. populations is that a major source of
monounsaturated fat (a signature component of the Mediterranean diet) is meat, which also con-
tains a large amount of saturated fat, possibly confounding the benefits of plant-source monoun-
saturated fats. To know whether translating the Mediterranean diet to additional cultural contexts
or substituting key ingredients will provide the same benefit observed in the primarily European
and European-descended populations examined to date will require randomized trials of CVD
outcomes in diverse regions of the world.
With respect to maintaining a healthy body weight, a meta-analysis of Mediterranean diet tri-
als found that while the dietary pattern was associated with lower weight overall (mean difference
between Mediterranean diet and control diet, −1.75 kg [95% CI −2.86 to −0.64 kg]), this difference
was stronger when combined with energy restriction or physical activity (Esposito et al. 2011).
Notably, the Mediterranean diet was not significantly associated with weight gain in any of the stud-
ies examined despite the high-fat content of the diet: in PREDIMED the intervention resulted in a
change in the type of fat rather than the quantity of fat consumed, as % energy from fat was >40% in
both intervention arms at follow-up due to the supplementation with olive oil and mixed nuts. There
are various suggested reasons for the lack of weight gain, including increased satiety (e.g., increased
protein and dietary fiber in the case of nuts) and possibly reduced energy storage (e.g., unsaturated
fats may increase oxidation) (Jackson and Hu 2014).
The PREDIMED trial reported clear benefits of the intervention on blood pressure (Estruch
et al. 2006) and on HDL cholesterol (HDL-C), total-C/HDL-C ratio, and TG (Estruch et al. 2013).
Similarly, a Cochrane meta-analysis of randomized trials of dietary interventions consistent with a
Mediterranean dietary pattern noted overall reductions in total and low-density lipoprotein (LDL)
cholesterol as well as blood pressure (Rees et al. 2013). With regard to inflammation, subanalyses of
the PREDIMED trial (Urpi-Sarda et al. 2012) and further research from the same group (Esposito
et al. 2004) suggest that a Mediterranean diet reduces circulating inflammatory markers such as
high-sensitivity C-reactive protein (hs-CRP) and interleukin (IL)-6, which have been implicated in
endothelial dysfunction and atherosclerosis.
Intervention trials of secondary prevention among participants with existing CVD are few;
prominent examples include the Lyon Diet Heart Study (de Lorgeril et al. 1999) and the Indo-
Mediterranean Diet Heart Study (Singh et al. 2002). Both compared a Mediterranean-style diet to a
low-fat dietary pattern and reported benefits for CVD mortality but have been criticized: important
concerns about data reliability were raised after the publication of the Indo-Mediterranean Diet
Heart Study (Horton 2005), while the Lyon Diet Heart Study and PREDIMED were both stopped
due to significant beneficial effects in interim analyses, which can exaggerate the magnitude of
intervention effect, particularly in small trials (Guyatt et al. 2012).
It bears mention that the Women’s Health Initiative observational study, an epidemiologic cohort,
has examined additional CVD outcomes not investigated in trial settings (e.g., heart failure and sud-
den cardiac death). For example, in the Women’s Health Initiative observational study, DASH scores
were not associated with sudden cardiac death, while higher Mediterranean diet scores predicted
lower sudden cardiac death risk (comparing highest to lowest quintile RR 0.64 [95% CI 0.43–0.94])
(Bertoia et al. 2014). For heart failure, results from Women’s Health Initiative showed a (nonsig-
nificant) trend toward a protective association with greater adherence to a Mediterranean-style diet
(Levitan et al. 2013).
OTHER COMMONLY USED A PRIORI DIETARY PATTERNS

Often, major indices of diet quality are calculated and compared in the same study; for example,
Fung et al. examined the association between change in diet quality indices and concurrent weight
change in a large sample of male health professionals and female nurses and found that a one
standard deviation increase in the aMed, AHEI-2010, and DASH adherence scores was associated
with significantly less weight gain over 4-year periods in both men and women (Fung et al. 2015).
Similarly, a recent meta-analysis found that diets of the highest quality, as assessed by the HEI,
AHEI, and DASH score, reduced by 22% the risk of T2D (RR 0.78, 95% CI 0.72–0.85) and CVD
incidence/mortality (RR 0.78, 95% CI 0.75–0.81) (Schwingshackl and Hoffmann 2015). These
findings are consistent with the standardized analyses conducted by the Dietary Patterns Methods
Project; while the highest scores (signifying higher diet quality) on each of the indices examined
(aMed, HEI-2010, AHEI-2010, and DASH) were associated with marked reductions in mortality,
the benefits were already evident at relatively lower levels of diet quality (Liese et al. 2015). This
suggests that even incremental improvements in overall diet quality—achievable through a vari-
ety of dietary modifications—could lead to clinically relevant reductions in risk. The flexibility
offered by a variety of healthful dietary patterns with a variety of components is an advantage in
the sense that healthful eating may be tailored to individual preferences and culture; for example,
an increase in the AHEI-2010 could be accomplished by eliminating sugar-sweetened beverages
or by reducing the intake of red and processed meats to <2.5 oz/day. However, a limitation of
these methods is the implication that an incremental improvement in diet quality, for example, ten
points on the AHEI-2010, is associated with a similar reduction in CVD risk regardless of how
it is accomplished. Thus, there is an inherent tension between describing the relationship of the
entirety of the diet and disease and the individual relationships of specific food groups that make
up the dietary pattern.
DASH DIET
As mentioned in the Introduction of this chapter, the DASH feeding trials (Sacks et al. 1995, Appel
et al. 1997, Sacks et al. 2001) and the variations of DASH with different macronutrient compositions
tested in the OmniHeart trial (Miller, Erlinger, and Appel 2006) are signature examples of random-
ized interventions testing the efficacy of a dietary pattern on CVD risk factors. The primary endpoint
in these trials was blood pressure, though benefits were also observed for blood lipids. The original
DASH intervention trials12 provided all food to 459 participants with mild hypertension for 8 weeks.
The diet provided was high in vegetables, fruits, low-fat dairy products, whole grains, poultry, fish,
and nuts; low in sweets and sugar-sweetened beverages; and lower in red and processed meats.
Further, the DASH dietary pattern is high in fiber, potassium, magnesium, calcium, and protein and
low in sodium, saturated fat, total fat, and cholesterol. When compared to a dietary pattern high in
saturated fat and sodium and low in vegetables and fruits, the DASH-style dietary pattern reduced
blood pressure by approximately 6/3 mmHg (systolic blood pressure/diastolic blood pressure)
across diverse age, sex, and race subgroups. Variations on the DASH diet also had marked benefits
for key CVD risk factors: the DASH sodium trials observed a greater benefit with the combined
intervention of sodium reduction plus the DASH diet than with either alone (Sacks et al. 2001).
With respect to the variations of the DASH dietary pattern, the OmniCarb trial found no difference
in CVD risk factors or insulin resistance comparing DASH diets with low and high glycemic index,
suggesting that carbohydrate intake and not glycemic index was the primary drive of lipid profiles
(Sacks et al. 2014). The OmniHeart trial (Miller, Erlinger, and Appel 2006) found favorable effects
of the DASH diet on LDL-C and total-C, and total-C/HDL-C ratio regardless of macronutrient
composition, with no appreciable effect on TG. Notably, the OmniHeart trial (Miller, Erlinger, and
Appel 2006) found the greatest improvements in CVD risk factors when carbohydrate was partially
replaced with monounsaturated fat or protein (Molitor et al. 2014). Overall, the DASH diet showed
marked benefits for blood pressure and blood lipids across populations defined by sex, race/ethnic-
ity, and hypertension status, with additional benefits when sodium was reduced or carbohydrate
replaced with high-quality fat or protein. Most recently, replacement of low-fat with high-fat dairy
was tested within the context of a DASH-like diet; the resulting higher intake of saturated fat and
lower intake of sugar achieved equivalent reductions in blood pressure as the original DASH, and
even greater reductions in plasma triglyceride and VLDL concentrations (Chiu et al. 2016).
While the DASH feeding trials provide robust evidence of the diet’s benefit on CVD risk fac-
tors, these trials were short in follow-up and never intended to evaluate clinical endpoints such as
CVD, CHD, stroke, and T2D; for this, we turn to prospective cohort studies that quantify adher-
ence to a DASH-style diet in a score or index. While multiple scores exist to quantify adherence to
a DASH-style dietary pattern, the most frequently used is the DASH score by Fung et al. (2008).
The summary DASH score ranges from 8 (nonadherence) to 40 points (perfect adherence). Based
on population-specific quintiles, individual DASH components are scored between 1 (worst) and
5 (best) with points awarded for higher intakes of eight food groups related to a lower risk of
hypertension (one point per higher quintile of fruits, vegetables, dairy, nuts and legumes, and whole
grains) and lower intakes of harmful foods (one point per lower quintile of sodium, red/processed
meats, and sweetened beverages).
Overall, the observational evidence is consistent with the CVD protection expected based on the
interventions’ impact on key CVD risk factors. For example, a recent meta-analysis of prospective
cohort studies observed a dose–response association between DASH-style diet adherence and over-
all CVD protection (Salehi-Abargouei et al. 2013). However, this is not without exception: despite
the consistent benefits on blood pressure in feeding trials, when long-term benefits were examined
in observational studies of community-dwelling populations, associations with higher scores on a
DASH-style dietary pattern and reduced blood pressure were small in magnitude and no association
was seen with incident hypertension, suggesting that consistent and strong adherence is a key factor
(Folsom, Parker, and Harnack 2007, Camoes et al. 2010).
Cohort studies have also been useful in evaluating additional CVD endpoints and risk factors
not examined in the DASH trials. For example, in the Swedish Mammography Cohort, a greater
adherence to a DASH-style diet was associated with a lower risk of heart failure (Levitan, Wolk,
and Mittleman 2009). Meanwhile, in the Cardiovascular Health Study, none of the dietary pat-
terns examined (AHEI, DASH, and an American Heart Association 2020 dietary goals score) were
associated with incident heart failure (Del Gobbo et al. 2015). Fung et al. found that, independent
of CVD factors including body mass index, adherence to the DASH-style diet was associated with
reduced CHD and stroke among middle-aged women during 24 years of follow-up, and in cross-
sectional analysis in a subgroup of women, the DASH score was inversely associated with lower
plasma levels of hs-CRP and IL-6, suggesting inflammation as a potential mechanism (Fung et al.
2008). A greater adherence to a DASH-style diet has also been associated with lower levels of
inflammatory markers in other studies (Nowlin, Hammer, and D’Eramo Melkus 2012).
With respect to body fatness, a recent meta-analysis of four prospective studies estimated that
the pooled RR comparing the highest DASH category to the lowest was 0.79 (95% CI 0.66–0.95)
(Schwingshackl and Hoffmann 2015). Not all studies have found this association; for example,
in the Multi-Ethnic Study of Atherosclerosis, higher DASH scores were associated with lesser
gain in waist circumference but not with reduced risk of T2D (de Oliveira Otto et al. 2015).
A recent meta-analysis of randomized controlled trials suggests that the DASH diet can signifi-
cantly reduce fasting insulin (mean difference −0.15 (95% CI −0.22 to −0.08) but not fasting blood
glucose or the homeostatic model assessment of insulin resistance (HOMA-IR) (Shirani, Salehi-
Abargouei, and Azadbakht 2013). This echoes the conclusion of another systematic review that
concluded that significant improvements in insulin sensitivity are observed only when adherence
to a DASH-style dietary pattern is part of lifestyle modification including exercise and weight loss
(Hinderliter et al. 2011).
HEALTHY EATING INDEX

The HEI was first developed to measure adherence to federal dietary guidelines in 1995 and later
adapted to reflect the revisions to the guidelines every 5 years. Contemporary studies use the
latest update and validation that reflects the 2010 Dietary Guidelines for Americans (Guenther
et al. 2013, 2014), though the HEI will likely be updated to reflect the recently released 2015
Guidelines. The HEI-2010 has 12 components expressed in terms of nutrient density (with the
idea of uncoupling nutrient quality from quantity), most weighted equally at 10 points. There are
nine adequacy components (all receive a score of zero for zero intake, other than fatty acids, for
which the 15th percentile of the 2001–2002 population distribution of one-day’s intake is set as
the minimum) and three moderation components (empty calories, sodium, and refined grains). For
moderation components, intakes at the level of the standard or lower receive maximum points (for
sodium, the maximum score is assigned to <1100 mg of sodium per 1000 calories). This score also
includes beans and peas under protein foods when protein requirements are not otherwise met to
enable healthy vegetarian dietary patterns to receive a perfect score. For additional components and
scoring criteria, consult Table 21.2.
Few prospective studies have evaluated the relationship of HEI-2010 to CVD, CHD, stroke,
diabetes, or CVD risk factors. The meta-analysis cited in Table 21.3 combined estimates from eight
studies for the highest versus lowest categories of HEI scores from various years and reported a
pooled RR of 0.82 (95% CI 0.76–0.89) for CVD incidence and mortality (Schwingshackl and
Hoffmann 2015). While no other review or meta-analysis provided information on subtypes of
CVD, a well-known prospective study by Chiuve et al. assessed associations among 71,495 female
nurses and 41,029 male health professionals during ≥24 years of follow-up and reported RRs com-
paring the highest to lowest categories of HEI-2005 of 0.79 (95% CI 0.71–0.88) for CVD, 0.76
(95% CI 0.68–0.84) for CHD, and 0.82 (95% CI 0.72–0.93) for stroke (Chiuve et al. 2012). Notably,
this study also updated the AHEI to its present form, the AHEI-2010, with additional chronic disease
risk factors and found similar associations as with HEI-2005 with stroke, but stronger association of
the AHEI-2010 than of the HEI-2005 with CHD and T2D.
Few prospective studies have evaluated the relationship of HEI-2010 to T2D or other CVD risk
factors. No meta-analysis has presented a pooled estimate of this relationship; thus, the best exist-
ing evidence is from the study described earlier in which Chiuve et al. report a RR of 0.82 (95% CI
0.76–0.89) for T2D comparing the highest to lowest categories of HEI-2005 (Chiuve et al. 2012).
Analyses with the updated HEI-2010 in the more racially and ethnically diverse populations in the
Women’s Health Initiative39 found similar results overall, while there was no association of HEI-
2010 with T2D in the Multiethnic Cohort.37 A potential reason for these disparate findings could be
culturally specific foods not captured through dietary assessment methods, such as the FFQ, leading
to misclassification of diet quality, or differences in the consumption of specific food groups, leading
to the same index score, but potentially with differing strengths of association with disease.
In the Multi-Ethnic Study of Atherosclerosis, higher HEI-2010 scores had a small but signifi-
cant inverse association with incident obesity, BMI, and waist circumference overall, with stronger
associations in non-Hispanic whites than in other racial/ethnic groups (Gao et al. 2008). Cross-
sectionally, higher HEI-2010 scores are also associated with lower odds of abdominal obesity in the
National Health and Nutrition Survey (NHANES) (Tande, Magel, and Strand 2010). Together, these
results provide weak but suggestive evidence that the long-term maintenance of a healthful diet as
characterized by the HEI-2010 could be protective against excessive weight gain.
Also in cross-sectional analyses in the NHANES, the total HEI-2005 score was not associated
with any CVD risk factors (blood pressure, fasting glucose and insulin, HOMA-IR, HDL-C, LDL-C,
TG, and hs-CRP) after adjustment for body mass index. The few studies that have examined HEI-
2010 scores have not observed significant benefits on blood lipid levels with greater adherence to
the 2010 Dietary Guidelines for Americans (Mertens et al. 2015). With regard to inflammation, Fung
et al. found no association of higher HEI-2010 scores with hs-CRP, IL-6, E-selectin, soluble inter-
cellular adhesion molecule 1, or soluble vascular cell adhesion molecule 1 (sVCAM-1), whereas in
the same study AHEI-2010 was inversely associated with all of these other than sVCAM-1 (Fung
et al. 2005).
ALTERNATE HEALTHY EATING INDEX

The AHEI is originally based on features of the HEI but adapted to incorporate scientific knowledge
of foods and nutrients predictive of chronic disease risk, such as the specific type of fat, carbohy-
drate, or protein consumed. By contrast to the HEI, which uses nutrient densities to express foods
and nutrients, the AHEI uses primarily absolute intakes. The most recent update is the AHEI-2010
by Chiuve et al. (2012), which ranges from 0 (nonadherence) to 110 (perfect adherence) with the ten
food components scored from 0 (worst) to 10 (best), and points awarded proportionally for inter-
mediate intakes. Points are allocated for higher intakes for healthful food groups (fruits, vegetables,
nuts and legumes, whole grains, long-chain n-3 fats, polyunsaturated fats), moderate consumption
of alcohol, lower intakes for unhealthy foods (red/processed meats and sweetened beverages), and
the lowest population-specific decile for sodium. While the contemporary AHEI-2010 and HEI-
2010 scores emphasize many of the same components (e.g., awarding points for greater consump-
tion of fruits and vegetables, consideration of fatty acid quality), these indices also differ not only in
the methods of scoring but also with respect to key components (e.g., alcohol, see Table 21.2). These
differences likely explain to some degree the differences in findings.
Cardiovascular Disease
In addition to strong observational evidence that higher-quality diets as measured via the AHEI
reduce the incidence of CVD, CHD, and stroke (Chiuve et al. 2012, Reedy et al. 2014, Liese et al.
2015, Wu et al. 2016), the dietary pattern also appears to have favorable effects on inflammation and
other intermediate markers of cardiovascular risk such as T2D (Fung et al. 2007, de Koning et al.
2011, Chiuve et al. 2012, Jacobs et al. 2014, Cespedes et al. 2016, Wu et al. 2016).
With respect to secondary prevention, among participants in the Nurses’ Health Study and Health
Professionals Follow-Up Study, greater increases in AHEI-2010 scores from pre- to post-myocardial
infarction were significantly associated with lower all-cause and cardiovascular mortality (Li et al. 2013).
CVD Risk Factors

Type 2 Diabetes
In the meta-analysis cited in Table 21.3, the pooled RR estimate across six studies comparing
the highest to lowest categories of the AHEI was 0.77 (95% CI 0.68–0.86) (Schwingshackl and
Hoffmann 2015). Two studies not included in this meta-analysis also found the AHEI-2010 to be
protective against T2D, though the strength of association was inconsistent across racial and ethnic
groups (Jacobs et al. 2014, Cespedes et al. 2016).
Body Fatness and Body Mass Index

As is the case for the HEI, evidence for an association of AHEI-2010 with the prevention of exces-
sive weight gain is suggestive but extremely limited. Aside from the Fung et al. study described
earlier that showed that adherence as measured by a variety of dietary scores (including AHEI-2010)
was associated with significantly less weight gain (Fung et al. 2015), few studies have examined the
association of AHEI-2010 scores with body fatness. One longitudinal analysis in the Whitehall II
study found long-term adherence to a healthful diet as measured by AHEI-2010 was associated with
reversal of the metabolic syndrome, driven by the association of AHEI-2010 among participants
with elevated TG and central obesity (waist circumference >102 cm in men or >88 cm in women)
rather than those with other metabolic derangements (Akbaraly et al. 2010).
Blood Pressure, Blood Lipids, and Inflammation

Similarly, very little research has examined AHEI-2010 in relation to biomarkers of CVD risk, and
most available studies examining the benefits of greater AHEI-2010 adherence with blood pressure
and blood lipids have focused on diabetic patients (Huffman et al. 2011, Wu et al. 2016). With regard
to inflammation, in the Whitehall II study in England, participants who maintained a high AHEI-
2010 score or who improved their score over time showed significantly lower mean levels of IL-6
(1.84 pg/mL [95% CI 1.71–1.98] and 1.84 pg/mL [95% CI 1.70–1.99], respectively) than those who
had a low AHEI score over the 6-year exposure period (2.01 pg/mL [95% CI 1.87–2.17]) (Akbaraly
et al. 2015).
Taken together, these results suggest that additional prospective research characterizing the rela-
tionship of various a priori dietary indices to intermediate biomarkers of CVD risk such as blood
lipids and blood pressure would further our understanding of the mechanisms underlying the asso-
ciations of each of these individual dietary patterns to CVD incidence and mortality.
OTHER POPULAR DIETS

In addition to the dietary patterns discussed here, there are many other a priori patterns that are
commonly consumed or commercially available, including vegetarian and vegan diets, defined by
the exclusion of select animal products, and the Atkins and Paleolithic diets, defined by the near-
elimination of carbohydrates and of processed foods, respectively. Vegetarian dietary patterns appear
cardioprotective, with a meta-analysis showing lower mortality from ischemic heart disease (29%)
and cerebrovascular (16%) and circulatory (12%) diseases compared to nonvegetarians (Huang et al.
2012). Importantly, some authors argue that the protective association is driven primarily by stud-
ies of Seventh Day Adventists (a Protestant sect that consumes a vegetarian diet rich in legumes,
whole grains, nuts, fruits, and vegetables), with less robust evidence of CVD reduction in other
populations (Kwok et al. 2014). Others argue that the benefits of a vegetarian diet are not unique, but
rather attributable to having a plant-based diet that limits (but does not necessarily eliminate) red and
processed meats, as this is associated with a reduced risk of coronary heart disease and T2D (McEvoy,
Temple, and Woodside 2012). A meta-analysis of prospective studies comparing an omnivorous diet
to loosely defined vegetarian diets found reductions in blood pressure also (Yokoyama et al. 2014),
but the evidence on vegetarian diets and other CVD risk factors has not been rigorously reviewed.
The Paleolithic diet takes its name from the nutritional habits of our pre-agricultural ancestors
of the Paleolithic era, whose diets likely varied substantially in the quantity of animal versus plant
foods depending on climate and latitude, but would have had in common heat as the main form of
food processing, very limited carbohydrate and a complete lack of dairy outside infancy (Cordain
et al. 2005). A recent meta-analysis including four randomized controlled trials testing the efficacy
of the Paleolithic diet compared to control diets based on dietary guidelines concluded that there was
moderate evidence that the Paleolithic diet resulted in greater short-term improvements in metabolic
syndrome components (i.e., significant reductions in waist circumference, TG, and blood pressure, and
nonsignificant reductions in fasting glucose and improvements in HDL-C) (Manheimer et al. 2015).
Importantly, these endpoints were all assessed at <6 months after intervention began. The restrictive-
ness of the Paleolithic diet (complete lack of dairy, very limited food processing) could pose a chal-
lenge to long-term adherence, but to date this is unknown; there is no evidence regarding the long-term
maintenance or efficacy of these diets compared to guidelines-based dietary patterns.
Unlike the Paleolithic diet, Atkins does not emphasize the consumption of unprocessed foods.
Atkins is a four-phase diet plan based on very-low-carbohydrate intake (it is a ketogenic diet, with
<20% of energy from carbohydrates) and unlimited fat and protein, which has only been assessed in
short-term interventions on intermediate endpoints (Atallah et al. 2014). With respect to weight loss,
while initially Atkins resulted in superior weight loss compared to low-fat controls, it had inconsis-
tent efficacy at 12–24 months. With regard to lipids, Akins had a suggestively beneficial effect in
short-term trials on HDL-C and TG. There was no effect on LDL-C at 12–24 months, despite the
early suggestion of an adverse influence. Effects on blood pressure were inconsistent compared to
control diets, and there was no evidence of a benefit for glycemic control (Atallah et al. 2014).
In sum, despite the scale of the weight loss industry in the United States, there is insufficient
evidence to compare the long-term maintenance or benefits of various popular commercial diets for
the prevention of CVD in the long term.
A POSTERIORI PATTERNS
Cardiovascular Disease
As described earlier, empirically derived a posteriori dietary patterns typically identify two broad
categories that explain the most variation in study participants’ consumption: healthy/prudent and
unhealthy/Western. While it strengthens the case for causality when similar dietary patterns show
consistent benefits in populations with different confounding structures and health behaviors, it
also complicates the task of summarizing a heterogeneous body of evidence. For example, diets
designated as healthy/prudent or unhealthy/Western using principal components analysis may have
different components or different factor loadings for individual components depending on the study
population. The meta-analysis cited in Table 21.3 illustrates the advantages and complications of
pooling results across studies (Rodriguez-Monforte, Flores-Mateo, and Sanchez 2015). The authors
evaluated results from cohort and case–control studies investigating the association between a pos-
teriori dietary patterns and CVD. Patterns designated as healthy/prudent had high-factor loadings
for vegetables, fruit, legumes, whole grains, fish, and poultry, and those designated as unhealthy/
Western had high-factor loadings for red and processed meat, refined grains, French fries, sweets,
desserts, high-fat dairy products, and alcohol. The authors found a lower risk of all CVD endpoints
with greater adherence to a healthy/prudent pattern, with the exception of stroke. Meanwhile, for the
unhealthy/Western, despite highly significant results in some individual studies, the overall pooled
estimate did not achieve statistical significance. As an explanation for why healthy/prudent dietary
patterns might be more consistently associated with (decreased) CVD risk than unhealthy/Western
patterns with (increased) CVD risk, the authors proposed, first, that dietary patterns are socially and
culturally mediated and, second, that the foods that explain the greatest variation in a dietary pattern
labeled as unhealthy may in fact have inverse relationships with select chronic disease outcomes,
even if they increased the risk of others. For example, a dairy product dietary pattern examined
among Japanese adults was associated with a reduced risk of stroke; despite including butter, the
pattern also included other types of dairy, fruits, and other items typically in the healthy patterns
(Maruyama et al. 2013). In another study focused on black Americans, a pattern defined by a high
intake of sweets and saturated fats was associated with a reduction in stroke risk, and the authors
hypothesized that perhaps adherence to the dietary pattern could be associated with a higher risk
of cancer or some kinds of CHD that might lead to death before a stroke could occur (Judd et al.
2013). Meanwhile, other studies characterizing a posteriori dietary patterns did find a protective
association of healthy/prudent dietary patterns with stroke: a meta-analysis assessing the influence
of food patterns identified via principal component analysis, cluster analysis, and/or factor analysis
concluded that the highest compared with the lowest categories of healthy/prudent dietary patterns
were associated with a decreased risk of stroke (summary RR 0.77 [95% CI 0.63–0.93]), whereas
unhealthy/Western dietary patterns were not associated with stroke (Zhang et al. 2015).
Often, dietary patterns derived via a hybrid method called reduced rank regression, which iden-
tifies food patterns that explain maximal variation in intermediate disease risk factors, are more
strongly associated with CVD risk than purely empirical patterns derived without regard to outcome.
For example, a recent study using data from nearly 35,000 participants in the European Prospective
Investigation into Cancer–Netherlands identified seven dietary patterns using reduced rank regres-
sion and principal components analysis that could be grouped into three broad categories: a Western,
prudent, and traditional pattern (Biesbroek et al. 2015). Despite deriving similar dietary patterns,
the reduced rank regression approach, which derived a pattern to explain maximal variation in body
mass index, total-C/HDL-C ratio, and systolic blood pressure, resulted in small differences in food
items that contributed to a stronger association with coronary artery disease than patterns derived
from principal components analysis: the reduced rank regression Western pattern included a fewer
number of important foods (high consumption of French fries, fast food, sausages, and soft drinks)
and was significantly associated to coronary artery disease, whereas the Western pattern derived
through principal components analysis (without the use of intermediate risk factors) was not associ-
ated with coronary artery disease despite having a larger number of components with higher-factor
loadings (e.g., alcohol, bread, sweets, and low-fiber cereal were included in the principal compo-
nents pattern) (Biesbroek et al. 2015). Similarly, in the Whitehall II study, researchers used reduced
rank regression to derive a dietary pattern associated with serum total-C and HDL-C and TG levels
as dependent variables. Among 7314 participants, researchers derived a diet characterized by “high
consumption of white bread, fried potatoes, sugar in tea and coffee, burgers and sausages, soft
drinks, and low consumption of French dressing and vegetables” that was associated with >50%
increased risk of CHD, even after an adjustment for the intermediates blood pressure and BMI (RR
for top versus bottom quartile, 1.57 [95% CI 1.08–2.27]) (McNaughton, Mishra, and Brunner 2009).
CVD Risk Factors

Type 2 Diabetes
Many empirically derived dietary patterns are associated with T2D risk (Alhazmi et al. 2014,
Maghsoudi, Ghiasvand, and Salehi-Abargouei 2015). A recent review meta-analysis of nine prospec-
tive cohort studies (totaling 309,430 participants and 16,644 incident cases) found a 15% lower T2D
risk for those in the highest category of healthy/prudent pattern compared with those in the lowest
category (RR 0.85, 95% CI 0.80–0.91) (McEvoy et al. 2014). Compared with the lowest category of
unhealthy/Western pattern, those in the highest category had a 41% increased risk of T2D (RR 1.41,
95% CI 1.32–1.52) (McEvoy et al. 2014). Another meta-analysis of cohort studies found a protective
RR of 0.79 (95% CI 0.74–0.86) for T2D comparing the highest to the lowest adherence to empirically
derived healthy/prudent dietary patterns, which emphasized whole grain products, fruits, and vegeta-
bles (Alhazmi et al. 2014). By contrast, when comparing the highest to lowest adherence to unhealthy/
Western dietary patterns, which emphasized red or processed meats, high-fat dairy, refined grains, and
sweets, an adverse RR of 1.44 was observed (95% CI 1.33–1.57) (Alhazmi et al. 2014).
Body Fatness and Body Mass Index

Empirically derived healthy/prudent dietary patterns characterized by the presence of vegetables,
fruit, whole grains, and reduced-fat dairy are also associated with a lower risk of obesity, healthier
weight or BMI, and lower weight and waist gain, whereas unhealthy/Western associated with a
higher risk of obesity were characterized by the presence of red meat and processed meats, sugar-
sweetened foods and drinks, and refined grains (Quatromoni et al. 2002, Newby et al. 2003, 2004a,
Newby, Muller, and Tucker 2004b, Schulze et al. 2006, McNaughton et al. 2007, Boggs et al. 2011,
Hosseini-Esfahani et al. 2012). However, the methods employed and study populations examined
were heterogeneous, and not all studies found significant associations.
Blood Pressure, Blood Lipids, and Inflammation

Very few prospective studies have examined blood pressure, blood lipids, or inflammation in rela-
tion to a posteriori dietary patterns (Jiang et al. 2015). With regard to inflammation, most available
studies have been relatively small and cross-sectional and show inconsistent results (Barbaresko
et al. 2013). For example, research conducted in the Nurses’ Health Study’s female participants
found an inverse association of a healthy/prudent diet with the markers of inflammation and endo-
thelial dysfunction such as hs-CRP and E-selectin (Lopez-Garcia et al. 2004); while among men in
the Health Professionals Follow-up Study, there was no association (Fung et al. 2001). While results
have not been wholly consistent, another recent narrative review concluded that studies have shown
an overall adverse association of unhealthy/Western dietary patterns with markers of inflammation
and endothelial dysfunction (Barbaresko et al. 2013).
Similarly, little research has addressed the association of a posteriori dietary patterns with blood
lipids and the available findings are inconsistent. For example, a study in the Cardiovascular Risk in
Young Finns Study suggesting that adherence to a health-conscious pattern (similar to the healthy/
prudent pattern and rich in fruit and vegetables, fish, legumes and nuts, tea, rye, cheese and other
dairy products, and alcoholic beverages) was associated with lower LDL-C and insulin in women
but not in men. Meanwhile, adherence to a traditional pattern (high consumption of potatoes, sau-
sages, milk, coffee, rye, and butter) was associated with higher LDL-C, apolipoprotein B, and CRP
concentrations in both men and women (Mikkila et al. 2007).
IMPLICATIONS AND DIRECTIONS FOR FUTURE RESEARCH

The overall body of evidence suggests that a healthful dietary pattern is strongly associated with a
decreased risk of CVD and associated risk factors, while unhealthy dietary patterns are associated with
an increased risk of CVD and associated risk factors. For example, meta-analyses indicate that diets
of the highest quality, as assessed by the HEI, AHEI, and DASH scores, reduce the risk of CVD (inci-
dence or mortality) by 22% (Schwingshackl and Hoffmann 2015). Variations in the strength of associ-
ations between dietary patterns with disease outcomes may be due to small differences in how dietary
patterns characterize an optimally healthy diet. For example, alcohol in moderation was included as a
positive component (e.g., Mediterranean style or AHEI-2010) and consumption of red and processed
meats as a negative component in some (e.g., Mediterranean or DASH) but not all patterns. How dairy,
poultry, or added sugars were treated also varied. While examining the associations of these individual
components with cardiometabolic disease may help in identifying the most active ingredients in a
healthful dietary pattern, isolating these foods and nutrients may not provide a realistic picture of what
people eat in combination and its health impact; it is likely the cumulative and interactive effects of
multiple components of diet that predict disease, and when one component of the diet changes, it is
typically substituted by another. This is where dietary pattern analyses become particularly useful: not
only do dietary patterns encompass the totality of diet but also they allow for multiple ways to achieve
a healthy diet. Thus, public health guidelines and recommendations may be most easily translated into
eating behaviors when described by the composite measure of diet quality encompassed in dietary pat-
terns. Increasingly, this is recognized; for example, the 2015 Dietary Guidelines Advisory Committee
(DGAC) focused its evidence review and recommendations on healthful dietary patterns instead of
individual nutrients or foods in its recently released scientific report. The DGAC noted remarkable
consistency in the findings over a wide range of disease outcomes, including cardiometabolic diseases,
and across different dietary pattern assessment methods (United States Department of Agriculture
2015b). This suggests that dietary modifications to improve CVD risk may also benefit other chronic
disease outcomes. Remarkably, despite different approaches to deriving dietary patterns, common ele-
ments—nutrients and foods—emerge over and over and are likely to be drivers of the observed effects.
In essence, dietary patterns associated with decreased risk of CVD feature fruits, vegetables, whole
grains, low-fat dairy, and fish as signature components. In these dietary patterns, red and processed
meat and added sugar were limited, while nuts and legumes and moderate consumption of alcohol
were often beneficial. Further, where specific nutrients are included in the dietary patterns, those low
in saturated fat and sodium but rich in fiber and potassium showed the greatest potential for reducing
CVD risk (United States Department of Agriculture National Evidence Library 2014, United States
Department of Agriculture 2015b).
Despite the consistency of the characteristics of a healthful dietary pattern across studies, there
remain significant gaps in the research literature and an urgent need to develop effective strategies
to improve diet quality at the population level. First, the vast majority of studies examined European
or European-descent populations. With this in mind, one consideration when developing guidelines
on the basis of dietary patterns research is that many signature foods vary by region or are culturally
specific. Already, there have been efforts to tackle this translational challenge; for example, a recent
report from a 3-day consensus workshop convened by the World Heart Federation argued that the
essence of the Mediterranean diet could be translated to regions and cultures and concretized this by
suggesting replacement with specific foods that represent staples of particular regions, for example,
“whole grains” might be sorghum or millet in East Asia, but brown or rye bread in Europe or North
America (Anand et al. 2015). Nevertheless, prospective research and large-scale trials of healthful
dietary patterns in diverse regions and populations of the globe would strengthen the evidence base
for relevant dietary guidelines at the country level.
Recently, there has been interest in testing the efficacy of national dietary guidelines on intermedi-
ate biomarkers of cardiovascular risk through short-term randomized controlled trials; this represents a
promising approach to evaluating the efficacy of national dietary guidance. For example, a recent trial of
165 adults found that the current UK dietary guidelines had favorable effects on blood pressure and lip-
ids compared to a traditional British diet (Reidlinger et al. 2015). In addition to randomized controlled
trials, continued analysis of adherence to healthful dietary patterns in the general population through
the nationally representative studies with repeated cross-sectional measures of diet (such as NHANES)
will be critical. One reason is that recent improvements in diet quality, while estimated to account for
substantial reduction in disease burden (13% fewer T2D cases and 9% fewer CVD cases [Wang et al.
2015]), have not been shared equally; despite steady improvement in AHEI-2010 scores in the U.S.
population overall from the 1999 to 2010 surveys by NHANES, absolute scores remain low and the gap
between low and high socioeconomic status widened over time (Wang et al. 2014). Monitoring changes
in diet quality in the population overall and among subgroups can inform public health and policy inter-
ventions to address these nutritional disparities and therefore mitigate disparities in cardiometabolic
diseases. Further, the use of diet quality indices provides a surveillance tool to evaluate the efficacy of
dietary guidelines for the prevention of cardiometabolic disease at the population level.
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22 The Mediterranean Diet
to Prevent Type 2 Diabetes
and Cardiovascular Disease
Michel de Lorgeril
CONTENTS
Introduction.................................................................................................................................... 421
The Mediterranean Diet Pattern: History, Epidemiology, and Randomized Trials....................... 423
The Mediterranean Diet in the Prevention of Type 2 Diabetes...................................................... 425
Conclusions.................................................................................................................................... 426
References...................................................................................................................................... 426
ABSTRACT
The traditional Mediterranean diet (MD) was shown to be associated with a lower incidence of cardio-
vascular disease (CVD) in the 1950s. Randomized controlled trials and epidemiological studies have
subsequently reported lower CVD rates in people following a traditional or a “modernized” form of MD.
In 1994 and 1999, the reports of the intermediate and final analyses of the trial Lyon Diet Heart Study
showed a striking protection of the MD against CVD complications. In 2003, a major epidemiological
study in Greece showed a strong inverse association between a “Mediterranean diet score” and the risk
of cardiovascular complications. In 2011–2012, several reports showed that even non-Mediterranean
populations can gain benefits from long-term adherence to the MD. In 2013, the PREDIMED trial dem-
onstrated a significant risk reduction of CVD complications with MD in a lower risk population than that
of the Lyon trial. Contrary to the pharmacological approach, the adoption of MD is also associated with
a significant reduction in new cancers and overall mortality. Thus, in terms of evidence-based medicine,
the full adoption of a modern version of the MD pattern should be considered as one of the most effec-
tive approaches for the prevention of CVD complications. On the other hand, there is a worldwide type 2
diabetes epidemic with no clear biological explanation. Lifestyle factors are probably important and
lifestyle interventions, including physical exercise and healthy nutrition, have been proposed to prevent
type 2 diabetes and its complications. The optimal dietary pattern seems to be again the MD model that
appears to also be the most appropriate to maintain the pleasure of eating—and therefore the long-term
adherence to the MD pattern—and thus protect against the main complications of type 2 diabetes, that is,
cardiovascular diseases and cancers, and to prolong survival with a good quality of life.
INTRODUCTION
A number of diets have received attention for their potential health effect. One of them, the
Mediterranean diet (MD), has been evaluated in many clinical and epidemiological studies. Before
examining these studies, we must define what the MD is.
The definition varies with experts and geography. The traditional dietary habits of the Italians are
not those of the Spaniards, and those of the Greeks are not those of the Moroccans, to make the story
short. All of them, however, live close to the Mediterranean Sea. An acceptable definition of the MD
could be that it is a modern nutritional pattern inspired by the traditional dietary habits of Greece
421
and southern Italy. The principal components include consumption of olive oil, legumes, unrefined
cereals, nuts, and vegetables; moderate consumption of fish and wine; and low consumption of ani-
mal products except fermented dairy products made from sheep and goat’s milk (see Box 22.1 for
additional comments).
Each aspect deserves comment, although a full description of that complex dietary pattern is not
possible here. We can take the dietary fat issue as an example of its complexity.
The Mediterranean dietary fat issue cannot be summarized—qualitatively and quantitatively—
with a single statement about olive oil. We must indicate the approximate amounts of each type of
fat provided by the (traditional or modernized) MD in comparison with the typical Western diet. It
is important to differentiate the monounsaturated fatty acid oleic acid provided by olive oil (or other
vegetable oils) and the oleic acid provided by animal fat, although it is the same chemical. Also, it is
important to separate the different categories of essential polyunsaturated fatty acids (omega-6 vs.
omega-3) and to differentiate the sources of omega-3 (plant vs. marine) and of omega-6 (plant vs.
meat). Finally, it is critical to separate industrial and natural (ruminant) trans fatty acids. All these
items are critical to understand the “modern” concept of MD. They are probably more important
than, for instance, the “total fat intake” when analyzing the MD concept compared to the Western
diet. Conventional issues such as “low fat” versus “high fat” are not relevant when comparing the
Western diet and the MD. In other words, it is quite clear that both “high fat” and “low fat” MD are
similarly protective against CVD complications.
Type 2 diabetes (T2D) is a devastating disease with rapidly increasing prevalence worldwide;
a similar evolution is seen with the metabolic syndromes [1,2]. They are characterized by insulin
resistance, defined as a decreased ability of insulin to exert its metabolic effects in key target tissues,
despite an elevation of insulin blood levels [3,4]. Besides cardiovascular, infectious, eye and kidney
diseases, other critical complications of T2D and insulin resistance are cancers [5–8]. The preven-
tion of T2D and metabolic syndromes is therefore critical to protect health in general. The biological
explanations of the worldwide T2D epidemic are unknown. However, it is usually accepted that life-
style factors are probably key to this issue. In contrast, it is important to recall that most of the drugs
prescribed to supposedly prevent CVD by reducing cholesterol levels (and blood pressure) increase
insulin resistance and the risk of T2D [9–23]. These major toxic side effects, in particular those of
statins, are usually underestimated by scientists working with the pharmaceutical industry [9–14].
In fact, most trial data (and meta-analyses) are derived from commercial studies entailing major con-
flicts of interest [19–21]. In contrast, studies independent from the industry clearly show that statins
greatly increase insulin resistance and the risk of T2D [15,16]. Biological mechanisms of statin toxic-
ity have been partly identified [9,17]. At the same time, the protective effects of statins against CVD
complications in diabetics remain debatable [18–22]. For instance, the main meta-analysis pooling
the results of studies examining the effects of statins in diabetics [22] was obviously flawed [18].
Finally, as T2D and insulin resistance increase the risk of many diseases, including cancer [5], the
claims that the protective effects of statins against CVD complications in T2D patients outweigh their
toxic effects appear terribly naive [11–14]. Finally, most drugs used to control blood glucose levels and
to prevent T2D, and the risk of CVD complications, may be considerably less e ffective—probably not
effective at all—than previously suspected [23–26]. Thus, it is critical to define nondrug treatments
to prevent T2D, insulin resistance, and their various—not only CVD—complications. Lifestyle inter-
ventions, including physical exercise and healthy nutrition, have been proposed [27–30]. Nutrition is
critical in preventing T2D, managing existing T2D, and preventing—or slowing—the development
of T2D complications [31]. According to the American Diabetic Association (ADA), the main goals
of nutrition in individuals with T2D are to maintain blood glucose levels in the “normal” range,
achieve a lipoprotein profile that reduces CVD risk (in accordance with the so-called “conventional”
targets), maintain blood pressure levels within the normal range, prevent (or slow) the development
of the various complications of T2D, address individual nutrition needs, and maintain the pleasure of
eating by only limiting food choices when indicated by scientific evidence [31].
The next question is: what is the best nutritional approach to achieve these goals?
The Mediterranean Diet to Prevent Type 2 Diabetes and Cardiovascular Disease 423
There are still controversies regarding the importance of reducing total energy intake, total fat intake,
specific fat intake, carbohydrate intake, and/or the benefits of foods with a low glycemic index [32–34].
So far, the optimal dietary pattern to meet the ADA goals [31] seems to be the MD model, which
is, on the other hand, the most appropriate to maintain the (gastronomic) pleasure of eating and thus
actually protect against the main complications of T2D—CVD and cancers—and to prolong sur-
vival with a good quality of life [35–38]. What is the scientific evidence for this claim?
THE MEDITERRANEAN DIET PATTERN: HISTORY,

EPIDEMIOLOGY, AND RANDOMIZED TRIALS
The term MD was first coined by a nonphysician American scientist (Ancel Keys) who observed a
lower mortality rate from CVD among people living in Greece—as well as certain parts of Italy and
the former Yugoslavia—than in Western cohorts (United States and the Netherlands, for instance),
while conducting large multinational studies in the 1950s through 1980s [39–41], in particular the
Seven Countries Study. As this study was observational and failed to demonstrate a cause–effect
relationship, the concept of MD was not widely recognized until the 1990s.
It was in 1994, when the preliminary results of the Lyon Diet Heart Study, a randomized con-
trolled trial testing a modern version of the MD in survivors of a prior acute myocardial infarction,
were reported [35] that the MD began to be accepted as a major scientific concept to prevent CVD
complications. The Lyon trial data were published in two steps. The first, in 1994, was the report of
intermediate results after an average follow-up of 27 months [35], upon the request of the Scientific
Committee whose members intended to stimulate the replication of the trial results in different condi-
tions (and countries). The second step was the final report, which was published in 1999 [36] after
a follow-up of about 4 years and 275 CVD endpoints. The delay—from 27 months to 4 years—
was due to the time needed to recontact and determine the health status of all the patients. Thus, the
total duration of the trial was ultimately close to the follow-up initially calculated to test the primary
hypothesis. Contrary to the claims of certain scientists, the Lyon trial was not prematurely terminated.
Not only was an impressive 50%–70% reduction in the risk of new heart attack or CVD complication
demonstrated among patients in the MD group, but fewer cancers were also observed, as well as a
significant reduction of all-cause mortality [36,37]. Because of these impressive data, experts of the
French National Institute for Health Research (INSERM) were commissioned to thoroughly review
the individual raw data, the whole dataset, and the analyses in the Lyon trial, but failed to identify
significant bias [42], which confirmed the validity of the trial results.
Curiously, there have been misunderstandings about the MD tested in the Lyon trial, the main one
related to the omega-3-rich rapeseed oil margarine that was used to replace butter and cream in the
MD group. Butter and cream were still intensively used at that time in the area around the city of Lyon,
where the patients were recruited. It was therefore essential to obtain good compliance of the patients
with the experimental MD by offering them an acceptable substitute, the rapeseed oil margarine. There
have been claims that the Lyon trial was in fact testing the effects of omega-3 fatty acids. This is wrong.
As shown in the Seven Countries Study when comparing the blood fatty acid patterns of the Greek and
Dutch cohorts [43], it was clear that the Greek diet was traditionally rich in the plant omega-3 alpha-
linolenic acid (ALA). At the time of the Lyon trial, however, the exact dietary sources of ALA in the
Greek diet were not known. Since then, Simopoulos and others have identified some of the ALA-rich
plants consumed in the Mediterranean areas [44,45], explaining the high blood ALA levels observed
in the Greek cohort, along with the low blood levels of omega-6 fatty acid linoleic acid [43]. Thus, to
best mimic the traditional MD, the patients of the Lyon trial were advised to eat some ALA-rich foods.
Olive oil being poor in ALA, rapeseed oil—with a saturated/monounsaturated fatty acid composition
close to that of olive oil—was chosen as a source of dietary ALA [35–37]. Efforts were made to avoid
increasing omega-6 intakes, as plasma omega-6 levels were lowest in the blood of the Greeks with the
lowest mortality rate from CVD in the Seven Countries Study [43]. Thus, rapeseed oil was identified as
the adequate edible oil, in association with olive oil, in the experimental group of the Lyon trial [35–37].
In summary, the Lyon Trial was the first trial to report health benefits of a “modernized” MD, even
though some confusion remained about the true characteristics of that dietary pattern [46–50].
About 10 years after the first results of the Lyon trial were published [35], a new wave of scien-
tific data about the health benefits of the MD appeared. In 2003, Trichopoulos published the first
modern epidemiological study examining the health effects of the MD [51]. In a large prospective
investigation, involving 22,043 adults in Greece, adherence to a MD pattern was measured through
the use of a MD score: the higher the score, the lower the mortality from CVD [51]. Both death due
to coronary heart disease and death due to cancer were inversely associated with greater compliance
with the MD, after adjustment for many confounders including physical activity and some socio-
economic factors [51]. The Greek study thus confirmed the results of the Lyon trial [35–37]. Lower
all-cause mortality also observed by Trichopoulos—including reduction of cancer mortality—sug-
gested that the MD is not associated with any major adverse effect. This is a critical issue in view of
the many side effects of drug-based CVD prevention [9–23]. Following the Greek study, and using
similar approaches with a MD score, several groups analyzed datasets from various populations
[52–54] and reported similar lower CVD and overall mortality. In brief, higher MD score was asso-
ciated with lower CVD complication rates and increased life expectancy, including after adjustment
for confounders such as physical activity and socioeconomic factors [52–54].
Despite these strong data, international organizations—such as the European Society of Cardiology
and the American Heart Association—still did not recommend at that time the MD for CVD preven-
tion. That “intellectual” resistance might have resulted from two factors: (1) the lack of a large ran-
domized trial clearly confirming the findings of the Lyon Diet Heart Study, and (2) the use of the
MD score in epidemiological studies that may have contributed to oversimplifying (and confusing)
the notion of MD—modernized versus traditional—in both theory and practice. It was in that context
that the PREDIMED study investigators reported the main results of their trial in February 2013 [55].
The Spanish investigators of the PREDIMED study reported that adopting a MD reduced the risk
of CVD complications by 30%, and specifically the risk of stroke by 40%, over a follow-up of about
5 years [55]. In this multicenter trial involving 7500 persons, three randomized groups were compared:
a control group advised to follow a low-fat diet and two experimental groups advised to follow a MD
supplemented with either extra-virgin olive oil or mixed nuts. While the sample size was large, the
total numbers of CVD complications were small in the three groups. This indicated that the recruited
population was at very low risk. Observing a significant protective effect in this context suggests that
the two dietary interventions tested were clearly effective. The analysis of the dietary habits in these
three Spanish groups is likely to explain the low rate of CVD complications in the entire study: in fact,
the average diet in the three groups, including the “low-fat” control group, was a Mediterranean-type
of diet known to be cardioprotective. As a consequence, the differences between the three groups for
most of the Mediterranean diet items were small; for certain items, such as the use of olive oil as main
culinary fat, wine drinking, the consumption of commercial wheat baked products, and others, the dif-
ferences were small and even not statistically significant [55]. Observing a significant protection in this
context suggests that the whole Mediterranean dietary pattern—and the interactions between various
nutrients—was the real biological source of protection, rather than any small difference in specific nutri-
ents. The PREDIMED study is thus a confirmation of the Lyon trial and of the Trichopoulos study data.
In terms of specific foods, the main differences explaining the different complication rates in the
three groups were essentially the wide use of extra-virgin olive oil in one experimental group and
the consumption of mixed nuts in the other one. Biomarkers of the consumption of these foods were
measured and higher levels of the plant omega-3 ALA in the mixed nuts group and a higher urinary
excretion of the olive polyphenol hydroxytyrosol in the extra-virgin olive oil group were found. This
is not unexpected as many studies have shown the protective effects of both omega-3 fatty acids
and polyphenols (see also the Box 22.1). Further studies are required to confirm that Mediterranean
polyphenols and plant omega-3 fatty acids are major factors of the health benefits of the MD pattern
and also, importantly, to determine whether the modern foods consumed by the Mediterranean (and
other) populations are still rich in these critical ingredients.
Thus, the PREDIMED study confirmed the findings of the Lyon Diet Heart Study [35–37] in a pri-
mary prevention setting, as well as those of the many epidemiological studies [52–54] published since
Trichopoulos’ 2003 report [51]. However, many questions have been raised. For instance, experts
stated that “The PREDIMED trial is neither a pure test of a Mediterranean-style diet … Interpretation
of the PREDIMED trial is similar in complexity to that of the Lyon Diet Heart Study, which tested
provision of a margarine rich in alpha-linolenic acid, coupled with brief advice to consume a
Mediterranean diet” [56]. Clarification about the use of rapeseed oil in the Lyon trial is given in the
previous section of this article. It is also noteworthy that the dietary intervention in the Lyon trial was
not “brief advice” [35–37]. In contrast, in the same issue of the New England Journal of Medicine, an
editorial stated that “the Mediterranean diet has become the standard for healthy eating” and that “the
PREDIMED results reinforce the Mediterranean diet’s value for health internationally, suggesting a
dietary template that may be of particular value as chronic disease becomes a global issue” [57].
THE MEDITERRANEAN DIET IN THE PREVENTION OF TYPE 2 DIABETES

It may be clinically important to differentiate the prevention of T2D itself, on one hand, and the manage-
ment of diabetic patients to prevent or delay the various complications resulting from T2D, in particular
CVD complications, on the other hand. However, this may be a very theoretical distinction as careful
analysis of the international literature clearly indicates that the MD prevents both the occurrence of
T2D and the complications of T2D in various populations [58–65]. Not only does the MD actually
reduce the risk of T2D, but this dietary pattern also reduces the rate of complications, in particular CVD
BOX 22.1 MEDITERRANEAN DIET AND POLYPHENOLS

Generally speaking, both the traditional and “modernized” Mediterranean diets are rich in
cereals (especially wheat), legumes, vegetables and fruits and quite poor in animal foods.
The consumption of seafood is variable, and the main everyday beverages (other than
water) are extremely rich in polyphenols, that is, wine on the Christian North bank of the sea,
tea on the Muslim South bank. This is an important aspect as plant polyphenols, especially
those found in typical Mediterranean foods such as olive products, may be useful for the
prevention of insulin resistance and T2D [1,2]. Other polyphenols—such as the anthocyanins
found in grapes and wine—have been shown to increase the endogenous synthesis (from the
plant precursor alpha-linolenic acid) of the marine very long-chain omega-3 fatty acids [3,4].
It is noteworthy that both plant and marine omega-3—which are major substances in the tra-
ditional Mediterranean diet—were shown to be inversely associated with the risk of T2D, in
particular alpha-linolenic acid found in plants [5]. As discussed in this chapter, alpha-linolenic
acid is a major component of the Mediterranean diet.
REFERENCES
1. de Bock M, Derraik JG, Brennan CM, Biggs JB, Morgan PE, Hodgkinson SC, Hofman PL, Cutfield
WS. Olive (Olea europaea L.) leaf polyphenols improve insulin sensitivity in middle-aged over-
weight men: A randomized, placebo-controlled, crossover trial. PLoS One. 2013;8(3):e57622.
2. Wainstein J, Ganz T, Boaz M, Bar Dayan Y, Dolev E, Kerem Z, Madar Z. Olive leaf extract as a hypo-
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3. de Lorgeril M, Salen P, Martin JL. Interactions of wine drinking with omega-3 fatty acids in patients with
coronary heart disease: A fish-like effect of moderate wine drinking. Am Heart J. 2008;155:175–181.
4. Toufektsian MC, Salen P, Laporte F, Tonelli C, de Lorgeril M. Dietary flavonoids increase plasma
very long-chain (n-3) fatty acids in rats. J Nutr. 2011;141:37–41.
5. Feskens EJ. The prevention of type 2 diabetes: Should we recommend vegetable oils instead of
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complications, associated with T2D. This is not very surprising, since the major foods that characterize
the MD are each individually linked with the risks of T2D and its complications [66–68].
One major issue that remains to be studied is whether typical Mediterranean foods with prebiotic
properties play a role in the modulation of the T2D risks. Actually, data in animals, but also obser-
vational studies in patients, have suggested that the composition of gut microbiota differs in patients
with diabetes versus patients without diabetes [69].
Finally, in addition to adhering to a MD, it is critical for the prevention of T2D in our societies
to stop taking (and prescribing) drugs with prodiabetic effects with the aim of reducing cholesterol,
blood glucose, or blood pressure [9–17].
CONCLUSIONS
Observational studies and randomized trials indicate that the MD may be considered as an effec-
tive nutritional approach to CVD reduction in particular in patients at high risk for diabetes.
Recommendations should be adapted depending on geographic location, the cultural or religious
background, and the available foods in each geographical area, in order to obtain long-term compli-
ance. This is what we call the “modernized” Mediterranean diet.
One difficulty is the fact that many foods offered to consumers at the present time do not contain
the nutrients expected to be present in those traditionally consumed by Mediterranean populations
because of the modern practices of crop farming and modern techniques of the food industry.
Some of our foods are not those that could be found in grocery shops and open-air markets
in the 1950s through 1970s. For example, the bread consumed today differs from the bread con-
sumed by our grandparents, because the wheat varieties that are grown and harvested today are
genetically and biologically very different from the wheat of the 1950s through 1970s [70,71].
Although this dietary pattern has been proposed as yielding long life expectancy, recent research
has found that certain Mediterranean populations are giving up their traditional dietary habits and
associated healthy lifestyles to adopt unhealthy Westernized food patterns instead. Recent studies
have shown that people with little education, overweight, or diabetes and those who were less physi-
cally active, single, divorced or separated, or smokers, were less likely to comply with the MD [72].
These groups will require special educational efforts.
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23 The DASH Diet
Catherine M. Champagne
CONTENTS
Development of the DASH Diet and Validation in the DASH-Sodium Study...............................431
The DASH (Dietary Approaches to Stop Hypertension) Trial...................................................431
The DASH-Sodium Trial...........................................................................................................434
Subsequent Studies with the DASH Diet Concept: PREMIER and Weight Loss Maintenance.....435
PREMIER Clinical Trial............................................................................................................435
Other Trials Using the DASH-Type Diet...................................................................................436
DASH Diet Compared to a High-Fat DASH Diet.................................................................436
DASH-Type Diet with Alternative Protein Sources..............................................................436
The Omni-Heart (Optimal Macronutrient Intake Trial to Prevent Heart Disease) Study....... 436
Effect of the DASH Diet on Cardiometabolic Health.....................................................................437
Conclusions.....................................................................................................................................445
Acknowledgments...........................................................................................................................445
References.......................................................................................................................................445
ABSTRACT
This chapter describes the Dietary Approaches to Stop Hypertension (DASH) feeding trials and subse-
quent studies undertaken in free-living individuals that evolved from DASH: the PREMIER and Weight
Loss Maintenance intervention trials. The DASH trials demonstrated efficacy of an intervention that
emphasized the intake of fruits, vegetables, and low-fat dairy products with higher protein and lower
dietary fat intake, in lowering blood pressure. This was followed by the PREMIER and Weight Loss
Maintenance trials that showed the effectiveness of the DASH diet in a more realistic clinical setting.
While the results were less robust, participants were able to lower their blood pressure in a free-living
environment. The DASH diet has been used in many other studies with modifications that show that other
sources of protein can be used and that full-fat dairy products are as effective as low-fat dairy products.
These primarily address cardiometabolic health and include improvements in lipid profiles and cardio-
vascular biomarkers, kidney disease, heart failure and stroke, all-cause mortality, and some cancers. The
DASH diet is effective in all age groups and in different ethnicities. A number of health organizations
have endorsed the DASH diet including it as a recommended dietary pattern in the Dietary Guidelines. It
has been selected as the best overall diet in the United States by U.S. News and World Report.
DEVELOPMENT OF THE DASH DIET AND VALIDATION

IN THE DASH-SODIUM STUDY
The DASH (Dietary Approaches to Stop Hypertension) Trial (Appel et al. 1997)
Hypertension is a major public health problem with significant cardiovascular risk. At the time
the DASH trial began, efforts to reduce the prevalence of hypertension had focused primarily on
nonpharmacologic approaches that lower blood pressure. Among these recommendations were
weight control, reduced intake of sodium chloride primarily as salt, reduced alcohol consumption,
and possibly increased dietary potassium and calcium.
431
Observational studies showed significant inverse associations of blood pressure with intake of
magnesium, potassium, calcium, fiber, and protein. However, in trials that tested these nutrients, often
as dietary supplements, the reduction in blood pressure has typically been small and inconsistent.
To test the concept that combining these nutrients through dietary patterns would be more effective,
the National Heart Lung and Blood Institute initiated a call for proposals to test the effect of dietary
patterns on change in blood pressure. Four institutions, including Harvard School of Public Health,
Johns Hopkins School of Public Health, Duke University, and the Pennington Biomedical Research
Center of Louisiana State University, were selected to develop and test different dietary patterns and
the coordinating center was located at the Kaiser Permanente Center for Health Research in Portland,
Oregon. The DASH (Dietary Approaches to Stop Hypertension) diet was the result of this multicenter
trial. Planning for this trial began in early 1994.
Prior observational and intervention studies had revealed that vegetarians had lower blood pres-
sures than their meat-eating counterparts (Sacks et al. 1974). Similarly, higher dietary potassium,
magnesium, calcium, and fiber were associated with lower blood pressure, although trials of these
agents individually had provided inconsistent results except for potassium (Vogt et al. 1999). Dietary
protein and polyunsaturated fat also affected blood pressure; and where excessive amounts of pro-
tein were consumed, dietary cholesterol and saturated fat were associated with higher blood pres-
sure (Vogt et al. 1999). Prior to the DASH study, trials of dietary protein and fat showed inconsistent
results on blood pressure (Vogt et al. 1999).
The goal of the DASH trial was to identify a dietary pattern that lowered blood pressure, yet
was palatable and acceptable to the general population. In the DASH trial, the nutrient targets for
potassium, magnesium, and calcium were set at the 25th percentile of average consumption for the
control diet and at the 75th percentile of average consumption as targets for the two “test” diets. No
micronutrient supplements were used to increase micronutrient targets—as the mandate was that
the difference in micronutrients was to come from natural food products. There was a 3-week run-in
period, after which the participants were randomized to one of three diets for a period of 8 weeks.
The first diet was a “standard American diet” with micronutrient levels for potassium, magnesium,
and calcium at the 25th percentile. The second diet was the fruits and vegetables diet that contained
fruits and vegetables in amounts sufficient to raise the potassium and magnesium intake to the
75th percentile of average intake; the third diet, known as the DASH, was designed with higher
fruits and vegetables and low-fat dairy products to raise potassium, magnesium, and calcium to the
75th percentile of average intake. This diet also had 18% protein and 27% total fat. Sodium intake
was constant across the three diets. Participants were recruited from the respective communities and
randomized to one of the three diets. Food was provided, with 1 or 2 meals eaten on-site and the
other as a takeout meal. Weekend food was also taken home on Friday.
The composition of the three diets is shown in the following:
1. The control diet was an average American diet, high in fat, with the following targets:
a. Total fat at 37% of energy, and high in saturated fat (16% of energy)
b. Moderate in protein at 15% of energy, with 48% of energy from carbohydrate and low
in fiber at about 9 g/2000 kcal
c. Fairly low in potassium—1700 mg/2000 kcal
d. Low in magnesium—165 mg/2000 kcal
e. Low in calcium—450 mg/2000 kcal
2. The fruit and vegetable diet (sometimes referred to as the intermediate diet) contained the
same macronutrient targets as the control diet, with the difference being that the micronu-
trient targets were obtained by the addition of fruits and vegetables:
a. Fiber was increased to about 31 g/2000 kcal.
b. Potassium and magnesium were almost three times that of the control diet:
i. Potassium was 4700 mg/2000 kcal
ii. Magnesium was 500 mg/2000 kcal
The DASH Diet 433
3. The DASH diet (sometimes referred to as the combination diet) was designed to be lower
in fat and saturated fat, with the same fiber, potassium, and magnesium targets as the fruit
and vegetable diet, but with the addition of a calcium target at the 25th percentile of aver-
age intake through the use of low-fat dairy products. The major differences between this
diet and the other two include the following:
• Total fat at 27% of energy, predominantly through the reduction of saturated fat to a
level of 6% of energy.
• Protein was higher, at 18% of energy, predominantly from low-fat and nonfat dairy
products.
• Carbohydrate was higher at 55% of energy.
• Calcium was increased to three times that of the control and fruit and vegetable
diets, containing approximately 1240 mg/2000 kcal, brought about primarily by
dairy products.
DASH Nutrient Targets at the 2100 kcal Level

Control F/V Combination
Fat (% kcal) 37 37 27
Sat fat (% kcal) 16 16 6
CHO (% kcal) 48 48 55
Protein (% kcal) 15 15 18
Fiber (g/day) 9 31 31
Potassium (mg/day) 1700 4700 4700
Magnesium (mg/day) 165 500 500
Calcium (mg/day) 450 450 1240
Sodium (g/day) 3–3.5 3–3.5 3–3.5
The main differences are shown in bold font.
Four hundred and fifty-nine (459) participants were randomized and 49% were women. Minorities
represented 66% of the sample. The mean age was 45 years. Most subjects were overweight (mean
BMI for women was 28.7 kg/m2; for men was 27.7 kg/m2). Mean blood pressure was 132/85 mmHg,
with approximately 29% of the population being hypertensive.
Both systolic and diastolic blood pressures were significantly lowered in those individuals consuming
both test diets, with the DASH diet (mean systolic −5.5 mmHg, mean diastolic −3.0 mmHg; compared to
control, P < 0.001) also producing a significantly greater reduction of blood pressure than the fruits and
vegetables diet. The blood pressure responses to the DASH diet are shown in Table 23.1.
The DASH diet lowered systolic and diastolic blood pressures significantly for both men and
women. Minorities consuming the DASH diet had a much greater drop in both systolic and diastolic
blood pressure than the nonminority groups.
The drop in blood pressure produced by the DASH diet in the hypertensive population was
comparable to drug therapy. Both hypertensive and nonhypertensive individuals had blood pres-
sure reductions that were significant with the DASH diet compared to the control diet. One impor-
tant message from the DASH trial and a basis for its popularity was the suggestion that if one
eats according to the DASH dietary pattern, blood pressure medications may be decreased or
discontinued.
The DASH diet affected serum lipids (Obarzanek et al. 2001). Although baseline lipid values did not
differ among the three treatment arms, individuals consuming the DASH diet had a reduction of 13.7
mg/dL in total cholesterol, a decrease in low-density lipoprotein cholesterol (LDL-C) of 10.7 mg/dL,
and a decrease in high-density lipoprotein cholesterol (HDL-C) of 3.7 mg/dL relative to the control
diet (P < 0.001 for each of the lipid values). This was probably due to the reduced saturated fat intake
in the DASH diet. For those individuals consuming the fruit and vegetable diet, there was no change
TABLE 23.1
Blood Pressure Changes in the Dietary Approaches to Stop Hypertension (DASH)
Trial and in the Dietary Approaches to Stop Hypertension (DASH)-Sodium Trial
Blood Pressure
Mean mmHg (95% CI)
Overall (Change with Control Group Subtracted Out) Systolic Diastolic
DASH Trial
DASH diet −5.5 (−7.4 to −3.7)*** −3.0 (−4.3 to −1.6)***
Fruit and vegetable diet −2.8 (−4.7 to −0.9)*** −1.1 (−2.4 to 0.3)
Gender effects of DASH diet
Males −4.9 (−7.3 to −2.5)*** −3.3 (−5.1 to −1.5)***
Females −6.2 (−9.2 to −3.3)*** −2.7 (−4.8 to −0.7)**
Ethnicity responses to the DASH diet
Minority population −6.8 (−9.2 to −4.4)*** −3.5 (−5.2 to −1.8)***
Nonminority population −3.0 (−5.9 to −0.1)* −2.0 (−4.2 to 0.2)*
Hypertensive status and the DASH diet
Hypertensives −11.4 (−15.9 to −6.9)*** −5.5 (−8.2 to −2.7)***
Nonhypertensives −3.5 (−5.3 to −1.6)*** −2.1 (−3.6 to −0.5)**
DASH-Sodium Trial
DASH vs. Average American Diet (Comparison Diet)
High sodium level −5.9 (−8.0 to −3.7)*** −2.9 (−4.3 to −1.5)***
Intermediate sodium level −5.0 (−7.6 to −2.5)*** −2.5 (−4.1 to −0.8)**
Low sodium level −2.2 (−4.4 to −0.1)* −1.0 (−2.5 to 0.4)
DASH Diet
Change from high to intermediate sodium −1.3 (−2.6 to 0.0)* −0.6 (−1.5 to 0.2)
Change from intermediate to low sodium −1.7 (−3.0 to −0.4)** −1.0 (−1.9 to −0.1)**
Average American diet at high sodium (comparison diet)
Change from high to intermediate sodium −2.1 (−3.4 to −0.8)*** −1.1 (−1.9 to −0.2)**
Change from intermediate to low sodium −4.6 (−5.9 to −3.2)*** −2.4 (−3.3 to −1.5)***
Source: Adapted from Champagne, C.M., Nutr. Rev., 64(2 Pt 2), S53, 2006.
* P < 0.05, **P < 0.01, ***P < 0.001.
in total cholesterol, LDL-C, or HDL-C. Women had smaller reductions in total cholesterol and
LDL-C than men (10.3 mg/dL; P = 0.052; 11.2 mg/dL; P < 0.02, respectively). African Americans
and non-African Americans had similar lipid responses to the DASH diet. Individuals with a higher
baseline plasma HDL responded to the DASH diet with greater reductions in HDL at a level of
3.7 mg/dL (P < 0.05). The investigators concluded that the DASH diet is likely to reduce coronary
heart disease risk by decreasing both total cholesterol and LDL-C in addition to lowering blood
pressure. The implications of the DASH results are significant since a 5 mmHg reduction in systolic
blood pressure is associated with 15% lower coronary heart disease risk and 27% lower risk of
stroke (Obarzanek et al. 2001).
The DASH-Sodium Trial (Sacks et al. 2001)

Sodium intake was held constant in the DASH diet, raising the question of whether dietary
sodium would affect the response to the DASH diet. The DASH-Sodium trial was conducted
by the same four centers that did the original DASH trial and was designed to test the effect
The DASH Diet 435
of three levels of dietary sodium on the response to the DASH dietary pattern or the average
American diet on changes in blood pressure.
The primary aim was to determine the effect on blood pressure of three levels of dietary sodium
set at 50, 100, and 150 mmol/day, in individuals consuming the average American diet (the control
diet) or the DASH diet.
Since this was an “efficacy” study, participants received their meals from the clinical centers
5 days a week with takeout food on the weekends. A total of 412 individuals were recruited. Women
comprised 56% of the participants versus 49% in the DASH trial and 56% of the subjects were
African Americans. The mean age was 49 years and the mean BMI was 29 kg/m2, which was slightly
higher than in the first DASH trial. The mean blood pressure was similar to the first DASH trial
(135/86 mmHg), but there was a higher proportion of people who were hypertensive (41% vs. 29%).
After a run-in period of 11–14 days on a control diet, participants were randomized to three periods
on either the control or DASH diet with the three levels of sodium presented in random order for
30 consecutive days each in a crossover design with a 7-day washout between each sodium level.
The effect of sodium intake on both systolic and diastolic blood pressure was fairly linear, but the
DASH diet, especially at the high and intermediate sodium levels, reduced blood pressure more than the
standard American diet. The difference between the low sodium levels on systolic blood pressure with
the average American (control) diet was less compared with the DASH diet at the lower sodium level;
diastolic blood pressure did not show a difference in response. This trial showed that the DASH diet
appears to be superior to the average American diet in lowering blood pressure at each sodium level.
In contrast to the original DASH study, the DASH-Sodium interventions did not affect total cho-
lesterol, LDL-C, HDL-C, or triglyceride concentrations (Harsha et al. 2004). Although there was no
dose response based on sodium intakes, at each level of sodium the levels of serum total cholesterol,
LDL-C, and HDL-C were lower on the DASH diet compared with the average American diet.
In summary, the DASH-Sodium trial showed that sodium reduction lowered blood pressure in
persons eating the DASH diet as well as those eating the average American diet. Blood pressure was
significantly higher with higher versus lower sodium intakes, and this response for the three levels
was linear. The DASH diet was associated with lower blood pressure at higher, intermediate, and
lower sodium intakes than the average American diet, but the combination of the DASH diet and
the lowest sodium level tested was superior in lowering blood pressure to the comparison diet. The
blood pressure results from the DASH-Sodium trial are displayed in Table 23.1.
SUBSEQUENT STUDIES WITH THE DASH DIET CONCEPT:

PREMIER AND WEIGHT LOSS MAINTENANCE
PREMIER Clinical Trial
The efficacy of the DASH diet in reducing blood pressure led to the question of whether this diet
could be translated into everyday practice. Over the long haul, people need to learn to prepare the
DASH diet on their own since they will not have their meals prepared for them on a daily basis with
strict attention to detail unless they are institutionalized. To test the effectiveness, as opposed to the
efficacy of the DASH diet, NHLBI funded the PREMIER study, which was designed to test the
effectiveness of the DASH diet in free-living populations.
A total of 810 individuals were randomized in the PREMIER study to one of the three condi-
tions: (1) advice only, (2) established strategies to lower blood pressure (EST), or (3) the established
procedures plus the DASH diet (EST + DASH). The primary outcome measures were assessed at
6 months (Appel et al. 2003), with end of intervention at 18 months (Elmer et al. 2006).
The PREMIER study suggested that individuals with suboptimal blood pressure and stage 1
hypertension can make multiple lifestyle changes when motivated to do so. These lifestyle changes
may lower their blood pressure and reduce cardiovascular disease risk, provided the changes are
sustained, but the DASH diet did not add to this effect.
Other Trials Using the DASH-Type Diet

DASH Diet Compared to a High-Fat DASH Diet
The DASH dietary pattern is high in fruits, vegetables, and low-fat dairy foods. Although it
significantly lowers blood pressure as well as low-density lipoprotein (LDL), it also lowers high-
density lipoprotein (HDL) cholesterol, which may be undesirable. This raises the question of
whether the low-fat dairy provides any advantage over regular dairy products. This study was
designed to test the effects of substituting regular (full-fat) dairy products for low-fat dairy prod-
ucts in the DASH diet (Chiu et al. 2015). The increased fat content was achieved by reducing
sugar intake. The trial was a three-period randomized crossover design in free-living healthy
individuals who consumed in random order a control diet, a standard DASH diet, and a higher-
fat, lower-carbohydrate modification of the DASH diet (HF-DASH diet) for 3 weeks each, sepa-
rated by a 2-week washout period. Lipoprotein particle concentrations were determined at the
end of each experimental diet by ion mobility. Thirty-six participants completed all three dietary
periods. Blood pressure was reduced similarly with the DASH and HF-DASH diets when com-
pared with the control diet. The HF-DASH diet significantly reduced triglycerides and large
and medium very-low-density lipoprotein (VLDL) particle concentrations and increased LDL
peak particle diameter compared with the DASH diet. The DASH diet, but not the HF-DASH
diet, significantly reduced LDL cholesterol, HDL cholesterol, apolipoprotein A-I, intermediate
density lipoprotein and large LDL particles, and LDL peak diameter compared with the control
diet. This study suggests that the improvements in blood pressure are comparable with the DASH
diet and the HF-DASH diet. The HF-DASH diet has the additional advantage of reducing plasma
triglycerides and plasma VLDL concentrations without significantly increasing LDL cholesterol
(Chiu et al. 2015).
DASH-Type Diet with Alternative Protein Sources

The protein sources in a DASH-type can be substituted without altering the effect on blood
pressure. In a study by Roussell et al. (2012), 36 hypercholesterolemic participants (with LDL-
cholesterol concentrations >2.8 mmol/L) were randomly assigned to consume each of the four
diets, HAD (33% total fat, 12% SFA, 17% protein, and 20 g beef/day), DASH (27% total fat,
6% SFA, 18% protein, and 28 g beef/day), BOLD (28% total fat, 6% SFA, 19% protein, and
113 g beef/day), and BOLD+ (28% total fat, 6% SFA, 27% protein, and 153 g beef/day), for
5 weeks. Blood pressure was reduced with the higher quantities of beef. LDL cholesterol and
TC decreased after consumption of the DASH, BOLD, and BOLD+ diets when the baseline
C-reactive protein (CRP) concentration was <1 mg/L; LDL cholesterol and TC decreased when
baseline CRP concentration was >1 mg/L with the BOLD and BOLD+ diets. Thus, lean beef
appears to improve the lipid response to the DASH diet without impairing the reduction in
blood pressure.
In a second study, Sayer et al. substituted pork for chicken and fish in a two-arm parallel study
in a randomized crossover study of 13 women and 6 men (mean age of 61 years; BMI of 31.2 and
elevated BP [130/85 mmHg]). Both pork and chicken plus fish reduced blood pressure to a similar
degree.
The Omni-Heart (Optimal Macronutrient Intake Trial to Prevent Heart Disease) Study
This three-period study examined the effect of replacing saturated fat with protein (25% vs. 15%)
or carbohydrate (58% vs. 48%) or unsaturated fat (predominantly monounsaturated fat 37%) in
a three-period crossover with the DASH diet as basic component of the dietary structure. The
carbohydrate diet used in this trial is similar to the DASH diet, except that the carbohydrate
intake of the DASH diet was 55% of kcal, versus 58% of kcal in the carbohydrate diet, and
the protein intake of the DASH diet was 18% of kcal, versus 15% of kcal in the carbohydrate
diet. The protein intake was reduced to 15% of kcal to achieve a 10% of kcal contrast with the
The DASH Diet 437
protein diet. Approximately two-thirds of the increase in protein from the carbohydrate to the
protein diets came from plants (legumes, grains, nuts, and seed). However, sources of protein
were varied and also included meat, poultry, egg product substitutes, and dairy products.
The protein diet included some soy products, but the amount was low, on average just 7.3 g/day.
The unsaturated fat diet emphasized monounsaturated fat. This diet included olive, canola, and
safflower oils, as well as a variety of nuts and seeds, to meet its target fatty acid distributions.
The type of carbohydrate in each diet was similar, as indicated by the total dietary glycemic index
(68 in carbohydrate diet, 71 in the protein diet, and 75 in unsaturated fat diet, relative to the white
bread index). Blood pressure, low-density lipoprotein cholesterol, and estimated coronary heart
disease risk were lower on each diet compared with baseline. The protein diet further decreased
mean systolic blood pressure by 1.4 mmHg (P = 0.002) and by 3.5 mmHg (P = 0.006) among those
with hypertension and decreased low-density lipoprotein cholesterol by 3.3 mg/dL (0.09 mmol/L;
P = 0.01), high-density lipoprotein cholesterol by 1.3 mg/dL (0.03 mmol/L; P = 0.02), and triglyc-
erides by 15.7 mg/dL (0.18 mmol/L; P = 0.001) compared to the carbohydrate diet. A comparison
of the carbohydrate diet showed that the unsaturated fat diet decreased systolic blood pressure by
1.3 mmHg (P = 0.005) and by 2.9 mmHg among those with hypertension (P = 0.02), increased
high-density lipoprotein cholesterol by 1.1 mg/dL (0.03 mmol/L; P = 0.03), and lowered triglycer-
ides by 9.6 mg/dL (0.11 mmol/L; P = 0.02) but had no significant effect on low-density lipoprotein
cholesterol. Compared with the carbohydrate diet, estimated 10-year coronary heart disease risk
was lower and similar on the protein and unsaturated fat diets. In the setting of a healthful diet,
partial substitution of carbohydrate with either protein or monounsaturated fat can further lower
blood pressure, improve lipid levels, and reduce estimated cardiovascular risk.
EFFECT OF THE DASH DIET ON CARDIOMETABOLIC HEALTH

A total of 466 publications were found when the term “DASH diet” was searched in PubMed,
214 of which were published in the last 5 years. Overviews of selected studies are presented in the
following table, which compares the (1) study objectives, (2) design, (3) study population, (4) out-
come measures, and (5) results (Table 23.2).
A systematic review and meta-analysis of the DASH diet on cardiovascular endpoints was
published in 2015 by Siervo et al. (2015). In their meta-regression analyses, they examined
the association between effect sizes, baseline values of the risk factors, BMI, age, quality of
trials, salt intake, and study duration. A total of 20 articles reporting data for 1917 participants
were included in the meta-analysis. The duration of interventions ranged from 2 to 24 weeks.
The DASH diet was found to result in significant decreases in systolic BP (−5.2 mmHg, 95%
CI −7.0, −3.4; P < 0.001) and diastolic BP (−2.6 mmHg, 95% CI −3.5, −1.7; P < 0.001) and in
the concentrations of total cholesterol (−0.20 mmol/L, 95% CI −0.31, −0.10; P < 0.001) and
LDL (−0.10 mmol/L, 95% CI −0.20, −0.01; P = 0.03). Changes in both systolic and diastolic BP
were greater in participants with higher baseline BP or BMI. These changes predicted a reduc-
tion of approximately 13% in the 10-year Framingham risk score for CVD. The DASH diet
improved cardiovascular risk factors and appeared to have greater beneficial effects in subjects
with an increased cardiometabolic risk. The DASH diet is an effective nutritional strategy to
prevent CVD.
If the DASH diet, which is recommended by several U.S. health organizations as a strategy for
preventing and managing blood pressure, were generally adopted, it would result in a reduction of
13% in the 10-year Framingham risk score for cardiovascular events based on the results of their
review and meta-analysis. These findings reinforce the evidence that adherence to the DASH dietary
pattern could make a significant contribution to the prevention of cardiovascular disease, above and
beyond lowering of blood pressure.
The original DASH diet lowered red meat consumption and raised protein intake to 18% with
chicken and fish. As described earlier, whether other meats would improve or impair the response
438
TABLE 23.2
Selected Studies That Have Utilized the DASH Diet as a Main or Comparative Focus
Article Objective Design Subjects (n) Outcome Measure(s) Results Conclusions
Asemi Z et al. “Effects To assess the effects of Randomized controlled 48 women with • Lipid profiles Adherence to the DASH The DASH diet group
of DASH diet on lipid the Dietary clinical trial PCOS. • Biomarkers of diet resulted in a resulted in a
profiles and Approaches to Stop • Women were oxidative stress— significant decrease in significant decrease in
biomarkers of Hypertension (DASH) randomly assigned plasma total weight, BMI, serum insulin,
oxidative stress in diet on lipid profiles to consume either antioxidant capacity decreased serum triglycerides and
overweight and obese and biomarkers of control or DASH (TAC) and total triglycerides and VLDL cholesterol and
women with oxidative stress in diet for 8 weeks— glutathione (GSH). VLDL cholesterol a significant increase
polycystic ovary overweight and obese both diets were levels compared to the in TAC and GSH
syndrome: A women with polycystic calorie-restricted. control group. levels. (Asemi et al.
randomized clinical ovary syndrome • Both diets consisted Increased TAC and 2014)
trial.” Nutrition 2014 (PCOS). of 52% GSH concentrations
November–December; carbohydrates, were also observed in
30(11–12): 18% proteins, and the DASH group.
1287–1293. 30% total fats.
Jacobs DR et al. “The To evaluate albumin Randomized, parallel 378 individuals • Albumin excretion The decrease in AER The DASH diet can be
effects of dietary excretion rate (AER) group, 8-week without diabetes rate (AER). after 8 weeks occurred especially beneficial
patterns on urinary while increasing controlled feeding: with in only those with in slowing disease
albumin excretion: protein intake in the • Followed DASH prehypertension or high-normal baseline progression for people
Results of the Dietary DASH trial. diet or control diet. stage 1 AER in the FV diet, in with early stage (1 or 2)
Approaches to Stop hypertension. a pattern distinct from kidney disease.
Hypertension (DASH) the blood pressure (Jacobs et al. 2009)
Trial.” Am J Kidney decrease. The DASH
Dis 2009; 53(4): diet did not increase
638–646. AER despite a 3%
increase in energy
from protein.
(Continued)
The DASH Diet
Sacks FM et al. To determine the effect Randomized crossover 163 overweight • Insulin sensitivity. Diets with low glycemic Low glycemic index
“Effects of high vs of glycemic index and controlled feeding trial: adults: • LDL cholesterol. index of dietary foods not needed for
low glycemic index of amount of total dietary • Given 4 diets based on • 85 (52%) • HDL cholesterol. carbohydrate, full cardiovascular
dietary carbohydrate carbohydrate on risk healthful DASH-type females. • Triglycerides. compared with high benefits with DASH
on cardiovascular factors for CVD and diet, each for 5 weeks • 78 (51%) males. • Systolic blood glycemic index of diet. DASH again
disease risk factors diabetes. Inclusion criteria: Race/ethnicity: pressure. dietary carbohydrate, proven to lower blood
and insulin sensitivity: • ≥30 years of age • Black 83 (51%). did not result in pressure and
The OmniCarb • Systolic BP 120–159 • Non-Hispanic improvements in cholesterol, benefits
randomized clinical mmHg; Diastolic BP white 66 (40%). insulin sensitivity, lipid independent of
trial.” JAMA 2014 70–99 mmHg • Asian 4 (2%). levels, or systolic glycemic index of
December 17; • BMI ≥25 • Other 5 (3%). blood pressure. foods in diet. (Sacks
312(23): 2531–2541. Exclusion criteria: et al. 2014)
• CV disease, DM, or
CKD
• Taking medication that
lowers BP or lipids
• FBG ≥ 125 mg/dL
Appel LJ et al. “Effects To compare the effects Randomized, 3-period, 164 adults with • Systolic blood In the setting of a Replacing
of protein, of 3 healthful diets, crossover feeding study prehypertension or pressure. healthful diet, partial added-sugars and
monounsaturated fat, each with reduced conducted in Baltimore, stage 1 • LDL cholesterol. substitution of refined starchy foods
and carbohydrate saturated fat intake, on Maryland and Boston, hypertension. carbohydrate with with either lean
intake on blood blood pressure and Massachusetts. either protein or protein-rich foods
pressure and serum serum lipids. • Each feeding period monounsaturated fat and/or foods rich in
lipids: Results of the was 6 weeks and body can further lower blood monounsaturated fats
OmniHeart weight kept constant. pressure, improve lipid improved blood
randomized trial.” • Diets: Diet rich in levels, and reduce pressure and blood
JAMA 2005 November carbs, diet rich in estimated lipid results in a
16; 294(19): protein (half from cardiovascular risk. DASH eating pattern.
2455–2464. plant sources), diet (Appel et al. 2005)
rich in unsaturated fat.
439
(Continued)
440

Couch SC et al. “The To examine the efficacy 57 adolescents with a 57 adolescents with • Blood pressure. DASH had a greater Teens that followed the
efficacy of a of a 3-month clinical diagnosis of prehypertension or decrease in systolic BP DASH diet,
clinic-based clinic-based behavioral prehypertension or hypertension. and a trend for a consuming more
behavioral nutrition nutrition intervention hypertension were greater decrease in fruits, vegetables,
intervention (the DASH diet) vs. randomly assigned to SBP over the study low-fat dairy, and
emphasizing a routine outpatient DASH or RC. SBP, period. Additionally, nuts, were more
DASH-type diet for hospital-based DBP, 3-day diet recall, DASH had a greater effectively able to
adolescents with nutrition care (RC) on weight, and height increase in the intake lower their blood
elevated blood diet and blood pressure were assessed at of fruits, potassium, pressure and improve
pressure.” J Pediatr in adolescents with pretreatment, and magnesium, and a their diets than those
2008 April; 152(4): elevated BP. posttreatment, and greater decrease in who followed the RC
494–501. 3 months later total fat, compared diet. (Couch et al.
(follow-up). to RC. 2008)
Blumenthal JA et al. To compare the DASH Randomized controlled 144 overweight or • Main outcome For overweight or obese Adding weight loss and
“Effects of the DASH diet alone or combined trial obese, measure: BP. persons with exercise to the DASH
diet alone and in with a weight • Assessments at unmedicated • Secondary outcome above-normal BP, the diet improves blood
combination with management program baseline and 4 outpatients with measures: Pulse addition of exercise pressure regulation
exercise and weight with usual diet controls months, with each high BP. wave velocity, and weight loss to the and showed
loss on blood pressure among participants patient assigned one flow-mediated DASH diet resulted in improvements in
and cardiovascular with prehypertension of the following dilation of the even larger BP other measures of
biomarkers in men or stage 1 diets: Usual diet brachial artery, reductions, greater cardiovascular health.
and women with high hypertension. controls, DASH diet baroreflex improvements in (Blumenthal et al.
blood pressure: The alone, and DASH sensitivity, and left vascular and 2010)
ENCORE study.” Arch diet plus weight ventricular mass. autonomic function,
Intern Med 2010 management. and reduced left
January; 170(2): ventricular mass, as
126–135. compared to the DASH
diet alone.
(Continued)
The DASH Diet

Levitan EB et al. To examine if the Followed participants 38,987 Swedish • Heart failure (HF). Men in the greatest Greater consistency
“Relation of DASH diet is over 7 years to men aged 45–79. quartile of the DASH with the DASH diet
consistency with the associated with lower examine consistency component score had a was associated with
dietary approaches rates of heart failure in with the DASH diet 22% lower rate of HF lower rates of HF
to stop hypertension men, as it has been (using DASH scores) events than those in the events in men aged
diet and incidence of shown in women. and rates of heart lowest quartile. 45–79 years. (Levitan
heart failure in men failure hospitalization et al. 2009b)
aged 45 to 79 years.” or mortality.
Am J Cardiol 2009b;
104(10): 1416–1420.
Levitan EB et al. To test the hypothesis Observational study 36,019 women aged • Heart failure (HF). Women in the top Women who followed
“Consistency with the that diets consistent conducted in the 48–83. quartile of the DASH the DASH diet were
DASH diet and with the DASH diet Swedish diet score based on associated with lower
incidence of heart would be associated mammography cohort ranking DASH diet incidence of heart
failure.” Arch Intern with a lower incidence • Diet measured using components had a 37% failure. (Levitan et al.
Med 2009a 169(9): of heart failure. FFQs and used lower rate of HF. 2009a)
851–857. DASH score to
assess consistency
with DASH diet;
also recorded rates
of HF-associated
hospitalization or
death.
(Continued)
441
442

Fung TT et al. To assess the association Diet assessed seven 88,517 women aged • Nonfatal myocardial Higher DASH scores Adherence to the
“Adherence to a between a DASH-style times during 24 years of 34 to 59 years infarction. were associated with DASH-style diet is
DASH-style diet and diet score and risk of follow-up with FFQs; with no history of • CHD death. lower risks of CHD, associated with a lower
risk of coronary heart coronary heart disease given DASH score; CVD or diabetes • Stroke. nonfatal myocardial risk of CHD and stroke
disease and stroke in (CHD) and stroke in recorded numbers of before 1980. infarction, fatal CHD, among middle-aged
women.” Arch Intern women. confirmed incident stroke, and lower plasma women during
Med 2008 April 14; cases of nonfatal levels of C-reactive 24 years of follow-up.
168(7): 713–720. myocardial infarction, protein and interleukin. (Fung et al. 2008)
CHD death, and stroke.
Harmon BE et al. To assess the ability of Prospective cohort: 156,804 adults • All-cause mortality. High DASH scores were Consuming a dietary
“Associations of key 4 diet-quality • Subjects completed 70,170 men: • CVD mortality. inversely associated pattern that has a high
diet-quality indexes Indexes: Healthy Eating a quantitative FFQ. • White (17,330). • Cancer mortality. with the risk of mortality diet-quality index
with mortality in the Index-2010 (HEI- • Scores for each • African American from all causes, CVD, score is associated
Multiethnic Cohort: 2010), Alternative dietary index were (9,014). and cancer in both men with lower risk of
The Dietary Patterns HEI-2010 (AHEI- computed. • Native Hawaiian and women. mortality from all
Methods Project.” Am 2010), alternate • Mortality was (4,992). causes, CVD, and
J Clin Nutr 2015 Mediterranean diet documented over • Japanese cancer in men and
March; 101(3): score 13–18 years of American women. (Harmon
587–597. (aMED), and the follow-up. (21,239). et al. 2015)
Dietary Approaches to • Latino (17,595).
Stop Hypertension 86,634 women:
(DASH) to predict the • White (20,653).
reduction in risk of • African American
mortality from all (16,072).
causes, cardiovascular • Native Hawaiian
disease (CVD), and (6,368).
cancer. • Japanese
American
(24,785).
• Latina (18,756).
(Continued)
The DASH Diet
Taylor EN et al. To examine the impact Used 14–18 years of 45,821 men. • Impact on kidney For participants in the There was a 45%
“DASH-style diet of the DASH diet on follow-up; constructed 94,108 older stone formation. highest compared with reduction in the risk of
associates with kidney stone a DASH score and women. the lowest quintile of kidney stones in men
reduced risk for formation. recorded incidents of 101,837 younger DASH score, the and 52% reduction in
kidney stones.” J Am kidney stones. women. multivariate relative women who were
Soc Nephrol October; risks for kidney stones consistent with the
20(10): 2253–2259. were 0.55 (95% CI, DASH diet. (Taylor
0.46–0.65) for men, et al. 2009)
0.58 (95% CI,
0.49–0.68) for older
women, and 0.60 (95%
CI, 0.52–0.70) for
younger women.
Shenoy SF et al. To evaluate the effects Prospective 12-week, 81 participants with • Weight loss in Those consuming juice Participants in a
“Weight loss in of a ready-to-serve 3-group parallel-arm metabolic individuals with lost more weight, program to promote
individuals with vegetable juice as part randomized controlled syndrome: metabolic syndrome. consumed more weight loss in people
metabolic syndrome of a calorie-appropriate trial 22 men. vitamin C, potassium, with metabolic
given DASH diet Dietary Approaches to • Participants assigned 59 women. and dietary vegetables syndrome lost more
counseling when Stop Hypertension to one of three than individuals who weight by adding
provided a low sodium (DASH) diet in an groups—0, 8, or 16 were in the group that low-sodium vegetable
vegetable juice: A ethnically diverse fl oz of low sodium only received diet juice, as a component
randomized controlled population of people vegetable juice; counseling (p < 0.05). of the DASH diet.
trial.” J. Nutr 2010 with metabolic educated on the (Shenoy et al. 2010)
February 23; 9: 8. syndrome on weight DASH diet and
loss and their ability to limited calorie
meet vegetable intake intake.
recommendations, and
on their clinical
characteristics of
metabolic syndrome.
(Continued)
443
444

Fung TT et al. To examine the • Obtained number of 86,621 women. A diet high in fruits and DASH diet is
“Low-carbohydrate association between incidences of breast vegetables, such as one associated with a
diets, dietary the DASH diet score, cancer, DASH diet represented by the lower risk of
approaches to stop overall, animal-based, score, FFQs. Dietary Approaches to developing Estrogen
hypertension-style and vegetable-based Stop Hypertension diet Receptor negative
diets, and the risk of low-carbohydrate-diet score, was associated breast cancer. In
postmenopausal breast scores, and major plant with a lower risk of particular, the
cancer.” Am J food groups and the ER-breast cancer. In increased intake of
Epidemiol 2011 risk of postmenopausal addition, a diet high in fruits and vegetables
September 15; 174(6): breast cancer. plant protein and fat associated with the
652–660. and moderate in DASH diet appeared
carbohydrate content to be beneficial. (Fung
was associated with a et al. 2011)
lower risk of
ER-cancer.
Fung TT et al. “The To prospectively assess • Followed participants 87,256 women and People following the
Mediterranean and the association for up to 26 years, 45,490 men DASH diet were less
Dietary Approaches to between the alternate calculated aMed, without a history likely to develop
Stop Hypertension Mediterranean diet DASH scores, and of cancer. colorectal cancer.
(DASH) diets and (aMed) and the colorectal cancer Following the
colorectal cancer.” Am DASH-style diet scores relative risks for each Mediterranean diet
J Clin Nutr 2010 and risk of colorectal participant that was did not show similar
December; 92(6): cancer in middle-aged assessed up to 7 times benefits. (Fung et al.
1429–1435. men and women. during follow-up. 2010)
The DASH Diet 445
has been tested by Roussell et al. (2012). They studied the effects on lipids, lipoproteins, and apo-
lipoproteins in a heart-healthy diet that contained lean beef compared to a DASH dietary pattern.
While this study investigated lipids and not blood pressure, the result was that incorporation of lean
beef in a low-saturated-fat, heart-healthy dietary pattern that mimicked the DASH macronutrient
targets elicited favorable cardiovascular effects comparable to DASH.
Other researchers studied a modification of the DASH diet when they substituted lean pork for
the poultry and fish recommendations of the DASH diet (Sayer et al. 2015). In assessing blood pres-
sure responses, DASH diets containing pork compared to those containing chicken and fish equally
reduced all measures of blood pressures. Their results suggest that lean pork within the DASH-style
diet is effective for blood pressure reduction.
CONCLUSIONS
From the material presented in this chapter, it is clear that a lifestyle that includes the DASH
eating plan is a good one. The DASH eating plan, developed from the findings of the DASH feed-
ing trials, is based on solid scientific evidence and has documented health benefits. The Dietary
Guidelines for Americans 2010 endorsed the DASH eating plan in the development of a healthy
eating pattern, indicating that “the USDA Food Patterns and the DASH Eating Plan apply these
Dietary Guidelines recommendations and provide flexible templates for making healthy choices
within and among various food groups.” The 2015 Dietary Guidelines have also described the
DASH dietary pattern as having many of the same characteristics as the Healthy U.S.-Style
Eating Pattern (USDA Dietary Guidelines 2015).
In January 2011, U.S. News and World Report began a column in their Health Section on the U.S.
News Best Diets. By convening a panel of diet and nutrition experts, this media entity unveils rank-
ings of many eating plans and/or diets. Since the launch in 2011, the DASH diet has now been ranked
Number 1 as the best overall diet every year this column has been published. Of the 38 diets evaluated in
2016, the DASH diet maintains the Number 1 position for the sixth consecutive year, truly an example
of science that has health benefits for Americans and perhaps other population groups.
ACKNOWLEDGMENTS
George Bray and the DASH, DASH-Sodium, PREMIER, and Weight Loss Maintenance Investigative
Teams.
No conflicts of interest to report.
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24 Nut Consumption and
Coronary Heart Disease
(CHD) Risk and Mortality
Christina Link, Alyssa Tindall, Jordi Salas-Salvadó,
Caitlin Lynch, and Penny Kris-Etherton
CONTENTS
Introduction.....................................................................................................................................450
Early Epidemiological Associations...............................................................................................450
More Recent Epidemiological Studies............................................................................................451
Nut Consumption and Risk of Type 2 Diabetes..............................................................................455
Nut Consumption and Metabolic Syndrome Risk..........................................................................456
Clinical Trials on Nut Consumption and Coronary Heart Disease.................................................457
CVD Events...............................................................................................................................457
Major CVD Risk Factors...........................................................................................................458
Other CVD Risk Factors............................................................................................................459
Lipoprotein Particles.............................................................................................................459
Lipoprotein Function.............................................................................................................466
Oxidation and Inflammation..................................................................................................467
Vascular Health.....................................................................................................................468
Insulin Resistance and Glycemic Response..........................................................................470
Body Weight..........................................................................................................................470
Summary.........................................................................................................................................472
References.......................................................................................................................................473
ABSTRACT
The purpose of this chapter is to review the evidence about the role of nuts in cardiovascular disease
(CVD) risk reduction. CVD risk reduction has been the target of dietary intervention for decades as
a result of the disease burden. Approximately 25% of all deaths in the United States are from heart
disease (CHD) each year, ranking CVD the number one cause of death, both in the United States and
worldwide. Epidemiological and clinical studies have been conducted that demonstrate the benefits
of nuts on CVD morbidity and mortality. In addition, there is impressive evidence demonstrating
beneficial effects of nuts on CVD risk factors.
Several landmark epidemiological studies, including the Adventist Study and The Nurse’s Health
Study, have reported beneficial associations between nut consumption and CVD risk, incidence, and
mortality with different populations. One seminal randomized, controlled trial (RCT), PREDIMED,
has also shown benefits of nut consumption on CVD risk, events, and mortality. This study, along
with many others, has reported favorable changes in blood lipids, inflammation, and other markers
of CVD with nut consumption.
The studies discussed in this chapter have added to the evidence base that informs Dietary
Guidelines. The 2015–2020 Dietary Guidelines recommend a healthful dietary pattern that includes
nuts, for health promotion, as well as the prevention of chronic diseases, including CVD.
449
INTRODUCTION
Cardiovascular disease (CVD) is the leading cause of death in both the United States and world-
wide. Approximately one in four Americans die from coronary heart disease (CHD) every year
(CDC, 2015). Moreover, a systematic analysis of descriptive epidemiology of 291 diseases in the
United States reported that ischemic heart disease (IHD) was one of the most prevalent diseases
with the largest number of years of life lost due to premature mortality (U.S. Burden of Disease
Collaborators, 2013). This report also found that dietary risks are the leading cause of prevent-
able mortality (U.S. Burden of Disease Collaborators, 2013). Authors of the 2010 Global Burden
of Disease Study used meta-regression to estimate the pooled effect of fruits, vegetables, nuts and
seeds, whole grains, fish, and dietary fiber on systolic blood pressure and LDL cholesterol, based on
controlled feeding studies (six treatment groups from three studies for blood pressure and six treat-
ment groups from two studies for cholesterol), and reported low nut and seed consumption was the
leading dietary risk factor attributable to IHD (Lim et al., 2012).
A healthy diet is the primary strategy for the prevention and treatment of CVD. The 2015–2020
Dietary Guidelines for Americans recommend a healthy dietary pattern that includes a variety of veg-
etables from all of the subgroups—dark green, red and orange, legumes (beans and peas), starchy, and
others; fruits, especially whole fruits; grains, at least half of which are whole grains; fat-free or low-fat
dairy, including milk, yogurt, cheese, and/or fortified soy beverages; a variety of protein foods, including
seafood, lean meats and poultry, eggs, legumes, and nuts, seeds, and soy products; and liquid vegetable
oils (http://health.gov/dietaryguidelines/2015/guidelines/). In addition, a healthy eating pattern limits
saturated fats, trans fats, added sugars, and sodium. The recommendation for nuts in the Healthy U.S.
Eating Pattern is 5 oz/week for a 2000-calorie diet. Other organizations also have issued dietary recom-
mendations for nuts (Lloyd-Jones et al., 2010; Sacco, 2011; Vannice and Rasmussen, 2014). The purpose
of this chapter is, thus, to review the evidence base in support of dietary recommendations for nuts and
the benefits they have on cardiometabolic health.
EARLY EPIDEMIOLOGICAL ASSOCIATIONS

Epidemiological evidence has demonstrated a consistent beneficial association between the con-
sumption of nuts and CHD risk and mortality (Albert et al., 2002; Fraser et al., 1992, 1995; Hu et al.,
1998). The Adventist Health Study, a landmark study, was the first to investigate nut consumption
and CHD risk in 31,208 healthy individuals (Fraser et al., 1992). In this study, there were fewer fatal
CHD events [relative risk (RR) = 0.52 (95% confidence interval [CI]: 0.36–0.76; P < 0.0001)] and
fewer nonfatal myocardial infarctions [RR = 0.49 (95% CI: 0.28–0.85; P < 0.005)] in individuals
who consumed nuts (type of nut undefined) ≥5 times/week (categories ranged from “never consume”
to “consume more than 1 oz/day”) compared to those who consumed nuts <1 time/week. Data from
this study were used to assess lifetime risk of developing CHD and a first coronary event in response
to nut consumption. Individuals who ate nuts ≥5 servings/week had a 12% lifetime reduction in risk
of developing CHD (P = 0.05) compared to those who rarely consumed nuts (Fraser et al., 1995).
In addition, men who developed the disease had a prolonged onset of CHD, approximately 5–6
years later, in comparison to men who rarely consumed nuts (Fraser et al., 1995). The Adventist
Health Study subgroups, oldest-old adults (≥84 years; n = 603) and blacks (n = 3229), were also
evaluated and similar reductions in the risk for CHD mortality and all-cause mortality were reported
(Fraser et al., 1997; Fraser and Shavlikm, 1997). Oldest-old adults who consumed nuts ≥5 times/
week had a RR of 0.82 (95% CI: 0.70–0.96; P < 0.01) for death and RR of 0.61 (95% CI: 0.45–0.83;
P < 0.001) for death from CHD compared with those consuming nuts <1 time/week (Fraser and
Shavlikm, 1997). Similarly, a decreased hazard ratio (HR) for death among black participants con-
suming nuts ≥5 times/week versus <1 time/week [HR = 0.6 (95% CI: 0.3–1.0)] has been observed
(Fraser et al., 1997). The Iowa Women’s Health Study examined the frequency of nut intake (type of
nut undefined) and CHD death in a population of healthy postmenopausal women (n = 34,486) over a
Nut Consumption and Coronary Heart Disease (CHD) Risk and Mortality 451
7-year period. Women in the highest nut consumption quartile (>4 servings/week, serving size unde-
fined) had a 40% reduction in risk of fatal CHD [RR = 0.60 (95% CI: 0.36–1.01; P = 0.016)] com-
pared to those who did not consume nuts (Kushi et al., 1996). The Nurses’ Health Study (NHS) was
another large, prospective study (n = 86,016) that evaluated the relationship between nut consumption
and CHD in middle-aged women (Hu et al., 1998). After a 14-year follow-up, a 39% decrease in the
risk of fatal CHD [RR = 0.61 (95% CI: 0.35–1.05; P = 0.007)] was observed in women consuming
≥5 servings nuts/week (1 serving = 1 oz nuts) compared to those consuming <1 serving nuts/month
(type of nut not defined) (Hu et al., 1998). In 2002, Albert and colleagues evaluated nut consumption
and the risk of CHD in 22,071 males enrolled in the Physicians’ Health Study (PHS) (Albert et al.,
2002). After a 17-year follow-up, men who consumed nuts (type of nut undefined) ≥2 times/week (1
serving = 1 oz nuts) had a reduced risk of total CHD death [RR = 0.53 (95% CI: 0.30–0.92; P = 0.01)]
compared to men who rarely or never consumed nuts. The reduction in CHD observed in this study
was primarily attributable to a reduction in sudden cardiac death. Relative risk of sudden cardiac
death was 47% lower in men who consumed nuts ≥2 times/week [RR = 0.53 (95% CI: 0.30–0.92;
P = 0.01]) (Albert et al., 2002). Furthermore, the reduced risk of CHD was independent of age, body
mass index, alcohol use, or presence of other CVD risk factors.
A pooled analysis of these four large U.S. studies demonstrated a 35% reduced risk of CHD inci-
dence for the group with the highest nut intake of ≥5 times/week [RR = 0.65 (95% CI: 0.47–0.89)]
(Kris-Etherton et al., 2008). The nonfatal CHD RR for persons consuming nuts ≥5 times/week was
0.68 (95% CI: 0.47–1.00). These studies demonstrate a beneficial relationship between frequency
of nut consumption and CHD risk, resulting in an 8.3% reduction in risk of CHD for each serving
weekly of nuts consumed (Kelly and Sabate, 2006). Collectively, the results from these studies con-
sistently demonstrate beneficial associations between nut consumption on CHD (Table 24.1 presents
an overview of the studies discussed).
MORE RECENT EPIDEMIOLOGICAL STUDIES

The association between nut consumption and the risk of CVD and all-cause mortality remains an
active area of research. A study published in 2015 assessed the association between nut consumption
and risk of 15-year total and CVD mortality in 2893 adults (Gopinath et al., 2015). Participants in
the second tertile for nut consumption (0.9–4.55 g nuts/day) versus those in the first tertile (0.00–0.5
g nuts/day) had a 24% reduced risk of total mortality (P-trend = 0.81), with reduced risk of
CVD [HR = 0.76 (95% CI: 0.61–0.94); P-trend = 0.43], and IHD mortality [HR = 0.77 (95% CI:
0.60–0.98); P-trend = 0.76] (Gopinath et al., 2015). Eslamparast et al. (2017) evaluated nut con-
sumption and all-cause and cause-specific mortality in 50,045 adults enrolled in the Golestan cohort.
The majority of prospective cohort studies have been conducted predominantly in Western coun-
tries, making this particular cohort unique because of the geographic location in northeastern Iran.
Researchers observed similar results in the Golestan cohort as those seen in Western cohorts. After
a 7-year period, the pooled multivariate-adjusted HRs for death among those who ate nuts, compared to
those who did not, were 0.89 (95% CI: 0.82–0.95) for consumption of <1 serving/week (1 serving = 28 g),
0.75 (95% CI: 0.67–0.85) for 1 to <3 servings/week, and 0.71 (95% CI: 0.58–0.86) for ≥3 servings/week.
This study provides further evidence of benefits of nut consumption and mortality in a cohort that differs
from others previously studied (Eslamparast et al., 2017).
Several recent epidemiological studies have explored nut consumption and CVD risk in popu-
lations that vary in socioeconomic status (SES). In a secondary analysis using data from the
NHS (a more affluent cohort), researchers assessed nut consumption and incidence of CVD in
6309 women with type 2 diabetes (Li et al., 2009). The authors reported that women who con-
sumed 5 servings/week of tree nuts or peanut butter [serving size = 28 g (1 oz) for nuts and 16 g
(1 tablespoon) for peanut butter] had a 44% lower risk [RR = 0.56 (95% CI: 0.36–0.89; P = 0.44)]
compared to women who rarely or never consumed tree nuts or peanut butter. In that study, a strong
inverse association with CVD in women who consumed ≥5 servings/week of nuts and peanut butter
452
TABLE 24.1
A Review of Observational Studies on Nut Consumption and Risk of Cardiometabolic Diseases
Follow-Up
Author/Year Study Population Nut Type (Years) Main Results
Fraser et al. (1992) The Adventist 31,208 healthy men Nuts (undefined) 6 RR = 0.52 (95% CI: 0.37–0.76; P < 0.05) for nonfatal and 0.49
Health Study and women (95% CI: 0.28–0.85; P = 0.005)
Comparison: >5 times/week compared to <1 time/week
Fraser et al. (1995) The Adventist 27,321 healthy men Nut (undefined) 6 12% lifetime reduction in developing CHD (P = 0.05)
Health Study and women Comparison: >5 servings/week compared to rare consumption
Kushi et al. (1996) Iowa Women’s 34,486 healthy Nuts (undefined) 7 RR = 0.60 (95% CI: 0.36–1.01; P = 0.016) of fatal CHD
Health Study postmenopausal Comparison: >4 times/week compared to 0 times/week
woman
Fraser et al. (1997) The Adventist 3,229 black California Nuts (undefined) 7–11 HR = 0.6 (95% CI: 0.3–1.0) for nut consumption for death
Health Study adults Comparison: >5 times/week compared to <1 time/week
Fraser and The Adventist 603 adults ≥84 years Nuts (undefined) 12 RR = 0.82 (95% CI, 0.70-0.96; P < 0.01) for death and RR =
Shavlikm (1997) Health Study 0.61 (95% CI, 0.45–0.83; P < 0.001) for death from CHD
Hu et al. (1998) Nurses’ Health 86,016 healthy 1. Nuts (undefined) 14 RR = 0.61 (95% CI: 0.35–1.05; P = 0.007) for CVD risk
Study middle-aged women 2. Peanuts Comparison: ≥5 servings/week of compared to <1 serving
RR = 0.92 (95% CI: 0.74–1.15; P = 0.094) for CHD risk
RR = 0.76 (95% CI: 0.51–1.15; P = 0.09) for fatal CHD
Comparison: ≥5 times/week compared to almost never
Jiang et al. (2002) Nurses’ Health 83,818 women with Nuts (undefined) 16 Nuts: RR = 0.73 (95% CI: 0.60–0.89; P = 0.001) for if type 2
Study type 2 diabetes Peanut butter diabetes
Comparison: ≥5 servings/week compared to never/almost
never
Peanut butter: RR = 0.79 (95% CI: 0.68–0.91; P < 0.001) for
risk of diabetes
Comparison: ≥5 servings/week compared to never
Albert et al. (2002) Physicians’ Health 22,071 healthy men Nuts (undefined) 17 RR = 0.53 (95% CI: 0.30–0.92; P = 0.01) for fatal and 47%
Study decrease in sudden death
Comparison: ≥2 servings/week compared to never or <1 time/
month
(Continued)
Follow-Up
Kochar et al. (2010) Physicians’ Health 20,224 healthy men Nuts (undefined) 1 No association for risk of diabetes. HR = 0.87 (95% CI:
Study 0.61–1.24; P = 0.99)
Lutsey et al. (2008) Atherosclerotic Risk 9,514 white and black Nuts (undefined) 9 No association for nut consumption and MetS RR = 0.99
in Community men and women (95% CI: 0.91–1.08; P = 0.52)
Study Comparison: Highest to lowest quintile
Li et al. (2009) Nurses’ Health 6,309 women with Tree nuts (undefined) 22 RR = 0.56 (95% CI: 0.36–0.89; P = 0.44) for CVD risk
Study II type 2 diabetes Peanuts Comparison: ≥5 servings/week compared to rarely or never
Peanut butter consumed
O’Neil et al. (2011) NHANES data 13,292 Nuts: peanuts, peanut butter, 6 Percentage of prevalence in MetS: (21.2% ± 2.1% vs.
1999–2004 tree nuts (i.e., almonds, Brazil 26.6% ± 0.7%; P < 0.05)
nuts, cashews, hazelnuts, Comparison: Consumers (≥1/4 oz/day compared to
macadamia nut, pecans, <1/4 oz/day
pistachios, walnuts, and pine
nuts), and tree nut butters
Bao et al. (2013) Nurses’ Health Study 76,464 healthy women Tree Nuts (undefined) 30 HR = 0.86 (95% CI: 0.82–0.89) for total nuts for cause death
Health Professionals 42,498 male peanuts HR = 0.88 (95% CI: 0.84–0.93) for peanuts for all-cause death
Follow-Up Study professionals HR = 0.83 (95% CI: 0.79–0.88) for tree nuts for all-cause death
Comparison: ≥7 servings/week compared to never consumed
Fernández-Montero Seguimiento 9,887 young men and Nuts (undefined) 6 32% lower risk of MetS
et al. (2013) Universidad de women Comparison: ≥2 servings/week compared to those who never/
Nut Consumption and Coronary Heart Disease (CHD) Risk and Mortality
Navarra (the SUN rarely consumed

study)
Ibarrola-Jurado PREDIMED 7,210 men and women Nuts (undefined) N/A OR = 0.75 (95%CI: 0.65–0.85; P < 0.001) for prevalence of MetS
et al. (2013) (cross-sectional) at high CVD risk Comparison: >3 servings/week compared to <1 serving/week
Pan et al. (2013) Nurses’ Health Study 58,063 healthy women Walnuts 10 HR = 0.76 (0.62–0.94; P = 0.002) for risk of type 2 diabetes
Nurses’ Health 79,893 healthy women Comparison: ≥2 servings/week compared to reference (HR =
Study II 1.00) never/rarely consumed
(Continued)
453
454

Follow-Up
Jaceldo-Siegl et al. Adventist Health 803 adults Total nuts, tree nuts, and N/A OR = (95% CI) were 0.77 (0.47, 1.28) for low tree nut/high
(2014) Study-2 peanuts peanut, 0.65 (0.42, 1.00) for high tree nut/high peanut and 0.68
(cross-sectional) (0.43, 1.07) for high tree nut/low peanut consumers, compared
to tree nut/low peanut consumers (P for trend = 0.056)
O’Neil et al. (2015) NHANES Data 14,386 adults ≥19 Tree nuts (i.e., almonds, Brazil 6 Associated with lower BMI, waist circumference, systolic
(2005–2010) years nuts, cashews, hazelnuts, blood pressure, HOMA-IR, and higher HDL
macadamia nuts, pecans, pine Comparison: Nut consumers (≥1/4 oz/day) and nonconsumers
nuts, pistachios, and walnuts) (<1/4 oz/day)
Gopinath et al. The Blue Mountains 2,893 participants ≥49 Nuts (undefined) 15 HR = 0.76 (95%CI: 0.65–0.89) for nut consumption for total
(2015) Eye Study (BMES) years mortality
Comparison: Second tertile and first tertile of nut intake
Luu et al. (2015) Southern Community 71,764 U.S. Peanuts and other nuts 5.4 HR = 0.62 (95% CI: 0.45–0.85; P = 0.01) in African descent
Cohort Study individuals with (undefined) 6.5 for ischemic heart disease
Shanghai Women’s African and European 12.2 HR = 0.60 (95% CI: 0.39–0.92; P = 0.007) in European descent
Health Study and descent, low SES for ischemic heart disease
Shanghai Men’s 134,265 men and HR = 0.70 (95% CI: 0.54–0.89; P = 0.001) in Asian descent for
Health Study women of Asian ischemic heart disease
descent Comparison: Highest and lowest quartile of nut intake
Eslamparast et al. Golestan cohort 50,045 participants Nuts (undefined) 7 HR = 0.89 (95% CI: 0.82–0.95) for nut consumption for death
(2017) study ≥40 years Comparison: Did not consume nuts and <1 serving/week
HR = 0.75 (95% CI: 0.67–0.85) for nut consumption for death
Comparison: Did not consume nuts and 1 to <3 servings/week
HR = 0.71 (95% CI: 0.58–0.86) for nut consumption for death
Comparison: Did not consume nuts and ≥3 servings/week
compared to women who consumed less was observed. The multivariate-adjusted analysis for an
incremental increase of 1 serving/day for nuts and peanut butter was associated with a significantly
lower CVD risk. Authors reported improvements in LDL-C (−6.56 mg/dL, P = 0.008), non-HDL-C
(−6.95 mg/dL, P = 0.014), TC (−7.34 mg/dL, P = 0.007), and ApoB (b coefficients) (−4 mg/dL,
P = 0.016) (Li et al., 2009).
Bao et al. (2013) investigated the relationship between nut consumption and total and cause-
specific mortality using data from the NHS (76,464 women) and the Health Professionals Follow-up
Study (HPFS) (42,498 men) (another more affluent cohort). Results demonstrated that participants
who consumed ≥7 servings/week of nuts (peanuts and tree nuts) had a 20% lower rate of mortality
compared to those who did not consume nuts (Bao et al., 2013). The pooled multivariate-adjusted
HRs for death from all causes were 0.86 (95% CI: 0.82–0.89) for total nuts, 0.88 (95% CI: 0.84–0.93)
for peanuts, and 0.83 (95% CI: 0.79 to 0.88) for tree nuts (Bao et al., 2013).
Recent studies targeted populations of lower SES, since most epidemiological studies on nut
consumption and CHD risk have been conducted primarily with U.S. populations of European
ancestry with a higher SES. Luu et al. (2015) examined nut consumption in Americans of
African and European descent that were predominantly of low SES and in Chinese individu-
als in Shanghai, China. The study showed an inverse association between nut intake and CVD
mortality for all three ethnic groups (all P < 0.001). The HRs for IHD were 0.62 (95% CI:
0.045–0.85; P = 0.01) in those of African descent; 0.60 (95% CI: 0.39–0.92; P = 0.007) in those
of European descent; and 0.70 (95% CI: 0.54–0.89; P = 0.001) in those of Asian descent for
the highest versus lowest quintile of intake, respectively. The associations for ischemic stroke
(HR = 0.77; 95% CI: 0.60–1.00 for the highest vs. lowest quintile of nut intake) and hemor-
rhagic stroke (HR = 0.77; 95% CI: 0.60–0.99 for the highest vs. lowest quintile of nut intake)
were significant only in Asians (P = 0.003 for both). The association between the frequency of
nut consumption and all-cause mortality and CVD mortality has also recently been observed in
the Netherlands Cohort Study, analyzing 120,852 men and women aged 55–69 years (van den
Brant and Schouten, 2015). In this study, peanuts and tree nuts were inversely related to mortal-
ity, whereas peanut butter was not.
Finally, a recent systematic review and meta-analysis including 20 prospective cohort studies and
467,389 participants has demonstrated that the frequency of nut consumption was significantly associ-
ated with a lower risk of all-cause mortality (10 studies; Risk Ratio [RR] = 0.81; 95% CI: 0.77–0.85 for
highest vs. lower quintile of intake), CVD mortality (5 studies; RR 0.73; 95% CI: 0.68–0.78), all CHD
(3 studies; RR 0.66; 95% CI: 0.48–0.91) and CHD mortality (7 studies; RR 0.70; 95% CI: 0.64–0.76),
as well as a statistically nonsignificant reduction in the risk of nonfatal CHD (3 studies; RR 0.71; 95%
CI: 0.49–1.03) and stroke mortality (3 studies; RR 0.83; 95% CI: 0.69–1.00) (Mayhew et al., 2016).
However, no evidence of association was found for total stroke. These findings demonstrate that nut
consumption is consistently associated with a decrease in all-cause mortality, total CVD and CVD mor-
tality, CHD and CHD mortality and sudden cardiac death in different ethnic groups, as well as ischemic
stroke in a Chinese cohort. Collectively, recent as well as earlier epidemiological studies demonstrate
that frequent nut consumption is associated with lower CVD risk on a population-wide basis. It is
important to note that epidemiological studies, and all studies to some extent, have limitations. Several
of the studies reviewed report associations between the frequency of nut consumption, rather than the
quantity of nuts consumed. However, the associations reported between frequent nut consumption and
reduced CVD risk have provided support for the clinical studies that have been conducted to better
understand cause-and-effect relationships (Covered in the next section).
NUT CONSUMPTION AND RISK OF TYPE 2 DIABETES

Jiang et al. (2002) analyzed data from the NHS to evaluate the relationship between tree nut and
peanut consumption on risk of type 2 diabetes. The RR ratios across the categories of nut consump-
tion (never/almost never, <once/week, 1–4 times/week, ≥5 times/week) were 1.0 (reference), 0.92
(95% CI: 0.85–1.00), 0.84 (95% CI: 0.76–0.93), and 0.73 (95% CI: 0.60–0.89) (P < 0.001), respec-
tively (Jiang et al., 2002). The authors also examined the relationship between peanut butter consump-
tion and diabetes risk. When comparing women who consumed peanut butter at least 5 times/week in a
multivariate analysis, the RR for diabetes for a 28 g serving was 0.79 (95% CI: 0.68–0.91 [P < 0.001])
compared to those who did not eat peanut butter (Jiang et al., 2002). However, subsequent studies
have reported conflicting results. For example, results from the Iowa Women’s Health Study indicated
that postmenopausal women who consumed nuts often (≥5 times/week) did not have a reduced risk
of diabetes compared to those who consumed nuts occasionally (never/almost never), after adjust-
ing for multiple confounding factors. Results, however, indicated an 18% reduction in RR between
highest and lowest categories of peanut butter consumption (Parker et al., 2003; Parker and Folsom,
2003). By contrast, in the Shanghai Women’s Health Study, an inverse association was demonstrated
between the frequency of peanut consumption and type 2 diabetes risk incidence. The PHS did not find
a significant relationship between nut consumption and incidence of type 2 diabetes (Kochar et al.,
2010). The multivariable adjusted HRs were 1.0 (reference), 1.06 (95% CI: 0.93–1.20), 1.10 (95%
CI: 0.95–1.26), 0.97 (95% CI: 0.82–1.14), 0.99 (95% CI: 0.76–1.30), and 0.87 (95% CI: 0.61–1.24)
from the lowest to the highest category of nut consumption, respectively (P-trend = 0.99) (Kochar
et al., 2010). Pan et al. (2013) studied the relationship of walnut intake and risk of type 2 diabetes
using the original NHS I & II database. Walnut consumption lowered the risk of type 2 diabetes (Pan
et al., 2013). Specifically, the HRs for individuals consuming 1–3 servings/month (1 serving = 28 g),
1 serving/week, and ≥2 servings/week of walnuts were 0.93 (95% CI: 0.88–0.99), 0.81 (95% CI:
0.70–0.94), and 0.67 (95% CI: 0.54–0.82) compared with women who never/rarely consumed wal-
nuts (P-trend < 0.001).
In a report published using data from the PREDIMED cohort (n = 3541 participants without
diabetes at baseline), 273 cases of new-onset diabetes occurred after 4.1 years follow-up: 80 in
the Mediterranean diet supplemented with extra virgin olive oil (MeDiet + EVOO) group, 92 in
the Mediterranean diet supplemented with nuts (MeDiet + nuts) group, and 101 in the control
group. After multivariable adjustment, hazard ratios for diabetes were 0.60 (95% CI: 0.43, 0.85)
for MeDiet + EVOO and 0.82 (95% CI: 0.61, 1.10) for MeDiet + nuts compared with the control
group (Martinez-Gonzalez et al., 2015).
Collectively, these studies suggest that consumption of nuts may elicit benefits on risk of diabe-
tes. The discrepancies among the studies conducted to date warrant further investigation (Table 24.1
summarizes the epidemiological studies reviewed).
NUT CONSUMPTION AND METABOLIC SYNDROME RISK

Few studies have evaluated tree nut and peanut consumption and the risk of Metabolic Syndrome
(MetS). The Atherosclerosis Risk in Community Study prospectively analyzed data from over 9500
healthy participants aged 45–64 years of white and black race (Lutsey et al., 2008) and reported no
association between nut intake and risk of MetS (RR = 0.99; 95% CI: 0.91–1.08; P = 0.52) (Lutsey
et al., 2008). Other studies have observed different outcomes when using individual diagnostic crite-
ria for MetS. O’Neil et al. (2011) evaluated the association of risk factors of MetS with consumption
of all types of nuts. Nuts were defined as peanuts, peanut butter, tree nuts (i.e., almonds, Brazil nuts,
cashews, hazelnuts, macadamias, pecans, pistachios, walnuts, and pine nuts), or tree nut butters.
Nut consumption was defined as intakes of at least ¼ oz (7.09 g) of nuts/day, while nonconsumers
were defined by an intake of <¼ oz/day (O’Neil et al., 2011). Nut consumption was associated with
benefits for the following MetS criteria: lower prevalence of abdominal obesity (43.6% ± 1.6% vs.
49.5% ± 0.8%; P ≤ 0.01), hypertension (31.4% ± 1.2% vs. 33.9% ± 0.8%; P ≤ 0.05), low HDL-C
(27.9% ± 1.7% vs. 34.5% ± 0.8%; P < 0.01), elevated fasting blood glucose (11.5% ± 1.4% vs.
15.0% ± 0.7%; P ≤ 0.05), and MetS (21.2% ± 2.1% vs. 26.6% ± 0.7%; P < 0.05) (O’Neil et al.,
2011). In the context of the Adventist Health Study, the frequency of tree nut consumption was also
inversely associated with metabolic syndrome prevalence (Jaceldo-Siegl et al., 2014).
The Seguimiento Universidad de Navarra (SUN) cohort study investigated nut consumption in
a Mediterranean population of university graduates (n = 9887) (Fernandez-Montero et al., 2013).
After a 6-year follow-up, researchers observed a 32% reduction in MetS risk with nut consumption
of >2 servings/week compared with those who never/almost never consumed nuts (adjusted OR =
0.68, 95% CI: 0.50–0.92; P = 0.075) (Fernandez-Montero et al., 2013). This inverse association was
stronger in women (RR = 0.29; 95% CI: 0.15–0.56; P < 0.001) and was not significant among men
(RR = 0.90; 95% CI: 0.63 = 1.29; P = 0.91), indicating that the association differed by sex. Ibarrola-
Jurado et al. (2013) reported a lower adjusted odds ratio (OR = 0.74, 0.65–0.85; P = 0.001) in
individuals consuming >3 servings/week of nuts compared to individuals consuming <1 serving/
week. Interestingly, the higher nut intake also was associated with a lower risk of abdominal obe-
sity (OR = 0.68, 95% CI: 0.60–0.79; P < 0.001) with no significant associations for hypertension,
dyslipidemia, or elevated fasting glucose (Ibarrola-Jurado et al., 2013). O’Neil et al. (2015) used
the National Health and Nutrition Examination Survey (NHANES) to investigate tree nut consump-
tion, and adiposity measures, CVD, and MetS diagnostics. The authors categorized participants
as nonconsumers (<¼ oz/day tree nuts) or consumers (>¼ oz/day tree nuts). Results showed tree
nut consumers had significantly lower BMI (27.9 ± 0.3 vs. 28.7 ± 0.1 kg/m2; P = 0.004) and waist
circumference (WC) (95.8 ± 0.7 vs. 98.1 ± 0.3 cm; P = 0.008) than nonconsumers. Systolic blood
pressure (SBP) was also lower in tree nut consumers (119.5 ± 0.8 vs. 122.1 ± 0.2 mmHg; P = 0.001).
This study demonstrates an association between tree nut consumption and better weight status in
addition to improvements in CVD and MetS risk factors (O’Neil et al., 2015). This epidemiologi-
cal study, in conjunction with others, provides further affirmation that consumption of tree nuts and
peanuts is associated with decreased risk of CVD, diabetes, and MetS. A dose–response relationship
has also been observed between nut consumption and improved diagnostic criteria for MetS.
A beneficial effect of nut supplementation on MetS status was also observed in the PREDIMED
trial (described in detail in the next section). In comparison with the control group, participants ran-
domized to either MeDiet were more likely to show reversal of MetS, with HR 1.35 (CI: 1.15–1.58)
for the MeDiet + EVOO, and HR 1.28 (CI: 1.08–1.51) for the MeDiet + nuts (Babio et al., 2014;
Salas-Salvadó et al., 2008). Of note, participants in the group supplemented with nuts showed a
significant decrease in central obesity.
The epidemiological evidence has continued to show a strong beneficial relationship between
nut intake and CVD risk in a diverse sample that is representative of the general public. However,
randomized controlled trials (RCTs) are needed to establish cause-and-effect relationships and iden-
tify the nutrients and/or bioactive compounds responsible for the beneficial associations reported for
tree nuts and peanuts and reduced risk of CVD, diabetes, and MetS.
CLINICAL TRIALS ON NUT CONSUMPTION AND CORONARY HEART DISEASE

CVD Events
The Prevención con Dieta Mediterránea (PREDIMED) trial was a major clinical nutritional inter-
vention designed to assess the efficacy of the Mediterranean diet in the primary prevention of CVD.
Participants (n = 7447) were 55–80 years of age with type 2 diabetes or with three or more major
CVD risk factors (hypertension, hypercholesterolemia, family history of heart disease, tobacco use, or
overweight/obesity) and were randomized to (1) a Mediterranean diet supplemented with extra-virgin
olive oil (1 L/week/family; 50 g/day per participant; 41.2% total fat, 9.4% SFA), (2) a Mediterranean
diet supplemented with nuts (30 g/day: 15 g walnuts, 7.5 g hazelnuts, 7.5 g almonds; 41.5% total fat;
9.3% SFA), or (3) a control diet (37.0% total fat; 9.1% SFA). The primary endpoint was rate of major
CVD events (myocardial infarction, stroke, or death from cardiovascular causes), which occurred
in 288 participants. The HRs were 0.70 (95% CI: 0.54–0.92) and 0.72 (95% CI: 0.54–0.96) for the
group supplemented with extra-virgin olive oil group (96 events) and the group supplemented with
nuts (83 events), respectively, versus the control group (109 events) (Estruch et al., 2013). Thus, a
Mediterranean diet supplemented with extra-virgin olive oil or nuts reduced the rate of major CVD
events compared with a lower-fat diet among individuals at high CVD risk.
Major CVD Risk Factors

The strong epidemiological evidence for benefits of nut consumption and chronic disease risk reduc-
tion has been the impetus for many clinical trials that have been conducted to evaluate the effects of
nuts on CHD risk factors and their underlying mechanism(s) of action. These studies have shown
beneficial effects on lipids and lipoproteins (Casas-Agustench et al., 2011a,b; Griel et al., 2007;
Nouran et al., 2010; Sheridan et al., 2007; Tey et al., 2011) and numerous other risk factors for
cardiometabolic diseases (Blanco Mejia et al., 2014; Viguiliouk et al., 2014). The first clinical trial
evaluating the effect of nut consumption on CVD risk factors was the Loma Linda University walnut
study (Sabate et al., 1993). In this study, healthy men were randomized to one of two cholesterol-
lowering diets: a diet providing 20% of energy from walnuts [total fat was 31% of energy, 6% from
saturated fatty acids (SFA), and 16% from polyunsaturated fatty acids (PUFA)] versus a Step-1
diet (total fat was 30% of energy, 10% SFA, and 10% PUFA). Total cholesterol (TC) and LDL-C
significantly decreased by 12% (P < 0.0001) and 16% (P < 0.001), respectively, from the walnut diet
versus the Step-1 diet.
Studies evaluating walnuts (Rajaram et al., 2009; Torabian et al., 2010), almonds (Berryman
et al., 2013; Jenkins et al., 2002), pistachios (Sauder et al., 2013; Sheridan et al., 2007), hazelnuts
(Mercanligil et al., 2007), peanuts (Lokko et al., 2007), and macadamia nuts (Griel et al., 2008) have
reported decreases in LDL-C ranging from 9% to 16%. A pooled analysis of 25 nut intervention
studies conducted in 7 countries with 583 men and women (normolipidemic and hypercholesterol-
emic) reported a dose–response improvement in lipids/lipoproteins with nut consumption (Sabaté
et al., 2010). The mean daily intake was 67 g of nuts (walnuts, almonds, pistachios, pecan, maca-
damia nut, and peanuts) with the following reductions achieved: TC (10.9 mg/dL [5.1% change]),
LDL-C (10.2 mg/dL [7.4% change]), LDL: HDL-C ratio (0.22 [8.3% change]), and TC: HDL-C
ratio (0.24 [5.6% change]) (P < 0.001 for all). No significant effect was reported for HDL-C or
triglycerides (TG), except in individuals with TG > 150 mg/dL, in whom there was a 20.6 mg/dL
(10.2%) decrease in TG. The effects of nut consumption were dose-related and different nuts had
similar effects on lipids/lipoproteins. The effects were greatest in those with higher LDL-C values (≥160
vs. <130 mg/dL), with a lower body mass (<25 vs. ≥25 BMI). Benefits were also greater when nuts
were part of a Western diet or lower-fat diet compared to a Mediterranean diet (Sabaté et al., 2010).
A meta-analysis of 13 clinical trials with 365 participants (healthy individuals and individuals at
high CVD risk) evaluated the effects of walnut consumption on lipids/lipoproteins and CVD risk fac-
tors (Banel and Hu, 2009). The diets were consumed for 4–24 weeks; walnuts provided 10%–24%
of total energy. Compared to the control diets, the walnut diets significantly decreased TC (−10.3
mg/dL, P < 0.001) and LDL-C (−9.2 mg/dL, P = 0.001). There was no significant effect of walnuts
on HDL-C or TG. This decrease in LDL-C (6.7%) is consistent with findings from a pooled analysis
of 25 intervention trials (Sabaté et al., 2010) in which a 7.4% decrease in LDL-C was reported with
consumption of different nuts (Sabaté et al., 2010). A recent systematic review and meta-analysis
was conducted to evaluate the dose–response effects of different nuts on lipids, lipoproteins, and
apolipoprotein B. Del Gobbo et al. (2015) evaluated 61 RCTs with 2582 participants (healthy indi-
viduals and individuals with comorbidities) who consumed different types and amounts of nuts for
3–26 weeks. Compared with control diets (habitual, American Heart Association, low-fat, high-
fat, and Mediterranean-type diets), consumption of tree nuts significantly decreased TC (weight
mean difference per 28 g/day: −4.7; 95% CI: −5.3, −4.0), LDL-C (−4.8%; 95% CI: −5.5, −4.2),
ApoB (−3.7; 95% CI: −5.2, −2.3), and TGs (−2.2; 95% CI: −3.8, −0.5). TC and LDL-C decreased
in a nonlinear fashion (P < 0.001); stronger effects were reported when consumption was ≥60 g/
day. Linear dose–response relationships were reported between nut intake and ApoB (r = −0.12) and
TG (r = −0.16). There were no significant differences among type of tree nuts. Stronger effects were
observed for ApoB in participants with type 2 diabetes (−11.5 mg/dL; 95% CI: −16.2, −6.8 mg/dL)
than in healthy individuals (−2.5 mg/dL; 95% CI: −4.7, −0.3 mg/dL) (P-heterogeneity = 0.015).
These findings demonstrate that the quantity of nuts consumed is a major determinant of the lipid/
lipoprotein effects rather than the type of nut.
A recent systematic review and meta-analysis of 12 RCTs lasting ≥3 weeks with 450 individu-
als with type 2 diabetes evaluated the effect of diets that included tree nuts (average amount was
56 g/day) compared to isocaloric control diets without nuts (i.e., high-fat, low-fat, ad libitum diet,
NCEP Step-2 diet) on HbA1c, fasting glucose, fasting insulin, and HOMA-IR (Viguiliouk et al.,
2014). The tree nut diets significantly lowered HbA1c (−0.07%; 95% CI: −0.10 to −0.03%;
P = 0.0003) and fasting glucose (−1.44 mg/dL; 95% CI: −4.86 to 0.36 mg/dL; P = 0.03) compared
to the control diets. No significant treatment effects were reported for fasting insulin and HOMA-IR
levels. Beyond this meta-analysis that evaluated the effects of nuts on endpoints related to diabetes
control, there have been few trials conducted that have reported beneficial effects of nuts on lipids
and lipoproteins in individuals with type 2 diabetes (Lovejoy et al., 2002; Sauder et al., 2015; Tapsell
et al., 2004). Of the three studies conducted, two reported decreases in TC (−6 to −9 mg/dL) after
consumption of almonds (10% of total fat or 2 oz/day) (Lovejoy et al., 2002) or pistachios
(20% of total energy) (Sauder et al., 2015). Tapsell et al. (2004) reported a 10% decrease in LDL-C
(P = 0.032) after consumption of 30 g walnuts/day incorporated into a lower-fat diet (<30% fat).
A systematic review and meta-analysis (Blanco Mejia et al., 2014) of 49 RCTs with 2226 par-
ticipants (healthy individuals or individuals diagnosed with dyslipidemia, MetS or type 2 diabetes)
assessed the effects of nut consumption on MetS criteria. A median intake of 50 g/day of nuts
(almonds, hazelnuts, cashews, walnuts, pistachio, macadamia, and mixed nuts) with a median fol-
low-up of 8 weeks improved MetS criteria with decreases in TG [(−5.30 mg/dL; 95% CI: −7.96 to
−2.56 mg/dL) with evidence of moderate heterogeneity (I2 = 34%, P = 0.02)], and fasting blood glu-
cose [(−7.08 mg/dL; 95% CI: −14.16 to −0.88) with evidence of moderate heterogeneity (I2 = 41%,
P < 0.05)] compared with the control intervention (i.e., usual, Step-1, average American diet, low-fat
diets). There was no effect on waist circumference, HDL-C, or blood pressure. In a review of six nut
intervention trials focusing on individuals with MetS (Salas-Salvado et al., 2014a,b), the authors
reported a 25.8% reduction in fasting insulin and 27.6% reduction in HOMA-IR in individuals who
received 30 g of nuts/day and advice on health compared to the control group. Wu et al. (2010) con-
ducted a study in which 283 Asian individuals with MetS were randomly assigned to one of three
treatments: (1) lifestyle counseling (LC) on the AHA guidelines, (2) LC + 30 g flaxseed/day, or
(3) LC + 30 g walnuts/day. After the 12-week intervention, prevalence of MetS decreased signifi-
cantly in all groups: −16.9% (LC only), −20.2% (LC + flaxseed), and −16.0% (LC + walnuts) (P <
0.05). In addition, there was a greater decrease in central obesity in the LC + flaxseed group (19.2%;
P = 0.008) and LC + walnuts (16.0%; P = 0.04) than in the LC-only group (6.3%) (Wu et al., 2010).
These findings suggest that nut supplementation decreases risk of MetS criteria and prevalence in
healthy individuals and individuals with MetS (Table 24.2 summarizes the studies discussed).
Other CVD Risk Factors

Lipoprotein Particles
Lipoprotein subfractions that are differentiated by particle diameter, density, and composition differ-
entially affect CVD risk (Carmena et al., 2004). While all LDL particles increase risk of CVD events,
there is evidence that small LDL particles are particularly atherogenic. The Quebec Heart Study
(Lamarche et al., 1995), Veterans Administration HDL Intervention Trial (VA-HIT) (Otvos et al.,
2006), Cardiovascular Health Study (Kuller et al., 2002), Women’s Health Study (Blake et al., 2002),
Atherosclerosis in Communities (ARIC) (Hallman et al., 2004), and Framingham Offspring Study
(Cromwell et al., 2007) have shown that higher concentrations of small, dense LDL (between 18.0
and 27.8 nmol/L) increase CVD risk. Some studies (e.g., the Strong Heart Study [Howard et al., 2000]
and Cholesterol and Recurrent Events (CARE) study [Campos et al., 2001]), however, do not agree
460
TABLE 24.2
A Review of Clinical Trials on Nut Consumption and Risk Factors for Cardiometabolic Diseases
Authors Study Design Participants Diet Design Endpoints Results
Fraser et al. Crossover Male and female Supplemented 320 kcal almonds or 0 kcal Weight Weight increased 0.40 kg (P approximately 0.09)
(2002) participants (n = 81) almonds for 6 months
Lovejoy RCT, controlled 30 individuals with Diets: Lipids/lipoproteins TC lowest in HFA diet (172.5 ± 0.14, 174.8 ±
et al. feeding type 2 diabetes 1. High-fat, high-almond (HFA; 37% 0.14, 179 ± 0.14, and 179 ± 0.14 mg/dL)
(2002) total fat, 10% from almonds) compared with HFA, HFC, LFA and LDC
2. Low-fat, high-almond (LFA; 25% (P = 0.0004)
total fat, 10% from almonds)
3. High-fat control (HFC; 37% total
fat, 10% from olive or canola oil)
4. Low-fat control (LFC; 25% total fat,
10% from olive or canola oil) for
4 weeks
Tapsell et al. RCT, given dietary 58 individuals with Diets: Lipids/lipoproteins TC: HDL-C ratio (+0.33 + 0.10 vs. 0.29 + 0.07
(2004) advice type 2 diabetes Low fat (control) and 0.26 + 0.06; P = 0.049) compared to the
Low fat/modified fat modified fat and control group, respectively.
Walnut (low fat/modified fat including LDL—10% reduction (P = 0.032)
30 g/day of walnuts) for 4 weeks
Banel and Hu Meta-analysis 365 participants 10%–24% energy of walnuts/day; diets Lipids/lipoproteins TC (−10.3 mg/dL, P < 0.001) and LDL-C
(2009) (healthy individuals lasting 4–24 weeks (−9.2 mg/dL, P = 0.001)
and individuals at high
CVD risk)
Sabaté et al. Pooled analysis 583 men and women 67 g/day of nuts (walnuts, almonds, Lipids/lipoproteins TC (−10.9 mg/dL [5.1% change])
(2010) (normolipidemic and pistachios, pecans, macadamia nut, and LDL-C (−10.2 mg/dL [7.4% change])
hypercholesterolemic) peanuts, with durations of 3–6 weeks LDL: HDL-C ratio (0.22 [8.3% change])
TC: HDL-C ratio (0.24 [5.6% change])
(P < 0.001 for all)
(Continued)
Wu et al. RCT 283 Asian individuals One of three treatments: Prevalence of MetS and Prevalence of MetS decreased significantly
(2010) with MetS 1. Lifestyle counseling (LC) on the MetS components in all groups: −16.9% (LC only), −20.2%
AHA guidelines (LC + flaxseed), and −16.0% (LC + walnuts)
2. LC + 30 g flaxseed/day (P < 0.05)
3. LC + 30 g walnuts/day for 12 weeks Reversion rate of central obesity was higher
in the LC + flaxseed group (19.2%;
P = 0.008) and LC + walnuts
(16.0%; P = 0.04) than in the LC-only
group (6.3%)
Baer et al. Randomized, Healthy adults (n = 16) Three test diets that included 0 g/day Measured energy (ME) Pistachios ME in the diet: (5.4 kcal/g);
(2012) crossover, pistachios, 42 g/day pistachios, or value 5% less than the calculated Atwater factors
controlled feeding 84 g/day pistachios for 3 weeks (5.7 kcal/g)
Foster et al. RCT Overweight and obese Supplemented two 28 g packages of Weight NFD lost more weight than AED (−7.4
(2012) individuals (n = 123) almonds per day for 18 months or compared with −5.5 kg; P = 0.04); No
nut-free diet significant differences at 18 months
Lipids and lipoproteins Significantly greater reductions in
triglycerides and TC in AED versus
NFD group at 6 months but not at
18 months
Novotny Randomized, Healthy adults (n = 18) Three diets that included 0 g/day whole Measured energy (ME) Almonds ME in the diet: 4.6 ± 0.8 kcal/g
et al. crossover, almonds, 42 g/day almonds, or 84 g/day value (equivalent to 129 kcal/28 g serving);
(2012) controlled feeding almonds for 18 days significantly less than 6.0–6.1 kcal/g
(168–170 kcal/serving), the energy
density determined by Atwater factors
(P < 0.001)
(Continued)
461
462

Zhang et al. Randomized, Healthy overweight Four test diets: 85 g of ground whole Lipoprotein function 85 g of whole increased cholesterol efflux by
(2012) controlled, participants (n = 15) walnuts, 34 g of ground de-fatted walnut 3.3% (P = 0.02) compared to the baseline
postprandial meat, 51 g of walnut oil or 5.6 g of
feeding study ground defatted walnut skins
Damasceno RCT PREDIMED cohort 1. Mediterranean diet supplemented Lipoprotein subparticles Traditional Mediterranean diet w/nuts
et al. (n = 169) with type 2 with extra-virgin olive oil (1 L/week/ increased large LDL (54 nmol/L; 95% CI:
(2013) diabetes or with 3 or family; 50 g/day per participant; 18–90) and decreased very small, dense LDL
more major CVD risk 39.1% total fat, 10.2% SFA) (−111 nmol/L: 95% CI: −180 to −42;
factors (hypertension, 2. A Mediterranean diet supplemented P = 0.017 for both values)
hypercholesterolemia, with nuts (30 g/day: 15 g walnuts, No significant differences in total LDL-C
family history of heart 7.5 g hazelnuts, 7.5 g almonds; among groups (P = 0.942)
disease, tobacco use, 37.4% total fat; 9.8% SFA)
or overweight/obesity) 3. A control diet (39.8% total fat;
10.6% SFA) for 1 year
Estruch et al. Randomized trial PREDIMED cohort 1. Mediterranean diet supplemented Rate of major CVD HR = 0.70 (95% CI: 0.54–0.92) for extra-
(2013) (n = 7447) with type 2 with extra-virgin olive oil (1 L/week/ events (MI, stroke, or virgin olive oil group (96 events) and 0.72
diabetes or with 3 or family; 50 g/day per participant; death from CVD (95% CI: 0.54–0.96) for nut group (83 events)
more major CVD risk 41.2% total fat, 9.4% SFA) causes) versus the control group (109 events)
factors (hypertension, 2. A Mediterranean diet supplemented
hypercholesterolemia, with nuts (30 g/day: 15 g walnuts,
family history of heart 7.5 g hazelnuts, 7.5 g almonds;
disease, tobacco use, 41.5% total fat; 9.3% SFA)
or overweight/obesity) 3. A control diet (37.0% total fat;
9.1% SFA) for 1 year
(Continued)
Babio et al. Randomized trial PREDIMED cohort 1. Mediterranean diet supplemented Incidence and reversion Incidence: HR = 1.10 (95% CI: 0.94–1.30,
(2014) with extra-virgin olive oil (50 g/day) of metabolic syndrome p = 0.231) for control versus extra-virgin
2. A Mediterranean diet supplemented olive oil; HR = 1.08 (95% CI: 0.92–1.27,
with nuts (30 g/day) P = 0.3) for control versus nuts
3. A control diet for 4.8-year follow-up Reversion: HR = 1.35 (95% CI: 1.15–1.58,
P < 0.001) for control versus extra-virgin
olive oil; HR = 1.28 (95% CI: 1.08–1.51,
P < 0.001) for control versus nuts
Blanco Review and 2226 participants Median nut (almonds, hazelnuts, MetS criteria TG (−0.06 mmol; 95% CI: −0.09 to −0.03
Mejia et al. meta-analysis (healthy individuals or cashews, walnuts, pistachio, mmol/L)
(2014) individuals diagnosed macadamia, and mixed nuts) dose was Fasting blood glucose (−0.08 mmol; 95% CI:
with dyslipidemia, 49.3 g/day (42–70.5 g/day). Median −0.16 to −0.01)
MetS or type 2 follow-up was 8 weeks (4–12 weeks)
diabetes) compared to control diets (usual, Step-1,
average American diet, low-fat diets)
Holligan RCT Individuals (n = 28) One of three diets: a lower-fat control Lipoprotein subclasses 10% and 20% pistachio diets reduced sdLDL
et al. with elevated LDL-C diet (control; 25% total fat and 8% levels (least square means [LSM] = 1.00
(2014) levels SFA); a diet that provided 10% of mmol/L [SE 0.03], P = 0.001) and (LSM = 0.86
energy from pistachios (32–65 g/day [SE 0.03], P = 0·03) compared to the control
[1PD]; 30% total fat and 8% SFA); a average American diet (LSM = 1.07 [SE] 0.03)
diet that provided 20% of energy from Cholesterol efflux No effect except in individuals with baseline CRP
pistachios (63–126 g/day [2PD]; 34% values < 0.2 mg/dL had significant increases in
total fat and 8% SFA) for 4 weeks ABCA1-mediated efflux and global efflux
following the 2 servings of pistachios compared
to 1 serving of pistachios (9.89% [SE 0.74] vs.
7.35% [SE 0.74], P = 0.016)
(Continued)
463
464

Salas-Salvado Review of RCTs 6 RCTs with 1,746 total 30 g of nuts/day compared to advice on Fasting insulin 25.8% reduction in fasting insulin
et al. individuals health compared to the control group HOMAR-IR 27.6% reduction in HOMAR-IR
(2014b) 8 weeks to 1 year
Salas- Randomized trial PREDIMED cohort 1. Mediterranean diet supplemented Diabetes incidence HRs = 0.60 (95% CI, 0.43–0.85) for the
Salvadó with extra-virgin olive oil Mediterranean diet supplemented with
et al. 2. A Mediterranean diet supplemented extra-virgin olive oil and 0.82 (CI, 0.61–1.10)
(2014a) with nuts for the Mediterranean diet supplemented with
3. A control diet for 4.1-year follow-up nuts versus control
Viguiliouk Systematic review 450 individuals with 56 g/day of tree nuts for 4–24 weeks HbA1c, fasting glucose, HbA1c (−0.07%; 95% CI: −0.10% to −0.03%;
et al. type 2 diabetes fasting insulin, P = 0.0003) fasting glucose (−1.44 mg/dL;
(2014) HOMA-IR 95% CI: −4.86 to 0.36 mg/dL; P = 0.03)
Berryman RCT Individuals (n = 48) Cholesterol-lowering moderate-fat diet Lipoprotein subparticles No effects for LDL1, LDL2, LDL3 or LDL4;
et al. with elevated LDL-C (26% calories from fat) with almonds Higher-fat almond diet decreased IDL1 (−0.06
(2015) (1.5 oz/day) was compared with an ± 0.33 vs. 0.76 ± 0.33 mg/dL; P = 0.01), total
isocaloric lower-fat (32%) diet with VLDL (0.15 ± 0.91 vs. 2.46 ± 0.91 mg/dL;
106 g banana muffin + 2.7 g butter for P = 0.02), VLDL3 (0.01 ± 0.49 vs. 1.18 ±
6 weeks 0.49 mg/dL; P = 0.02), and apoB (−9.7 ± 1.8
vs. −5.5 ± 1.8 mg/dL; P = 0.01)
Del Gobbo Systematic review 2,582 (healthy 28 g/day of nuts for 3–26 weeks Lipids/lipoproteins TC (−4.7; 95% CI: −5.3, −4.0)
et al. and meta-analysis individuals and LDL-C (−4.8%; 95% CI: −5.5, −4.2) ApoB
(2015) individuals with (−3.7: 95% CI: −5.2, −2.3) TGs (−2.2; 95%
comorbidity) CI: −3.8, −0.5)
(Continued)
Hernandez- RCT Individuals (n = 54) Pistachio diet (PD, 50% carbohydrates, Lipoprotein subclasses Greater decrease in small, dense LDL from the
Alonso with prediabetes 33% fat, including 57 g/day of higher-fat pistachio diet compared to the
et al. pistachios) or a control diet (55% lower-fat control diet (−28.07 nM [95%CI:
(2015) carbohydrates, 30% fat) for 4 months −60.43, 4.29] versus −16.49 nM [95% CI:
−14.19, 47.18] P = 0.023)
Sauder et al. RCT, controlled 30 individuals with Matched diets with either incorporations Lipids/lipoproteins TC (−5.8 mg)
(2015) feeding type 2 diabetes of 20% pistachios into the diet or no TC: HDL ratio (−0.31)
pistachios for 4 weeks (P < 0.05)
Baer et al. Randomized, Healthy adults (n = 18) Two test diets that included: 0 g/day Measured energy (ME) Walnuts ME in the diet: 5.22 ± 0.16 kcal/g
(2016) crossover, walnuts or 42 g/day walnuts value (146 kcal/28 g serving); significantly lower
controlled feeding than calculated Atwater factors (6.61 kcal/g;
185 g/serving); overestimating by 21%
Estruch et al. Randomized trial PREDIMED cohort 1. Mediterranean diet supplemented Bodyweight and waist Mediterranean diet with extra-virgin olive oil
(2016) with extra virgin olive oil circumference group was −0.43 kg (95% CI −0.86 to −0.01;
2. A Mediterranean diet supplemented P = 0.044) and in the nut group was
with nuts −0.08 kg (−0.50 to 0.35; P = 0.730) versus
3. A control diet for 4.8-year follow-up control group
Changes in waist circumference were −0.55 cm
(–1.16 to −0.06; P = 0.048) in the

Mediterranean diet with extra-virgin olive oil
group and −0.94 cm (–1.60 to −0.27;
P = 0.006) in the nut group versus
control group
465
with these results. This discrepancy may be due to differences in study populations and different meth-
ods used to quantify lipoprotein particle size. The Strong Heart Study included individuals with type 2
diabetes and the CARE study included individuals with history of myocardial infarctions, while the
previous studies were conducted in persons with no history of CVD. In addition to the predictive
value of LDL size, HDL subpopulations have also been shown to predict CHD risk. The Framingham
Offspring Study reported that every 1 mg/dL increase in apoA-I in very large α-1 HDL was associated
with a 26% (P < 0.0001) decrease in CHD risk (Asztalos et al., 2004). In the VA-HIT trial, logistic
regression indicated each 1 mg/dL decrease in α-1 increased the odds of CHD 13% (P < 0.0001) in
participants with low HDL-C, after adjustment for lipid and nonlipid CHD risk factors (Asztalos et al.,
2005). Thus, evaluation of lipoprotein particles may provide further insight into CVD risk.
In a subset of the PREDIMED study cohort (n = 169), consumption of a traditional Mediterranean
diet with nuts increased large LDL (54 nmol/L; 95% CI: 18–90) and decreased very small, dense
LDL (−111 nmol/L; 95% CI: −180 to −42; P = 0.017 for both values) (Damasceno et al., 2013). There
were no significant differences in total LDL-C among groups (P = 0.942). In a study conducted by
Berryman et al., the effects of a cholesterol-lowering moderate-fat diet (32% calories from fat) with
almonds (1.5 oz/day) were compared with an isocaloric lower-fat (26%) diet with 106 g banana muf-
fin + 2.7 g butter for 6 weeks (Berryman et al., 2015), in individuals (n = 48) with elevated LDL-C
(149 ± 3 mg/dL). There were no treatment effects for LDL1, LDL2, LDL3, or LDL4; however, the
higher-fat almond diet significantly decreased IDL1 (−0.06 ± 0.33 vs. 0.7 6 ± 0.33 mg/dL; P = 0.01),
total VLDL (0.15 ± 0.91 vs. 2.46 ± 0.91 mg/dL; P = 0.02), VLDL3 (0.01 ± 0.49 vs. 1.18 ± 0.49
mg/dL; P = 0.02), and apoB (−9.7 ± 1.8 vs. −5.5 ± 1.8 mg/dL; P = 0.01) compared with the lower-fat
diet. Pistachios also have been studied for their effects on lipoprotein subclasses. One randomized,
crossover controlled-feeding study was designed to evaluate the effects of pistachio consumption on
lipoprotein subclasses in individuals (n = 28) with elevated LDL-C levels (≥110.6 mg/dL) (Holligan
et al., 2014). There were three experimental diets: a lower-fat control diet (control; 25% total fat and
8% SFA); a diet that provided 10% of energy from pistachios (32–65 g/day (1PD); 30% total fat and
8% SFA); and a diet that provided 20% of energy from pistachios (63–126 g/day (2PD); 34% total
fat and 8% SFA). Both the 10% and 20% pistachio diets significantly reduced small, dense LDL
levels [least square means [LSM] = 1.00 mmol/L (SE 0.03), P = 0.001 and LSM = 0.86
(SE 0.03), P = 0.03, respectively], compared to the lower-fat control diet [LSM = 1.07 (SE) 0.03].
A similar effect also has been reported in individuals at risk for diabetes. In a study of 54 adults
with prediabetes, subjects were randomized to a diet with pistachios (PD, 50% carbohydrates, 33%
fat, including 57 g/day of pistachios) or a control diet (55% carbohydrates, 30% fat) for 4 months.
There was a significantly greater decrease in small, dense LDL from the higher fat pistachio diet
compared to the lower-fat control diet [−28.07 nM (95% CI: −60.43, 4.29) vs. −16.49 nM (95% CI:
−14.19, 47.18); P = 0.023, respectively] (Hernandez-Alonso et al., 2015). Collectively, these studies
demonstrate a beneficial relationship between nut consumption and reduced CVD risk in relation to
lipoprotein subclass distribution within the context of a moderate-fat diet compared with a lower-fat
control diet without nuts.
Lipoprotein Function
Reverse cholesterol transport (measured as cholesterol efflux) is the process by which cholesterol
is removed from macrophages. In a U.S. cohort study of 2924 healthy individuals, there was a 67%
reduction in cardiovascular risk in the highest quartile of cholesterol efflux capacity versus the lowest
quartile [HR = 0.33; (95% CI: 0.19–0.55)]. Efflux capacity was measured using fluorescence-labeled
cholesterol and assayed, evaluating cholesterol efflux as mediated by ATP-binding cassette transporter
A1 (ABCA1) (Rohatgi et al., 2014). Studies have shown that dietary intervention can impact cho-
lesterol efflux. Walnuts, walnut oil, and alpha-linolenic acid (ALA), a component of walnuts, have
been shown to increase cholesterol efflux (Berryman et al., 2013; Zhang et al., 2011, 2012). Zhang
et al. (2011) demonstrated that consumption of 51 g walnut oil incorporated in diet Jell-O™ increased
cholesterol efflux from macrophage-derived foam cells by 17% in participants with low C-reactive
protein (CRP, a marker of systemic inflammation) levels (<2 mg/L), but not in those with higher CRP
(≥2 mg/L) compared to baseline. In addition, consumption of 85 g of whole walnuts incorporated
into diet Jell-O increased cholesterol efflux by 3.3% (P = 0.02) compared to the baseline (fasted state)
(Berryman et al., 2013). In the study by Holligan et al. (2014), consumption of one serving of pista-
chios (10% energy from pistachios) or two servings of pistachios (20% energy from pistachios) did not
affect ABCA1-mediated cholesterol efflux or global serum efflux. However, individuals with baseline
CRP values <0.2 mg/dL had significant increases in ABCA1-mediated efflux and global efflux follow-
ing the two servings of pistachios compared to one serving of pistachios treatment [9.89% (SE 0.74)
vs. 7.35% (SE 0.74), P = 0.016]. Thus, the effects of diet on efflux capacity may be blunted in persons
with chronic inflammation. These studies suggest that one cardioprotective benefit of nuts may be due
to an increase in cholesterol efflux capacity in the absence of low-grade inflammation.
Oxidation and Inflammation

Nuts are a rich source of bioactive compounds that contain antioxidants including tocopherols, phe-
nolic compounds, phytosterols, melatonin, and selenium that may contribute to improved oxidative
status (Ros, 2009). Early in vitro studies using nut extracts demonstrated reductions in lipid peroxi-
dation and oxidative DNA damage (Anderson et al., 2001; Chen et al., 2005, 2007; Gentile et al.,
2007). Acute and long-term animal studies reported improved oxidative biomarkers and beneficial
effects on lipid peroxidation, antioxidant enzymatic activity, and cholesterol oxidation products
(Aksoy et al., 2007; Davis et al., 2006; Hatipoglu et al., 2004; Iwamoto et al., 2002; Reiter et al.,
2005). RCTs conducted in humans consuming various tree nuts (i.e., almonds, walnuts, pistachios,
cashews, pecans, and Brazil nuts) and peanuts, with intakes ranging from 17 to 168 g/day for 3–12
weeks, reported an improvement in oxidative stress markers (i.e., antioxidant capacity, serum oxi-
dized LDL, urinary isoprostanes, malondialdehyde concentration, DNA damage) (Lopez-Uriarte
et al., 2009). In a postprandial study conducted by Berryman et al. (2013), the ferric-reducing anti-
oxidant potential was significantly greater (P < 0.01) after consumption of walnut oil (51 g) and
walnuts skins (56 g) compared to the nutmeat group. In addition, nuts have also been shown to
decrease oxidized LDL. In the PREDIMED trial, a subsection of individuals (n = 372) was evaluated
at 3 months to measure the effects of the dietary interventions on lipid oxidative damage. There were
significant decreases in oxidized LDL after consumption of the Mediterranean diet with extra-virgin
olive oil (−10.6 U/L; 95% CI: −14.2 to −6.1; P = 0.02) and nonsignificant decreases following the
Mediterranean diet with nuts (−7.3 U/L; 95% CI: −11.2 to −3.3), compared to a lower-fat diet (−2.9
U/L; −7.3 to 1.5) (Fito et al., 2007). The Mediterranean diet with nuts also significantly decreased
adhesion molecules IL-6 by 97 μg/L (P < 0.018), sICAM-1 by 167 μg/L (P < 0.003), and sVCAM
by 167 μg/L compared to the lower-fat diet (P < 0.003) (Estruch et al., 2006).
In the pistachio study described earlier, pistachio intake of 1 and 2 servings/day reduced oxidized
LDL compared to a lower-fat control diet (1 serving/day pistachio diet = 46.57 ± 3.03 U/L; 2 servings/
day pistachio diet = 43.43 ± 3.02 U/L; vs. control = 48.57 ± 3.02 U/L, P < 0.05) (Kay et al., 2010). This
study also reported greater serum antioxidant levels following the 2PD (421.89 ± 21.89 nmol/L) and
1PD (337.77 ± 22.03 nmol/L) compared to the lower-fat diet (239.37 ± 21.89 nmol/L), with greater
increases following the 2PD indicating a dose effect (P < 0.001). After controlling for the change in
LDL-C, increases in serum lutein and γ-tocopherol following the 2PD period were modestly associ-
ated with decreases in oxidized LDL (r = −0.36, P = 0.06 and r = −0.35, P = 0.08, respectively). This
beneficial effect has also been shown in individuals with MetS. In a study conducted in 50 individuals
with MetS (Lopez-Uriarte et al., 2010), 30 g mixed nuts (15, 7.5, and 7.5 g/day of walnuts, almonds,
and hazelnuts, respectively) incorporated into a healthy dietary pattern (defined by meeting both nutrient
and food-based dietary recommendations) had no effect on oxidized LDL (Lopez-Uriarte et al., 2010),
but DNA damage evaluated by 8-oxo-7,8-dihydro-2’-deoxyguanosine was reduced significantly in the
nut group (−6.35 nmol/mmol creatine; 95% CI: −7.20 to −5.51; P < 0.001). These findings suggest that
nut consumption decreases both oxidative stress and markers of inflammation, which may contribute to
their CVD protective effects.
Inflammation plays a key role in the progression of CHD from the initial lesion to the later-stage
thrombotic complications (Libby, 2006). Studies evaluating nut consumption on inflammation have
reported favorable effects on inflammatory markers and mediators (Estruch et al., 2006; Ros et al., 2004).
Zhao et al. (2007) reported a decrease in CRP in participants who consumed 37 g of walnuts + 15 g
walnut oil/day for 6 weeks (<0.08). Furthermore, the study diet decreased adhesion molecules ICAM-1
by 18% (P < 0.05) and E-selectin by 6% (P < 0.05%). In healthy individuals (n = 25), after 4 weeks of
consuming almonds corresponding to 10% or 20% of total energy, serum CRP levels were significantly
lower both in the low-almond group (1.40 mg/dL) and high-almond group (1.47 mg/dL) compared to
control (1.54 mg/dL) (P < 0.05). Walnuts incorporated into a meal also have been shown to reduce post-
prandial mRNA expression of Il-6 in circulating blood mononuclear cells (Cortes et al., 2006) and con-
sumption of a breakfast meal with walnuts reduced monocyte expression of pro-inflammatory ligands
(Jimenez-Gomez et al., 2009) compared to a breakfast meal containing butter. The consumption of tree
nuts and peanuts and the resulting decreases in oxidative and inflammatory markers suggest this may be
an important mechanism by which tree nuts and peanuts provide CHD protection.
Vascular Health
The effect of nut consumption on blood pressure has been evaluated as a secondary outcome measure (Casas-
Agustench et al., 2011a,b; Chisholm et al., 2005; Edwards et al., 1999; Estruch et al., 2006; Fito et al., 2007;
Iwamoto et al., 2002; Jenkins et al., 2002; Llorente-Cortes et al., 2010; Mukuddem-Petersen et al., 2007;
Olmedilla-Alonso et al., 2008; Ros et al., 2004; Sabate et al., 1993; Sari et al., 2010; Schutte et al., 2006;
Sheridan et al., 2007; Spaccarotella et al., 2008; Wien et al., 2003, 2010; Wu et al., 2010). These trials have
been conducted with healthy participants and individuals at risk of CVD. Daily intakes ranged from 30 to
108 g/day of different types of nuts (almonds, walnuts, hazelnuts, pistachios, cashews, and mixed nuts).
Of these studies, four reported a decrease in SBP and diastolic blood pressure (DBP) in the nut consump-
tion group compared to control (Estruch et al., 2006; Fito et al., 2007; Llorente-Cortes et al., 2010; Wien
et al., 2003), and the remaining studies found no effect on resting blood pressure (Casas-Agustench et al.,
2011a,b; Chisholm et al., 2005; Edwards et al., 1999; Iwamoto et al., 2002; Jenkins et al., 2002; Mukuddem-
Petersen et al., 2007; Olmedilla-Alonso et al., 2008; Ros et al., 2004; Sabate et al., 1993; Sari et al., 2010;
Schutte et al., 2006; Sheridan et al., 2007; Spaccarotella et al., 2008; Wien et al., 2010; Wu et al., 2010). A
recent systematic review and meta-analysis of 21 clinical trials with 1652 adults found that nut consump-
tion (i.e., walnuts, almonds, pistachios, cashews, hazelnuts, macadamia nuts, pecans, peanuts, and soy nuts)
resulted in a significant reduction in SBP (−1.29 mmHg; 95% CI: −2.35–0.22; P = 0.02) in individuals not
diagnosed with type 2 diabetes, but not in the total population (Mohammadifard et al., 2015). A subgroup
analysis of different types of nuts suggests that pistachios, but not other nuts, significantly decrease SBP
(−1.82 mmHg; 95% CI: −2.97, −0.67; P = 0.002), while both pistachios and mixed nuts significantly
reduce DBP (−0.80 mmHg, P = 0.01; −1.19 mmHg, P = 0.04), respectively.
Other techniques that have been used to measure vascular health include ambulatory blood
pressure, central blood pressure, and flow-mediated dilation (FMD). Ambulatory blood pressure
is a technique used to measure blood pressure at regular intervals over 24 hours. This method
avoids the effect of “white coat hypertension” which is the increase in blood pressure due to anxi-
ety caused by the examination process (Jhalani et al., 2005; Ogedegbe et al., 2008). Ambulatory
blood pressure monitoring over a 24-hour period also assesses nocturnal blood pressure with a
dip in blood pressure during the night being considered normal and desirable (Pickering et al.,
2005). Absence of this dip is associated with poorer health outcomes, including increased mortal-
ity (Minutolo et al., 2011). In a randomized, crossover controlled-feeding study, individuals with
type 2 diabetes consumed either a low-fat diet (27% fat) containing a low-fat/fat-free carbohy-
drate snack (i.e., pretzels, string cheese, etc.) or a moderate-fat diet containing pistachios (33%
total fat; 20% of energy from pistachios) for 4 weeks (Sauder et al., 2014). The pistachio diet
resulted in no significant changes in resting SBP (P = 0.76) or DBP (P = 0.28). However, systolic
ambulatory blood pressure was significantly decreased (−3.5 mmHg, P = 0.046), with the greatest
reduction observed during sleep (−5.7 mmHg, P = 0.052). Similarly, in the PREDIMED study,
consuming a Mediterranean-style diet supplemented with 30 g/day of mixed nuts (15 g walnuts,
7.5 g hazelnuts, and 7.5 g almonds) for 1 year decreased ambulatory SBP (−2.6 mmHg; 95%
CI: −4.3 to −0.9; P < 0.001) and DBP (−1.2 mmHg; 95% CI: −2.2 to −0.2; P = 0.017) compared
with a lower-fat diet (Domenech et al., 2014). These results provide further evidence for multiple
mechanisms by which nut consumption decreases CVD risk.
Arterial stiffness is a consequence of biological processes such as aging and atherosclerosis. It is
often measured by pulse wave velocity (PWV), the rate at which pressure waves move down the vessel
and reflect back (Hansen et al., 2006). To date, only one study has evaluated nut consumption on arterial
stiffness. The study was designed to assess measures of arterial stiffness after a 30-month intervention of
a lifestyle modification (physical activity and adherence to the “therapeutic lifestyle change” diet) with
or without the addition of 80 g/day of pistachios in adults with mild dyslipidemia (Kasliwal et al., 2015).
The lifestyle modification with pistachios lowered carotid PWV (770.9 ± 96.5 vs. 846.4 ± 162.0 cm/s;
P = 0.08), left brachial-ankle (baPWV) (1192.4 ± 152.5 vs. 1326.3 ± 253.7 cm/s; P = 0.05), and average
baPWV (1208.2 ± 118.4 vs. 1295.8 ± 194.1 cm/s; P = 0.08) compared with the lifestyle modification
group without nuts. Because arterial stiffening typically precedes atherosclerosis, these findings are
important as they suggest a protective role of tree nuts in the prevention of CVD.
Endothelial dysfunction is a pathological state that represents an imbalance between vasodilation
and vasoconstriction (Deanfield et al., 2005), which also can precede the onset of atherosclerosis (Aoki
et al., 2006). FMD is a noninvasive method to measure endothelial function quantified by brachial
artery ultrasound imaging (Peretz et al., 2007). This technique works by inducing reactive hyperemia
via temporary arterial occlusion and measuring the relative increase in blood vessel diameter through
ultrasound (Quinaglia et al., 2015). A growing number of clinical trials have demonstrated that nuts
favorably affect endothelial function (Cortes et al., 2006; Ma et al., 2010; Ros et al., 2004; West et al.,
2010). In hypercholesterolemic men and women (n = 18), consumption of a walnut-rich diet (i.e., 18%
of energy) for 4 weeks compared with a Mediterranean diet without walnuts significantly improved
endothelial-dependent vasodilation from 3.6% to 5.9% (P = 0.043) (Ros et al., 2004). This effect was
also observed when pistachios were included in a Mediterranean diet (~20% energy) for 4 weeks. The
pistachio diet increased FMD compared with the traditional Mediterranean diet without pistachios
(10.29% ± 2.76% vs. 7.86% ± 2.28%; P = 0.002) in healthy participants (Sari et al., 2010). This effect
was also reported when hypercholesterolemic subjects consumed a step-2 diet that replaced 18%–21%
of energy with hazelnuts daily for 4 weeks; there was a significant increase in FMD (56.6%; P < 0.001)
compared to a control diet (step-2 diet without hazelnuts) (Orem et al., 2013). Other studies have also
reported this effect in individuals diagnosed with type 2 diabetes. When individuals with type 2 diabe-
tes consumed an ad libitum diet with walnuts (56 g/day) FMD significantly increased compared to an
ad libitum diet without walnuts (2.2% ± 1.7% vs. 1.2% ± 1.6%; P = 0.04) (Ma et al., 2010).
The benefits reported in long-term nut interventions (treatment periods lasting 4 weeks to
3 months) have also been reported in postprandial studies. Researchers have demonstrated acute
improvement in dilation after consumption of nuts, particularly walnuts (Berryman et al., 2015; Cortes
et al., 2006). In a study by Cortes et al. (2006), healthy individuals (n = 12) and hypercholesterolemic
individuals (n = 12) consumed two high-fat meals (80 g of fat; 35% saturated fat) to which either
walnuts (40 g) or olive oil (25 mL) was added. Postprandial FMD was reduced after the olive oil meal
in both healthy (−17%) and hypercholesterolemic subjects (−36%) compared to the control group.
However, after the walnut-enriched meal, FMD was unchanged in the healthy subjects and increased
by 24% in the hypercholesterolemic subjects (P < 0.006). In order to understand the contribution of
individual components of walnuts, Berryman et al. (2013) studied the effects of acute consumption
of walnut skin, defatted walnut meat and walnut oil compared with whole walnuts on reactive hyper-
emia (measured by pulse amplitude tonometry). Walnut oil (51 g) improved reactive hyperemia index
(RHI) compared to walnut skins (5.6 g) (P = 0.01). The authors explained this finding as being due to
a greater bioavailability of bioactives in walnut oil compared with the nutmeat and skins.
The beneficial effects of nut consumption have also been reported at the microvascular level,
although the number of studies is limited (Holt et al., 2015; Huguenin et al., 2015; Maranhao et al., 2011).
Microvascular reactivity is associated with CVD risk factors (Baumbach et al., 1991). In a paral-
lel design study with 38 hypercholesterolemic, postmenopausal women, consumption of 40 ver-
sus 5 g/day of walnuts significantly increased the fasting reactive hyperemia index (RHI) (2.63 ±
0.10 vs. 2.23 ± 0.13, respectively, P = 0.025). However, two studies assessing Brazil nuts (13–25 g/
day) with treatment durations between 3 and 4 months reported no effect on microvascular parameters
(i.e., capillary diameter) (Huguenin et al., 2015; Maranhao et al., 2011). The conflicting results and
limited number of studies highlight the need for additional research of nuts on microvascular function.
Insulin Resistance and Glycemic Response

Decreasing postprandial glycemia lowers the risk of developing hypertension, diabetes, and CHD
in high-risk individuals (Chiasson et al., 2002, 2003). Post-meal hyperglycemia is also an indepen-
dent risk factor for CVD (Levitan et al., 2004). There is no postprandial glycemic response when
nuts are consumed alone, likely due to their very low carbohydrate content. When consumed with
carbohydrate-rich foods, nuts have shown to blunt postprandial glycemia (Jenkins et al., 2006; Josse
et al., 2007; Kendall et al., 2011a,b). In a postprandial study of 15 healthy subjects, glycemic indices
were measured after consumption of three test meals: almond meal, 60 g almonds + 97 g of bread;
parboiled rice meal, 68 g cheese + 14 g butter + 60 g parboiled rice; and mashed potato meal, 62 g
cheese and 16 g butter and 68 g mashed potatoes (Jenkins et al., 2006). Glycemic indices for the
rice (38 ± 6) and almond meals (55 ± 7) were less than for the potato meal (94 ± 11) (P < 0.003),
as were the postprandial areas under the insulin concentration time curve (P < 0.001). A subsequent
study with nine healthy participants evaluated the effects of varying amounts of almonds on the
postprandial blood glucose response to a carbohydrate meal. Each test meal contained bread as the
source of carbohydrate (50 g) to be eaten alone or with 30, 60, and 90 g of almonds. The addition
of almonds to white bread resulted in a dose-dependent reduction in the glycemic index for the 30 g
(105.8 ± 23.3), 60 g (63.0 ± 9.0), and 90 g (45.2 ± 5.8) doses of almonds (r = −0.524, P = 0.001)
(Josse et al., 2007). Pistachio nuts also have a beneficial effect on postprandial glycemia. In 10
healthy volunteers, the consumption of 28 g of pistachios with white bread (50 g) resulted in reduced
relative glycemic response (89.1 ± 6.0; P = 0.100), with a greater reduction achieved when 84 g of
pistachios were added (51.5 ± 7.5; P < 0.001) compared to the white bread control (100 g) (Kendall
et al., 2011a,b). An expansion of this study conducted with subjects with the MetS found
50 g of white bread +85 g of pistachios significantly blunted the postprandial glucose response
compared to consuming white bread alone (Kendall et al., 2014), but no differences were observed
for postprandial insulin levels. In a study by Kendall et al. (2011a,b), the effects of 30, 60, and 90 g
of mixed nuts (almonds, macadamias, walnuts, pistachios, hazelnuts, and pecans in equal portions
by weight) alone and in combination with white bread on postprandial glycemia were examined in
14 healthy subjects and 10 subjects with type 2 diabetes. The relative glycemic response was signifi-
cantly lower with the three doses of mixed nuts compared to the white bread control in both normo-
glycemic subjects (2%–6% of control; P < 0.001) and in subjects with type 2 diabetes (4%–8% of
control, P < 0.001). In normoglycemic subjects, the addition of 30, 60, and 90 g of nuts to the bread
reduced the glycemic response by 11.2% ± 11.6%, 29.7% ± 12.2%, and 53.5% ± 8.5% (P = 0.35,
P = 0.031, and P < 0.001, respectively). However, in subjects with type 2 diabetes, the reduction in
glycemic response of 30, 60, and 90 g nuts was half that observed for the normoglycemic subjects
(6.6% ± 8.8%, 16.6% ± 9.3%, 30.8% ± 7.6%; P = 0.474, P = 0.113, and P = 0.015, respectively).
Interestingly, a study by Casas-Agustench et al. (2011a,b) found that subjects with the MetS were
less responsive to the cholesterol-lowering effect of mixed nuts compared to previous reports with
healthy individuals. Collectively, these studies demonstrate that individuals with metabolic condi-
tions are less responsive to the cardioprotective benefits of nuts on some CVD risk factors.
Body Weight
Cross-sectional studies have reported an inverse association between nut consumption and BMI
(Almario et al., 2001; Alper and Mattes, 2002; Fraser et al., 1992; Hu et al., 1998; Jackson and Hu,
2014; Kris-Etherton et al., 1999; Martinez-Gonzalez and Bes-Rastrollo, 2011; O’Byrne et al., 1997).
Studies also have reported reduced waist circumference with increased nut consumption (Casas-
Agustench et al., 2011a; Martinez-Gonzalez et al., 2012; O’Neil et al., 2011). Smith et al. (2015)
reported results from a prospective investigation involving three cohorts that included 120,877
healthy U.S. women and men from the NHS I (n = 50,422 women) NHS II (n = 47,898 women) and
the HPFS (n = 22,557 men). After 4 years, weight change was inversely associated with the intake
of nuts (−0.57 lb; 95% CI: −0.97 to −0.17; P = 0.005).
A meta-analysis of 33 clinical trials (n = 1888 participants) evaluated the effects of tree nut
(almonds, walnuts, pistachios, hazelnuts) and peanut consumption on body weight in diets that
include nuts versus controlled diets (i.e., habitual, lower-fat, American Diabetes Association, Step-2,
low-calorie diets) (Flores-Mateo et al., 2013). Pooled results indicated a nonsignificant effect on
body weight (−0.47 kg; 95% CI: −1.17, 0.22 kg; I2 = 7%), BMI (−0.40 kg/m2; 95% CI: −0.97,
0.17 kg/m2; I2 = 49%), and waist circumference (−1.25 cm; 95% CI: −2.82, 0.31 cm; I2 = 28%) of
diets including nuts compared with control diets. These epidemiological and clinical trial findings
indicate no increase and perhaps a decrease in body weight following nut consumption.
The mechanism is unclear for the association between nuts and body weight (Almario et al., 2001;
Alper and Mattes, 2002; Flores-Mateo et al., 2013; Fraser et al., 1992; Hu et al., 1998; Kris-Etherton
et al., 1999; Martinez-Gonzalez and Bes-Rastrollo, 2011; O’Byrne et al., 1997). One possible expla-
nation is the recent discovery related to the discrepancy between the Atwater energy factor predicted
for almonds, pistachios, and walnuts and the empirically measured energy value when the nuts are
consumed (Baer et al., 2012, 2016; Novotny et al., 2012). Atwater-specific factors are a series of energy
values for macronutrients that take digestibility and heat of combustion into account. Three studies were
conducted to measure the available energy in almonds, pistachios, and walnuts compared to the cor-
responding calculated Atwater factor value. Each of the studies used a controlled diet, crossover design
with one or two treatment diets and a control diet. Almonds were studied in 18 healthy adults fed three
different diets for 18 days each, including 0, 42, and 84 g almonds/day. The authors reported the energy
content of almonds in the diet as 4.6 ± 0.8 kcal/g (equivalent to 129 kcal/28 g-serving), which is signifi-
cantly less than 6.0–6.1 kcal/g (168–170 kcal/serving), the energy density determined by the Atwater
factors (P < 0.001). This study concluded that the Atwater factors overestimate the measured energy
content of almonds by 32% (Novotny et al., 2012). Similarly, in a study examining the energy value
of pistachios, 16 healthy adults consumed 0 g/day pistachios, 42 and 84 g/day for 3 weeks. Baer et al.
(2012) reported a measured energy value for pistachios (5.4 kcal/g) that was 5% less than the calculated
Atwater factors (5.7 kcal/g). The researchers found similar results for walnuts in 18 healthy adults. The
measured energy value of walnuts [42 or 0 g/day was 5.22 ± 0.16 kcal/g (146 kcal/28 g-serving)] was
significantly lower than the calculated Atwater factors (6.61 kcal/g; 185 g/serving), resulting in a 21%
overestimation of energy content (Baer et al., 2016).
The measured versus absorbed energy content of almonds and walnuts may be a mecha-
nism, in part, to explain the relationship between nut consumption and reduced body weight.
However, some studies (Foster et al., 2012; Fraser et al., 2002) have shown weight gain (albeit
very small) with nut consumption. Using a crossover study design, Fraser et al. (2002) tested the
effects of providing a free daily supplement (averaging 320 kcal) of almonds or no supplement to
81 male and female participants for six months. After the almond feeding period, average body
weight increased only 0.40 kg (P ~ 0.09), suggesting there was not significant weight change
after almonds were incorporated isocalorically in the diet for other foods. Weight change was
dependent on baseline BMI (P = 0.05). Only those who were initially in the lower BMI tertiles
experienced a small weight gain (0.40 kg) with the almonds. Foster et al. (2012) reported similar
results in 123 overweight and obese individuals who were randomly assigned to consume an
almond-enriched diet (AED) or nut-free diet (NFD) for 18 months and instructed in traditional
behavioral methods of weight control, such as self-monitoring and stimulus control. Participants
in the AED group lost slightly, but significantly, less weight than did those in the NFD group at
6 months (−5.5 compared with −7.4 kg; P = 0.04), but there were no differences at 18 months.
No significant differences in body composition were found between the groups at 6 or 18 months.
It is important to address the difference between the addition of nuts to the diet compared to the
isocaloric substitution of nuts in the diet for other foods.
In a recent analysis of the PREDIMED trial, the long-term effects of an ad libitum, lower-fat
control diet, a Mediterranean diet supplemented with extra-virgin olive oil, and a Mediterranean
diet supplemented with mixed nuts on body weight and waist circumference were assessed in older
people at risk of cardiovascular disease, most of whom were overweight or obese. After a median
4.8 years of follow-up, participants in all three groups had a marginally reduced body weight and
increased waist circumference (Estruch et al., 2016). The adjusted difference in 5-year changes in
body weight in the Mediterranean diet supplemented with extra-virgin olive oil group was −0.43 kg
(95% CI 0.86, −0.01; P = 0.044) and in the nut group was −0.08 kg (−0.50, 0.35; P = 0.730), com-
pared with the control group. The adjusted difference in 5-year changes in waist circumference was
−0.55 cm (−1.16, −0.06; P = 0.048) in the Mediterranean diet supplemented with extra-virgin olive
oil group and −0.94 cm (−1.60, −0.27; P = 0.006) in the nut supplemented group, compared with
the control group. This study suggests that a long-term intervention with a Mediterranean diet that
includes extra-virgin olive oil or nuts was associated with decreases in body weight and less gain in
central adiposity compared with a control diet.
Collectively, the majority of research conducted to date demonstrates benefits of nut consumption
on body weight, as well as measures of visceral adiposity. These findings provide further support for includ-
ing nuts in a healthy eating pattern that meets energy needs for body weight control.
SUMMARY
Many health benefits of nut consumption have been reported in both epidemiological and clinical
studies. Landmark population studies, such as the Adventist Health Study, the Iowa Women’s Health
Study, the NHS, and the PHS have demonstrated that the consumption of nuts was associated with
decreased risk for CHD. A pooled analysis of these four large U.S. studies demonstrated a 35%
reduced risk of CHD incidence for the highest nut intake of >5 times/week (Kris-Etherton et al.,
2008). Studies in populations of different geographical regions and socioeconomic status have also
reported similar benefits (Eslamparast et al., 2017; Gopinath et al., 2015; Luu et al., 2015). In addi-
tion, a beneficial relationship has been reported for nut consumption and risk reduction of type 2
diabetes and the MetS (O’Neil et al., 2011; Pan et al., 2013).
In the groundbreaking PREDIMED trial, a Mediterranean dietary pattern with nuts (30 g/day),
or extra-virgin olive oil, decreased cardiovascular events by approximately 30% and decreased
risk of stroke by almost 50% in men and women at high risk for CVD. Many clinical trials have
reported benefits of nut consumption on numerous CVD risk factors including blood lipids/lipopro-
teins, inflammation, oxidation, vascular function, insulin resistance, and glycemic response. Studies
evaluating walnuts (Rajaram et al., 2009; Torabian et al., 2010), almonds (Berryman et al., 2013;
Jenkins et al., 2002), pistachios (Sauder et al., 2013; Sheridan et al., 2007), hazelnuts (Mercanligil
et al., 2007), peanuts (Lokko et al., 2007), and macadamia nuts (Griel et al., 2008) have reported
decreases in LDL-C ranging from 9% to 16%. Additionally, a pooled analysis of 25 nut interven-
tion studies conducted in 7 countries with 583 men and women reported a dose–response improve-
ment in lipids/lipoproteins with nut consumption (Sabaté and Wien, 2010). In summary, the clinical
studies demonstrate multiple cardiovascular benefits of nut and peanut consumption when they are
isocalorically substituted for other foods. In addition, recent evidence from PREDIMED and other
studies supports no restriction on intake of healthy fats (such as those provided by nuts) as appropri-
ate for body weight maintenance and overall cardiometabolic health, as recently acknowledged by
the Dietary Guidelines Advisory Committee (2015).
The 2015–2020 Dietary Guidelines recommend specific food substitutions for a healthy diet.
The guidelines state: “choose nutrient-dense foods and beverages across and within all food
groups in place of less healthy choices” (U.S. Department of Health and Human Services and
U.S. Department of Agriculture, 2015). The 2015–2020 Dietary Guidelines further recommend
“use oils rather than solid fats … when cooking, increasing the intake of foods that naturally con-
tain oils, such as seafood and nuts, in place of some meat and poultry, and choosing other foods,
such as salad dressings and spreads, made with oils instead of solid fats”.
Three healthy food-based dietary patterns are recommended to implement the 2015–2020 Dietary
Guidelines (U.S. Department of Health and Human Services and U.S. Department of Agriculture,
2015). Two of the USDA Food Patterns (Healthy U.S.-Style Pattern and Healthy Mediterranean-
Style Pattern) recommend 5 oz equivalents/week of nuts/seeds and the third (Healthy Vegetarian
Pattern) recommends 14 oz equivalents/week. The U.S. diet falls short of meeting all food-based
dietary recommendations, including those made for nuts/seeds. Achieving these recommendations on
a population-wide basis is expected to confer many health benefits. In addition, based on the evidence
summarized herein, adoption of just the recommendations for nuts/seeds by the U.S. population
would confer marked benefits on cardiometabolic risk. At this juncture, it is important to increase
consumer and health care professional awareness of the many health benefits of nuts/seeds, and teach
implementation strategies for incorporating them in a healthy dietary pattern. Future research on nuts
will advance our understanding of their mechanisms of action as well as provide better behavior
change strategies for incorporating nuts into a heart-healthy diet.
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25 Dairy Product
Consumption, Dairy Fat,
and Cardiometabolic Health*
Benoît Lamarche
CONTENTS
Abbreviations................................................................................................................................. 482
Introduction.................................................................................................................................... 482
Cardiometabolic Risk..................................................................................................................... 483
Dairy and Plasma Lipid Levels................................................................................................. 483
Dairy and Low-Grade Systemic Inflammation......................................................................... 485
Dairy and Glucose–Insulin Homeostasis.................................................................................. 485
Dairy, Blood Pressure, and Vascular Function.......................................................................... 486
Dairy Fat and Cardiometabolic Risk.............................................................................................. 488
Conclusions.................................................................................................................................... 488
References...................................................................................................................................... 489
ABSTRACT
Because regular-fat dairy products contribute significantly to dietary intake of saturated fatty acid
(SFA), and because of the well-known cholesterol-raising effects of SFA, most dietary guidelines
advocate consumption of low-fat dairy products as opposed to regular/high-fat dairy foods. Yet,
results from numerous randomized controlled trials (RCTs) have reported inconsistent effects of
dairy consumption, including regular/whole-fat dairy, on blood lipid levels and on many other car-
diometabolic risk factors, such as blood pressure and inflammatory biomarkers. Thus, the recom-
mendation to have low-fat dairy integrated as part of healthy eating guidelines in 2015 needs to be
revisited. This review suggests that consumption of dairy foods and dairy fat appears to have a null
effect on a large spectrum of cardiometabolic risk factors. Data also suggest that the purported detri-
mental effects of SFA on cardiometabolic health may be attenuated when provided in complex food
matrices such as cheese. Despite the fact that there are still numerous research gaps to be addressed,
available data suggest no potential harmful effects of dairy consumption, irrespective of dairy fat,
on cardiometabolic risk. Thus, the focus on low-fat dairy products in current guidelines may need to
be revisited in light of the current evidence.
* Disclosures
The author is Chair of Nutrition at Laval University. This Chair is supported by unrestricted endowments from Royal
Bank of Canada, Pfizer, and Provigo/Loblaws. The author has received funding in the last 5 years for his research from the
Canadian Institutes for Health Research (CIHR), Natural Sciences and Engineering Research Council of Canada (NSERC),
Agriculture and Agrifood Canada, the Canola Council of Canada, Dairy Farmers of Canada (DFC), Dairy Research Institute
(DRI), Atrium Innovations, the Danone Institute, Merck Frosst. The author has received speaker honoraria over the last
5 years from DFC, DRI, the Dairy Council of Northern Ireland, and the International Chair on Cardiometabolic Risk.
The author is Chair of the Expert Scientific Advisory Panel of DFC, and of the ad hoc committee on saturated fat of Heart
and Stroke Foundation of Canada.
481
ABBREVIATIONS
AIx Augmentation index
BP Blood pressure
CRP C-reactive protein
FMD Flow-mediated dilation
HDL-C High-density lipoprotein cholesterol
HOMA Homeostatic model assessment
IL Interleukin
LDL-C Low-density lipoprotein cholesterol
MCP-1 Monocyte chemoattractant protein 1
MetS Metabolic syndrome
NCEP National Cholesterol Education Program
RCT Randomized controlled trial
sICAM-1 Soluble intercellular adhesion molecule-1
SSB Sugar-sweetened beverage
sVCAM Soluble vascular cell adhesion molecule-1
T2D Type 2 diabetes
TFA trans fatty acids
TG triglycerides
TNF-α Tumor necrosis factor-α
INTRODUCTION
Dairy product consumption is recommended in most dietary guidelines around the world. This is
in large part justified by the contribution of dairy foods to the intake of key nutrients for health,
primarily high-quality protein and calcium, but also magnesium, potassium, phosphorus, vitamin
B12, riboflavin, vitamin A, and vitamin D in countries where dairy is fortified (Huth et al. 2013).
In general, dietary guidelines recommend consuming low-fat/reduced-fat dairy products to limit
the intake of saturated fat (SFA), to which dairy foods contribute significantly. Indeed, dietary SFA
increases plasma LDL-cholesterol (LDL-C), a key risk factor for coronary heart disease (CHD),
compared with dietary carbohydrates, monounsaturated fat (MUFA), and polyunsaturated fat
(PUFA) (Mensink et al. 2003).
However, fairly recent meta-analyses of observational cohort studies have failed to show a sig-
nificant association between dietary SFA and CHD risk (Siri-Tarino et al. 2010; Chowdhury et al.
2014), thus igniting a new controversy in the area of nutrition and health. While the recommendation
to limit the intake of dietary SFA remains relevant until the debate is put to rest, this whole con-
troversy also raises the pertinence of focusing on whole foods rather than on single nutrients when
crafting dietary guidelines for health. This may particularly be the case for dairy products.
We have recently performed a systematic review to ascertain the association between dairy prod-
uct consumption and the risk of clinical outcomes, including cardiovascular disease, type 2 diabetes
(T2D), and metabolic syndrome (Drouin-Chartier et al. 2016). The review was based on the highest
level of evidence from meta-analyses of prospective cohort studies as well as on more recent cohort
studies not included in these meta-analyses. This extensive review revealed no evidence of unfavor-
able associations between dairy intake, irrespective of product type and fat content, and the risk of
CHD-related clinical outcomes. For some outcomes, dairy product intake showed no association
Dairy Product Consumption, Dairy Fat, and Cardiometabolic Health 483
with risk, while for others the associations were favorable. For example, total dairy, low-fat dairy,
and milk intakes were each associated with reduced risk for hypertension, while total dairy, low-fat
dairy, yogurt, and cheese intakes were associated with a reduced risk for T2D. Total dairy and milk
consumption was inversely associated with metabolic syndrome (MetS).
Although reducing LDL-C concentrations along with proper management of high blood pressure
are considered key targets for prevention and treatment of CHD (Weintraub et al. 2011), considering
the impact of diet on a larger spectrum of risk factors may provide additional insight on how diet
modification is likely to affect risk of clinical outcomes in the future. This is important because a
large RCT on dairy with hard endpoints is highly implausible in the future. This chapter is a nar-
rative review of the data from clinical trials in humans that have documented the impact of dairy
intake, with particular focus on dairy fat, on several cardiometabolic risk factors that are involved in
the etiology of atherosclerosis and CHD.
CARDIOMETABOLIC RISK
Raised LDL-C concentrations represent only one of several risk factors to consider in the etiology
of atherosclerosis leading to clinical outcomes such as CHD. There are indeed a plethora of other
cardiometabolic risk factors that contribute to modulating CHD risk, including vascular dysfunction
with high blood pressure, plasma lipid risk factors other than elevated LDL-C such as high triglycer-
ides and low HDL-C concentrations, increased LDL particle number and small dense LDL particles,
systemic inflammation, and insulin resistance (Despres et al. 2008). While documenting the impact
of dairy intake and dairy fat on LDL-C is of great interest from a CHD prevention perspective,
appreciating the changes in other cardiometabolic risk factors also is of key importance.
Dairy and Plasma Lipid Levels

Data from a meta-analysis of RCTs conducted among healthy individuals have shown that intakes
of total dairy and low- and whole-fat dairy products had no significant impact on plasma LDL-C
or HDL-C concentrations (Benatar et al. 2013). Results from recent studies by our group are con-
sistent with these observations. In a crossover RCT, postmenopausal women with abdominal obe-
sity were fed two National Cholesterol Education Program (NCEP) diets for 6 weeks each, one
including 3.2 servings/day of 2% fat milk per 2000 kcal and one without milk or any other dairy
products. Diets were matched for calories as well as most nutrients, with the exception of calcium
and vitamin D. There was no significant difference in plasma LDL-C, HDL-C and TG levels, the
total cholesterol/HDL-C ratio, and LDL particle size between the milk diet and the milk-free diet
(Drouin-Chartier et al. 2015). In a larger crossover RCT, 101 men and women with hs-CRP values
>1 mg/L consumed 3 servings/day of dairy (375 mL low-fat milk, 175 g low-fat yogurt, and 30 g
regular-fat cheddar cheese) versus energy-matched control products (fruit juice, vegetable juice,
cashews, and 1 cookie) as part of prudent diets (Abdullah et al. 2016). A slight but significant
increase in plasma LDL-C was observed after the dairy diet (+3.3%, P = 0.02 vs. the control dairy-
free diet), with no difference, however, in plasma HDL-C, TG and apoB concentrations, or in LDL
particle size between the two diets (Abdullah et al. 2016). Interestingly, DNA sequence variants in
two genes (ABCG5 and CYP7A1) identified among a list of 13 genes known to regulate cholesterol
metabolism were found to significantly modulate the LDL-C response to dairy (Abdullah et al.
2016). This is a good example of the heterogeneous response to dietary changes among individuals,
hence explaining to some extent the inconsistencies often seen in the literature regarding the impact
of diet on cardiometabolic risk features.
There is increasing evidence that the food matrix influences the cardiometabolic response to vari-
ous nutrients, including SFA (Siri-Tarino et al. 2015). In that respect, the LDL-C raising effect of
dietary SFA may vary depending on its food of origin and background diet. For example, consump-
tion of a Gouda-type 27% fat cheese (Norvegia® 80 g/day) for 8 weeks did not significantly increase
plasma LDL-C levels compared with baseline values (Nilsen et al. 2015). In a meta-analysis of
4 RCTs combining data from a total of 100 individuals, intake of SFA from cheese reduced plasma
LDL-C and HDL-C levels compared with similar amounts of SFA from butter, while effects on
plasma TG concentrations were similar between cheese and butter (de Goede et al. 2015). The
calcium content of cheese as well as the phospholipids present in the milk fat globule membranes
of cheese fat have been evoked as potential mechanisms explaining the difference between cheese
and butter in modulating plasma lipid levels, despite similar SFA content (de Goede et al. 2015).
Chiu et al. (2016) have recently compared the impact of DASH diets that comprised either full-fat
or low-fat dairy products on plasma lipids and other cardiometabolic outcomes. The high-fat DASH
diet (14% of calories as SFA) resulted in significantly lower plasma triglycerides, large and medium
VLDL concentrations, and significantly higher LDL particle size compared with the low-fat DASH
diet (8% of calories as SFA). There were no differences between diets in LDL-C, apoB, and HDL-C
concentrations.
The lower plasma HDL-C concentrations with SFA from cheese versus butter is puzzling from a
CHD health perspective, considering that SFA intake is generally associated with increased HDL-C
compared with carbohydrates and other dietary fats (Mensink et al. 2003). While increased plasma
HDL-C concentrations are generally associated with a reduced risk of CHD (Gordon et al. 1977;
Austin 1991), recent data from large clinical trials have challenged the importance of this risk factor
in the broader scheme of CHD prevention (Barter et al. 2007; Briel et al. 2009; Schwartz et al. 2012).
An emerging concept suggests that HDL structure and functionality, beyond simple “static” choles-
terol measures, need to be considered when assessing the HDL-related risk of CHD (Asztalos et al.
2011). For example, cholesterol efflux capacity—a key step in reverse cholesterol transport mea-
sured ex vivo in cultured macrophages—has been inversely correlated with carotid intima-media
thickness and with coronary disease status even after adjustment for HDL-C concentrations (Khera
et al. 2011). This is a good example of how a metric of enhanced HDL function may better predict
the risk of vascular disease than variations in plasma HDL-C concentrations (Khera et al. 2011).
Very little is known regarding the impact of dairy and dairy fat on various metrics of HDL function
and structure; this represents an exciting and novel area of research.
Finally, the impact of consuming fermented dairy products on plasma lipid levels has been of
interest for more than 30 years. Agerholm-Larsen et al. (2000a) published a meta-analysis of six
RCTs on Gaio®, a commercially available yogurt fermented with one strain of Enterococcus
faecium and two strains of Streptococcus thermophiles. Data indicated that consumption of
the fermented yogurt significantly reduced plasma LDL-C compared with consumption of control
yogurts, which were of identical composition to the test yogurt, but chemically fermented with an
organic acid instead of a live bacterial culture. Such data have been reproduced in some (Anderson
and Gilliland 1999; Andrade and Borges 2009) but not all (de Roos et al. 1999; Sadrzadeh-Yeganeh
et al. 2010) of the more recent studies of fermented dairy intake. Finally, consumption of fermented
dairy of any form seems to have very little impact on plasma TG (Schaafsma et al. 1998; Bertolami
et al. 1999; St-Onge et al. 2002; Hansel et al. 2007), while the effects on HDL-C concentrations have
not been investigated thoroughly. In a small parallel RTC, we have shown that 4-week consumption
of yogurt products containing different doses of Bifidobacterium animalis subsp. lactis (BB-12) and
Lactobacillus acidophilus (LA-5) had no significant impact on plasma LDL-C, TG, and HDL-C
concentrations compared with a control yogurt (Savard et al. 2011). This is an area that certainly
deserves intensified research.
In summary, fairly robust data from meta-analyses as well as from additional RCTs suggest rela-
tively neutral effects of dairy intake and of dairy fat at least when consumed as part of a dairy food
such as cheese on plasma lipid levels, including LDL-C. The effect of dairy in general and of dairy
fat more specifically on other lipid risk factors such as apoB also appears to be relatively neutral
(Drouin-Chartier et al. 2015; Chiu et al. 2016), while the impact on LDL particle size is mixed, with
studies showing neutral effects (Drouin-Chartier et al. 2015) and others showing increase in LDL
size with dairy fat (Chiu et al. 2016). Most of the existing studies on this topic have been conducted
among healthy individuals and therefore such evidence needs to be further substantiated among
patients with CHD or T2D, who may show different responses to dietary changes. Data pertaining
to the effect of fermented dairy on plasma lipid risk factors are mixed and additional studies in this
area are also warranted.
Dairy and Low-Grade Systemic Inflammation

Low-grade systemic inflammation plays a key role in the etiology of atherosclerosis and related
clinical outcomes (Libby 2002, 2006). Investigating the impact of diet on systemic inflammation is
challenging. Indeed, a wide array of surrogate markers have been used to reflect different aspects
of pro- and anti-inflammatory processes, and this creates a lot of heterogeneity among studies.
Nevertheless, some of these markers like C-reactive protein (CRP) and serum amyloid A are con-
sidered good surrogates of low-grade systemic inflammation, while others like interleukin(IL)-6,
TNF-alpha, and adiponectin may reflect localized inflammation (Libby 2007).
We have recently reviewed RCTs that have assessed the impact of dairy consumption on markers
of low-grade systemic inflammation (Labonte et al. 2013). Four of the eight retrieved RCTs pointed
toward a null impact of dairy consumption on inflammation, while the other four studies suggested
favorable effects. However, we have stressed in our review that only one of the retrieved RCTs was
designed primarily to investigate the impact of dairy intake on inflammation as a primary outcome,
while all other reports are based on secondary analyses, which is a significant shortcoming. The
study in which change in inflammation markers was the main outcome showed a significant favor-
able effect of dairy consumption on plasma levels of CRP, TNF-α, and MCP-1. In most of the avail-
able studies, dairy included a combination of different types of products, thereby limiting our ability
to distinguish the effects of isolated dairy products. It must also be stressed that results in some of
these studies may have been confounded by weight loss, which was part of the intervention in addi-
tion to the dairy component of the study (Labonte et al. 2013).
Partly consistent with our findings, Benatar et al. (2013) in their meta-analysis of data from seven
RCTs have reported no significant impact of low- or full-fat dairy intake on plasma CRP levels. This
is also consistent with recent data from a large crossover RCT by our group, in which 112 men and
women with subclinical inflammation (CRP > 1 mg/L) consumed 3 servings/day of dairy products
(low-fat milk, low-fat yogurt, and regular cheddar cheese) or energy-matched control products (fruit
juice, vegetable juice, cashews, and 1 cookie) as part of prudent 4-week diets (Labonte et al. 2014).
The study was specifically designed to assess change in biomarkers of inflammation as the primary
outcome. Consumption of dairy had no significant impact on serum CRP or adiponectin concentra-
tions but significantly reduced IL-6 concentrations compared with baseline values. The reduction in
serum CRP versus baseline values with the dairy-free control diet was greater than with the dairy
diet, while the reductions in serum IL-6 concentrations were similar between the two diets. These
variations in surrogates of systemic inflammation were not correlated to variations in the expression
level of key inflammatory genes and transcription factors in whole blood cells (Labonte et al. 2014).
In summary, evidence available to date suggests a relatively neutral effect of consuming low- or
full-fat dairy products on inflammatory biomarkers. As already emphasized, most of this evidence
is based on studies that were not specifically designed to investigate inflammation as a primary
outcome, with sample sizes that were more appropriate to examine changes in plasma lipid levels or
weight loss. Thus, more studies are warranted to characterize better the impact of dairy consumption
(any form and any fat content) on low-grade, systemic inflammation.
Dairy and Glucose–Insulin Homeostasis

Insulin resistance is one of several key cardiometabolic features associated with abdominal obesity
and MetS (Despres and Lemieux 2006). Elevated plasma insulin levels, which reflect to some extent
a higher degree of insulin resistance, have been associated with an increased risk of hypertension
and CHD (Xun et al. 2013). It remains unclear if insulin is involved etiologically or is simply a
partner in crime in processes leading to atherosclerosis.
Turner et al. (2015) have systematically reviewed RCTs having assessed the effects of dairy
intake on various measures of glucose–insulin homeostasis. The literature search focused on trials
with minimal dietary change other than the dairy component, as well as weight stable conditions.
Only 10 RCTs were retrieved. Four of the dairy interventions showed a positive effect on insulin
sensitivity as assessed by HOMA, one was negative, and five had no effect. Study duration appeared
to be an important confounding factor. Indeed, trials of less than 8 weeks’ duration saw no signifi-
cant changes in insulin sensitivity, while those between 12 and 24 weeks showed a beneficial effect
of higher dairy consumption. Results from the 6-month interventions were mixed, dairy intake lead-
ing to improvement in HOMA values in one study but to no change in two other studies (Turner et al.
2015). The authors concluded their review by emphasizing the importance of relying on more and
larger studies on this topic to better assess the impact of dairy on insulin and glucose metabolism.
We have recently shown in an RCT among postmenopausal women that milk consumption
(3.2 servings/day, 2% fat) for 6 weeks had no impact on fasting glucose and insulin levels and
on Cederholm and Matsuda insulin sensitivity indices compared with a macronutrient-matched,
milk-free control diet (Drouin-Chartier et al. 2015). The Cederholm index represents mainly periph-
eral insulin sensitivity and muscle glucose uptake, while the Matsuda index is a composite of both
hepatic and peripheral tissue insulin sensitivity. Finally, in a small RCT among 14 overweight post-
menopausal women, cheese consumption (96–120 g/day) for 3 weeks compared with a macronu-
trient-matched nondairy, high-meat control diet and a nondairy, low-fat, high-carbohydrate control
diet had no effect on the HOMA-insulin resistance index and on fasting and postprandial insulin and
glucose levels (Thorning et al. 2015).
While results from prospective cohort studies suggest that dairy intake, particularly cheese and
yogurt intake, may be associated with a reduced risk of T2D (Drouin-Chartier et al. 2016), RCTs
provide limited and mixed results regarding the impact of dairy consumption on insulin resistance.
As emphasized in the systematic review by Turner et al. (2015), additional high-quality studies with
insulin resistance as primary outcome are needed in this area. Longer-term interventions may be
considered, while the confounding effects of weight loss in many of the individual studies so far
need to be addressed in future research efforts. Finally, the extent to which population characteristics
(men vs. women, healthy vs. diabetic, lean vs. obese) influence the impact of dairy intake on insulin
sensitivity also needs to be considered in future studies.
Dairy, Blood Pressure, and Vascular Function

The impact of dairy consumption on blood pressure (BP) regulation has been extensively studied.
Data from epidemiological studies suggest favorable associations between total dairy and milk
intake and the risk of hypertension (Drouin-Chartier et al. 2016). Surprisingly, data from shorter-
term RCTs are not entirely supportive of data from observational studies. In the meta-analysis of
existing RCTs by Benatar et al. (2013), total dairy intake had no significant impact on systolic and/
or diastolic BP. This was true for both low-fat as well as whole-fat dairy. These data have been
corroborated by additional RCTs of various dairy products, including low-fat and nonfat milk,
yogurt, and full-fat cheese (Agerholm-Larsen et al. 2000b; Hilpert et al. 2009; Hjerpsted et al.
2011; Maki et al. 2013; Rideout et al. 2013; Schlienger et al. 2014; Ivey et al. 2015). We have
recently examined the impact of dairy intake (low-fat milk, low-fat yogurt, and full-fat cheese, for
a total of 3 servings/day) versus no dairy on ambulatory BP change in 76 mildly to moderately
hypertensive patients. Dairy intake significantly reduced mean daytime systolic BP in men but not
in women in comparison to the dairy-free control diet (Drouin-Chartier et al. 2014). Interestingly,
Chiu et al. (2016) have shown that the BP-lowering effects associated with the DASH diet were
preserved even when low-fat dairy products were replaced by high-fat dairy. Finally, the original
DASH study has shown that consumption of low-fat dairy products as part of a healthy diet rich in
fruits and vegetable and low in SFA led to further reduction in systolic and diastolic BP compared
with the dairy-free healthy diet (Appel et al. 1997).
RCTs assessing the impact of fermented dairy on BP have recently been meta-analyzed by
Dong et al. (2013). Pooled data from 13 RCTs indicated that consumption of fermented milk (100–
450 g/day) compared with a milk-based placebo significantly reduced systolic and diastolic BP.
Hypertensive patients appeared to be more responsive to the BP-lowering effect of fermented milk
than normotensive individuals. The analysis was based on a total of 702 normo- and hypertensive
subjects aged 35–75 years, using antihypertensive drugs or not. These results have been partly repro-
duced in the Cochrane meta-analysis by Usinger et al. (2012), according to which consumption of
fermented milk was found to significantly reduce systolic BP but not diastolic BP. However, authors
have emphasized that effect sizes were small and heterogeneous among individual RCTs, thus limit-
ing the generalizability of the results.
Thus, there are apparent discrepancies between results from RCTs and from prospective cohort
studies regarding the impact of dairy intake on BP regulation. This can, of course, be attributed to a
number of factors. In epidemiological studies, residual confounding (i.e., unmeasured confounding
factors associated with both dairy intake and BP variations) can influence the observed associations.
Limitations inherent to estimation of self-reported dietary intake from FFQ and 24 h recalls should
also be pointed out. Such confounding is less likely to occur in well-controlled RCTs. One the other
hand, it is possible that dairy consumption per se may attenuate the deterioration in BP generally
seen with aging and with weight gain, thereby being associated with more favorable BP outcomes in
the longer term. This hypothesis needs to be pursued and validated in future studies.
Measures of vascular function such as flow-mediated dilation (FMD) as well as augmentation
index (AIx) and pulse wave velocity, which both reflect arterial stiffness, have been proposed to be
more holistic markers of vascular health and predictor of cardiovascular events and mortality than
BP per se (Vlachopoulos et al. 2010; Ras et al. 2013). Vascular function can also be assessed using
surrogate markers in the blood such as the soluble adhesion molecules (e.g., intercellular adhesion
molecule-1 [sICAM-1], vascular cell adhesion molecule-1 [sVCAM-1], and E-selectin). However,
the extent to which these adhesion molecules predict the risk of CHD independent of other known
risk factors is unclear (Page and Liles 2013).
In the Caerphilly prospective cohort study of 2512 men, aged 45–59 years, who were followed
up at 5-year intervals for a mean of 22.8 years, dairy intake in quartiles was inversely related to AIx
(Livingstone et al. 2013), suggesting favorable impact from a cardiovascular health perspective.
However, other measures of arterial stiffness such as pulse wave velocity showed no association
with dairy intake. Thus, the significance of these results from a cardiovascular health perspective
therefore remains unclear. Such epidemiological data are not entirely supported by data from RCTs.
On the one hand, intake of 4 servings/day of nonfat dairy for 4 weeks has been shown to reduce
carotid–femoral pulse wave velocity with a concomitant increase in brachial FMD and cardiovagal
baroreflex sensitivity compared with baseline values among patients with elevated BP (mean BP
134 ± 1/81 ± 1 mm Hg) (Machin et al. 2015). The control, dairy-free diet (4 servings/day of fruits
products) had no such effects on these measures of vascular function. On the other hand, consump-
tion of low-fat dairy products (500 mL low-fat milk and 150 g low-fat yogurt) daily for 8 weeks had
no impact on plasma levels of sICAM-1 and sVCAM-1 compared with a control dairy-free diet (600
mL fruit juice and three fruit biscuits), suggesting no effect of low-fat dairy on endothelial func-
tion (van Meijl and Mensink 2010). In an RCT from our group, consumption of 3 servings of milk,
yogurt, and cheese for 4 weeks versus a dairy-free control diet had no significant impact on reactive
hyperemia index in a sample of 76 men and women with stage 1 hypertension (Drouin-Chartier
et al. 2014).
In general, acute and short-term (≤2 weeks) effects of dairy intake in RCTs have been associated
with favorable changes in various measures of vascular function, even in the absence of concomitant
changes in BP (Ballard and Bruno 2015). Longer-term interventions have provided less consistent
results (Ballard and Bruno 2015). From a mechanistic perspective, the vasoprotective properties of
dairy and its constituents have been suggested to be mediated through improvements in nitric oxide
bioavailability as well as to potential changes in oxidative stress, inflammation, and insulin resis-
tance (Ballard and Bruno 2015). However, the extent to which dairy foods per se versus the foods
they replace in the diet are responsible for the beneficial changes in vascular function seen in some
studies needs further investigation.
DAIRY FAT AND CARDIOMETABOLIC RISK

Dairy foods, particularly whole-fat dairy, contribute significantly to the dietary intake of SFA (Huth
et al. 2013) and this has prompted most health organizations to promote consumption of low-fat
dairy in place of regular fat dairy for optimal cardiovascular prevention. However, as already indi-
cated, the impact of SFA on plasma lipids, including LDL-C, may be influenced and partly mitigated
by the food matrix through which it is consumed (Siri-Tarino et al. 2015). This might explain to
some extent why several epidemiological studies have failed to observe a significant association
between SFA intake and CVD or CHD risk (Siri-Tarino et al. 2010; Chowdhury et al. 2014).
As reviewed in Chapter 12 of this book, dairy fats also contribute to the dietary intake of naturally
occurring trans fatty acids (TFA). Indeed, the production of milk fat by ruminants involves bacteria
that hydrogenate polyunsaturated cis fatty acids into TFA in the rumen of these animals, mostly
in the form of vaccenic acid, but also conjugated linoleic acid (CLA). Evidence from studies in
animal models has suggested potential benefits of vaccenic acid and CLA on cardiometabolic risk
(Blewett et al. 2009; Gebauer et al. 2011; Jacome-Sosa et al. 2014). While epidemiological studies
have reported unequivocal positive associations between intake of industrially produced hydroge-
nated TFA and CHD risk (Ascherio et al. 1999), neutral associations have been reported regarding
naturally occurring TFA (Gebauer et al. 2011). It has been argued that the lack of a significant asso-
ciation between intake of naturally occurring TFA and CHD risk may be due, among others, to the
very low intake of such fat in the human diet. Indeed, TFA represents only 3%–8% of total milk fat
(Stender and Dyerberg 2003). In that context, very high intakes of naturally occurring TFA are virtu-
ally unattainable in the current dietary scheme of Western countries, even those with high intake of
dairy foods (Stender et al. 2008). Nevertheless, we have quantified the dose–response relationship
between intake of naturally occurring TFA and changes in plasma lipid levels based on data from
13 RCTs (Gayet-Boyer et al. 2014). Consistent with data from epidemiological studies, we found
no relationship between intake of naturally occurring TFA of up to 4% of daily energy and changes
in cardiovascular risk factors such as the total cholesterol/HDL-C and LDL-C/HDL-C ratios. On
the other hand, Gebauer et al. have recently investigated in a double-blind, crossover feeding RCT
involving 106 healthy adults the impact of high intakes of vaccenic acid (corresponding to 3.9% of
daily energy) on blood lipids. They have shown that such high intakes of vaccenic acid significantly
increased LDL-C, apoB, HDL-C, apoAI, and Lp(a) concentrations compared with a low vaccenic
acid control diet (Gebauer et al. 2015). Once again, such high intakes of vaccenic acid are improb-
able as current estimates are well below 1% of daily energy in most countries (Gebauer et al. 2011).
It has been proposed that TFA from dairy is unlikely to have adverse effects on key lipid CHD risk
markers in healthy individuals at current dietary intake levels (Gayet-Boyer et al. 2014), but this is
not a view shared by all (Brouwer et al. 2013).
CONCLUSIONS
This chapter has highlighted a number of key points related to intake of dairy foods and cardio-
metabolic health. Firstly, consumption of dairy products, even in their whole-fat versions, appears
to have a neutral effect on plasma LDL-C concentrations. This is consistent with data from several
epidemiological studies having failed to report significant associations between SFA intake and risk
of CHD. More research is warranted to better characterize the impact of dairy consumption on other
cardiometabolic risk factors such as elevated plasma apoB levels, small dense LDL particles, HDL
function, markers of systemic inflammation, and glucose–insulin homeostasis. The impact of dairy
intake per se on BP based on data from RCTs is mixed and this topic needs further research as well,
with emphasis on longer-term studies.
Secondly, research pertaining to the impact of dairy fat per se on cardiometabolic health needs to
be undertaken with caution. Indeed, dairy fat with the exception of butter is not consumed in isolation.
Dairy fat is consumed as part of complex matrices that may modulate its impact on health. Research
should really focus on documenting the impact of whole foods rather than individual nutrients on health.
In that regard, research comparing the impact of low-fat and whole-fat versions of different dairy prod-
ucts on cardiometabolic health will be extremely insightful to better inform future dietary guidelines.
Thirdly, one aspect that contributes to confusion in the literature pertains to the fact that dairy intake
can hardly be studied in isolation. Integrating dairy foods in the diet inevitably displaces other foods
that may have more or less favorable effects on health. For example, studies have shown that isocaloric
replacement of milk by sugar-sweetened beverages (SSB) leads to increased visceral adiposity and
hepatic fat deposition (Maersk et al. 2012). Dairy foods in published RTCs have been replaced by a
wide variety of nondairy foods, which may have intrinsic beneficial or adverse health effects of their
own. Future research should be designed to compare dairy foods with other foods that they are most
likely to replace in the diet, such as milk versus juice or SSBs, or cheese versus other types of snacks.
In sum, the data available to date support the inclusion of dairy as part of a healthy diet, with virtu-
ally no evidence of potential harmful effects. The extent to which dairy intake is healthy per se remains
unclear, as dairy intake may simply be a marker of good quality diets. Further research is needed to
better define optimal dietary recommendations regarding dairy intake in terms of servings per day, and
whether there is a difference between low-fat and whole-fat dairy in terms of health outcomes.
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26 Paleolithic Diets
Staffan Lindeberg, Maelán Fontes Villalba,
Pedro Carrera-Bastos, and Lynda Frassetto
CONTENTS
Introduction.....................................................................................................................................494
Nutrition during Human Evolution.................................................................................................494
Carbohydrate-Rich Foods...............................................................................................................495
Other Plant Foods of the Paleolithic Diet.......................................................................................496
Meat................................................................................................................................................496
Fish and Shellfish............................................................................................................................497
Insects and Larvae...........................................................................................................................497
Alcohol............................................................................................................................................497
Foods with No or Minimal Contribution in Paleolithic Diets.........................................................497
Paleolithic Diets and Absence of Grains.........................................................................................498
Paleolithic Diets and Absence of Dairy..........................................................................................500
Nutritional Characteristics of Paleolithic Diets..............................................................................501
Contemporary Hunter–Gatherers and other Non-Western Groups.................................................502
The Kitava Study............................................................................................................................503
Effects of Urbanization...................................................................................................................504
Controlled Trials of “Paleolithic Diets”..........................................................................................505
Conclusion......................................................................................................................................507
References.......................................................................................................................................507
ABSTRACT
If, as indicated by some studies, prudent diets such as the Mediterranean or DASH diets can be
further improved, an evolutionary approach may be helpful. Paleolithic diets represent the food
habits during more than two million years of hominid and human evolution before the development
of agriculture. Fruits, tubers, nuts, lean meat, larvae, insects, fish, shellfish, eggs, honey, and a large
variety of vegetables have been staple foods. Contemporary non-Western populations with similar
lifestyles have shown exceptionally low rates of cardiovascular disease, obesity, insulin resistance,
type 2 diabetes mellitus, and hypertension.
Available evidence lends some support in favor, and less against, the notion that Paleolithic diets
are an appropriate template in the dietary prevention and treatment of cardiometabolic diseases.
In Memoriam
Staffan Lindeberg, MD, PhD
The authors of this chapter will always be grateful for Dr. Lindeberg’s invaluable contribu-
tions to the clinical science behind the chapter we wrote. Dr. Lindeberg died shortly after we
submitted it. May he rest in peace.
Maelán Fontes Villalba, Pedro Carrera-Bastos, and Lynda Frassetto
493
INTRODUCTION
Paleolithic* diets are sometimes seen as templates for healthy diets, based on the following
arguments:
1. very low age-adjusted rates of cardiovascular disease and other nutrition-related disorders
among contemporary hunter–gatherers and other populations minimally affected by mod-
ern habits have been reported (Eaton and Konner 1985; Lindeberg 2010).
2. hunter–gatherers and traditional horticulturalists exhibit better body composition, physical
fitness, and health biomarkers, when compared to Westernized populations and even mod-
ern rural populations (Lindeberg 2010; Carrera-Bastos 2011).
3. the majority of Westerners are affected by atherosclerosis and associated metabolic abnor-
malities, and our understanding of the main underlying causes is very limited (Lindeberg
2005, 2010).
4. among the several lifestyle factors that are very different from those of modern urban and
rural populations, diet appears to stand out (Eaton and Konner 1985; Cordain et al.
2005; Lindeberg 2010; Carrera-Bastos 2011).
5. intervention studies with Paleolithic-type diets in patients with the metabolic syndrome and/or
glucose intolerance (Lindeberg et al. 2007; Jönsson et al. 2009; Manheimer et al. 2015) show
superiority in various cardiometabolic biomarkers when compared to prudent diets.
6. the main characteristics of human physiology are essentially the same in all human popu-
lations and are obviously the result of a long evolution in Africa. Therefore, if there is a
healthier diet for humans in general, irrespective of ethnicity, it makes sense to consider
evolution and to focus on the time period up to the emergence of fully modern humans
around 200,000 years ago, well before some of them left Africa some 60,000 years ago and
different ethnic groups emerged (Campbell and Tishkoff 2010).
Accordingly, this review relates recent evidence of Western diseases, as well as common concepts
of healthy nutrition, to probable food patterns of our Paleolithic ancestors before they left Africa.
In the blogosphere and popular press, Paleolithic diets are often described as low-carbohydrate
diets with lots of meat. In the scientific discourse, Paleolithic diets represent much more. They can
even be high in carbohydrate and low in meat (Lindeberg 2010).
NUTRITION DURING HUMAN EVOLUTION

It could be argued that the Miocene vegetarian-like habitats, between 23 and 5 million years ago,
provide a proper reference for human nutrition and that we have not changed much since then
(Jenkins and Kendall 2006). In contrast, others argue that later habitats exerted strong selection pres-
sures and that humans became adapted to a high intake of meat (Brand Miller and Colagiuri 1994;
Finch and Stanford 2004; Zink and Lieberman 2016). However, neither position excludes the other
since we may be adapted to a food item and thrive on it without necessarily being dependent on it
for high reproductive success.
It is often impossible to determine, for any particular habitat and certainly over longer time
periods, the percentage of food that came from each of the available foodstuffs. The staple food
items typically consumed by our bipedal ancestors in Africa is a matter of debate, but the principal
foods available included sweet and ripe fruits and berries, shoots, flowers, buds and young leaves,
muscle meat, bone marrow, organ meats, fish, shellfish, insects, larvae, eggs, roots, bulbs, nuts, and
non-grass seeds (Gräslund 2005; Ungar 2007). In principle, these were the only types of foods that
* The Paleolithic (or Palaeolithic) is the time period of roughly 2.5 million years when hominids and humans were using
stone tools and until the development of agriculture. The first part, until around 200,000 years ago, is sometimes called the
Lower Paleolithic. Thereafter, the evolution of different ethnic groups started.
Paleolithic Diets 495
were available during human evolution, which now only provide about one quarter of the caloric
intake for the typical Westerner (Cordain et al. 2005). Most of us now get the greater part of our
energy from grains (grass seeds), dairy products, refined fat and sugar, and legumes (Cordain et al.
2005; Statens Livsmedelsverk 2012; U.S. Department of Agriculture). In addition, we have very
little variation among plant foods today.
CARBOHYDRATE-RICH FOODS
Our primate ancestors may have consumed fruits more or less regularly during the 50 million years
until they became bipedal around 6 million years ago (Bloch and Boyer 2002; Gräslund 2005; Ungar
2007). For chimpanzees, bonobos, and orangutans, fruit makes up more than 75% by weight of the
diet. Fruit was also the most common plant food among twentieth-century hunter–gatherers, as
described in the Ethnographic Atlas (n = 229) (Cordain et al. 2000a). Fruits differ from other edible
plants in that they contain appreciable amounts of fructose, a monosaccharide that typically consti-
tutes 20%–40% of available carbohydrates in wild fruits (Ko et al. 1998; Milton 1999; Dzhangaliev
et al. 2003) and 10%–30% in cultivated fruits (National Food Administration 1986). Honey, where
50% of the carbohydrate is fructose, may have been consumed in considerable amounts in some
habitats, at least for a few months per year (Allsop and Miller 1996; Marlowe et al. 2014).
A high intake of fructose, in particular from sodas and processed foods, has been proposed to
cause abdominal obesity and associated metabolic disturbances, including diabetes type 2, high
blood pressure, blood lipid disorders (high triglycerides and low HDL cholesterol), hyperuricemia,
and fatty liver (Johnson et al. 2009; Park and Yetley 1993; Sanchez-Lozada et al. 2008). However,
strictly controlled experiments suggest that the amounts of fructose that may be harmful are consid-
erably higher than what is possible to get from whole fruit (Sievenpiper et al. 2009, 2012; Cozma
et al. 2012; Kuzma et al. 2015). There is much evidence suggesting that any concerns with fructose
are outweighed by beneficial attributes of whole fruit, such as high nutrient density, fiber content,
low glycemic index, and high water content lending volume. Notably, fruit has been an essential part
of dietary patterns for which there are data for cardiovascular benefit. For comprehensive reviews of
dietary fructose, see Chapters 13 through 15.
Starchy underground storage organs (roots, tubers, bulbs, corms) may have become staple foods
during periods of repeatedly dry and cool climates perhaps around one million years ago (Luca et al.
2010) or even before that (Laden and Wrangham 2005). Humans have a relatively high activity of
salivary amylase in comparison with other primates (Samuelson et al. 1990; Perry et al. 2007) and our
tooth morphology, including incisal orientation, seems well adapted to chewing tubers (Lucas et al.
2006). In order to increase the caloric yield per workload, roots and tubers may often have been an
adequate choice (O’Connell et al. 1983), especially after cooking (Wrangham and Conklin-Brittain
2003). It has been suggested that the multiplication of the salivary amylase gene became selectively
advantageous in terms of evolutionary fitness only when cooking became widespread (Hardy et al.
2015). The period for this change in human subsistence with regular use of fire and habitual cooking is
a matter of speculation, but it might have been more than 300,000 years before the present or possibly
long before that (Hardy et al. 2015).
The excellent health status among starch-eating ethnic groups (Sinnett 1977; Lindeberg 2010),
including the Kitavans described subsequently, contradicts the notion that a high intake of starch
would be associated with obesity and type 2 diabetes (Spreadbury 2012; Mann et al. 2014; Naude
et al. 2014). Although a high starch load undoubtedly raises blood sugar after a meal, the main
causes of an individual’s inability to limit blood sugar rise after eating carbohydrates (i.e., glucose
intolerance) remain obscure, and it is questionable whether dietary starch plays a causative role
(Reaven 2005; Due et al. 2008; Brinkworth et al. 2009). Low-carbohydrate diets can sometimes
decrease fasting glucose more than low-fat diets (Samaha et al. 2003; Krebs et al. 2013; de Luis
et al. 2015), but it is uncertain if this is due to the reduction of carbohydrate per se or some associ-
ated dietary changes (Gannon and Nuttal 2006; Lindeberg 2010).
In addition to the questionable effect on glucose tolerance, the proportion of carbohydrate in

the diet has not been proven to materially affect body weight (Clifton et al. 2014). The marginally
greater weight loss with carbohydrate restriction in obesity, as opposed to restriction of fat or protein,
does not suggest that dietary starch is a major cause of obesity (Clifton et al. 2014).
OTHER PLANT FOODS OF THE PALEOLITHIC DIET

Tree nuts provide a high amount of energy even when they must be collected and cracked one by one
for consumption, and were probably an essential part of the diet in some of the occupied habitats. Tree
nuts are typically rich in unsaturated fat, protein, soluble fiber, and various micronutrients, while low in
saturated fat (Lindeberg 2010). Despite their high energy density, in Western populations the effect of
tree nuts on caloric intake and adiposity appears to be neutral (Flores-Mateo et al. 2013) and their effect
on cardiovascular risk factors appears to be beneficial (Del Gobbo et al. 2015). Nuts have also been an
important component of the DASH diet (Appel et al. 1997), Mediterranean diets (Estruch et al. 2013),
and the Portfolio diet (Jenkins et al. 2003), all of which have shown beneficial effects in randomized
controlled trials. Observational studies also suggest that increased intake of tree nuts is inversely associ-
ated with ischemic heart disease and type 2 diabetes (Afshin et al. 2014).
A large quantity of nonstarchy vegetables (leafy vegetables, flowers, buds) from many different
plant species were also staple items of hunter–gatherer diets with a small contribution to caloric intake
(Isaacs 1987; Kuhn and Stiner 2001; Terashima and Ichikawa 2003; Termote and van Damme 2010).
MEAT
Another food that could provide a high-energy yield for preagricultural humans is meat, which is
even consumed in considerable amounts by the chimpanzee (Stanford 1999; Pruetz et al. 2015). In
one observational study, adult chimpanzees consumed an average of 65 g meat per day in the dry
season (Stanford 1999). For humans, available paleontologic evidence is consistent with, but does not
prove, regular high meat intake in the last 2 million years (Finch and Stanford 2004; Ungar 2007).
Contemporary hunter–gatherers have generally been able to eat large amounts of meat or fish, although
the figures are based on rather imprecise ethnographic data (Cordain et al. 2000a; Panter-Brick et al.
2001). Of the 229 hunter–gatherer populations studied during the twentieth century, the majority
(73%) were estimated to get more than half their caloric intake from meat, fish, and shellfish (Cordain
et al. 2000a). Among five African populations, for which more exact quantitative data were available,
meat and/or fish constituted on average 26%, 33%, 44%, 48%, and 68% of the food (Cordain 2006).
Contemporary hunter–gatherers have had exceptionally favorable levels of serum cholesterol,
blood pressure, and other cardiovascular risk factors, even with very high meat consumption
(Lindeberg 2010). For instance, Truswell found no evidence of sudden, spontaneous death when
interviewing 96 adults among the San tribe, hunter–gatherers with a high meat intake in the Kalahari
desert, Botswana, South Africa (Truswell and Hansen 1976). However, wild game meat has a lower
fat content, a higher percentage of omega-3 fatty acids and a lower omega-6/omega-3 ratio than
domestic meat (Naughton et al. 1986; Mann 2000; Cordain et al. 2002).
Observational studies among Western populations have found that a high intake of red meat, and
in particular processed meats, is associated with an increased risk of cardiovascular disease and dia-
betes type 2 (Chen et al. 2013; Mozaffarian 2016). There is substantial controversy and uncertainty
as to whether unprocessed red meats increase cardiometabolic risk and the extent to which residual
confounding explains any increase (Micha et al. 2010; Mozaffarian 2016). Randomized controlled
trials have shown that, within the pattern of a healthy diet, there is no difference between red and
white meat effects on LDL-cholesterol (Davidson et al. 1999; Maki et al. 2012; Roussell et al. 2012).
Moreover, animal experiments do not suggest that meat causes atherosclerosis and the notion that
“animal protein” causes atherosclerosis is based on studies with milk proteins, typically casein (Foo
et al. 2009; Lindeberg 2010). For instance, one study in hamsters found that meat protein (from
bison and beef, respectively) resulted in less pronounced atherosclerosis than soy protein and casein
(Wilson et al. 2000).
Furthermore, red meat is a rich source of high-quality protein and high content of bioavailable
essential nutrients, many of which are lacking in the diets of some population groups (Wyness
2016). Therefore, lean nonprocessed red meat can hypothetically be a healthy option in the context
of a Paleolithic diet. In addition, Paleolithic diets are not necessarily high in meat. In fact, due to its
high bioavailable essential nutrients, a moderate intake (70 g/day) can be sufficient to meet nutri-
tional requirements (Wyness et al. 2011; Wyness 2016).
FISH AND SHELLFISH

Paleolithic African humans appear to have lived at times by the shores of lakes, rivers, and, presum-
ably, the sea, where they could catch fish and shellfish, although their dependence on marine food
has not been proven (Richards 2002; Kuipers et al. 2010). Omega-3 fatty acids from fish have been
suggested to prevent coronary heart disease, although randomized controlled trials suggest that the
effect is limited (Hooper et al. 2004, 2006). Observational studies have found an approximately 20%
lower cardiovascular risk among Westerners who eat fatty fish at least twice a week, compared with
those who do not (Schmidt et al. 2000; Hu et al. 2001). Possibly, this only applies to high-risk popula-
tions (Marckmann and Gronbaek 1999). The notion that fish is protective originally emerged from an
observed very low incidence of myocardial infarction and sudden cardiac death among the Inuits of
Greenland and Canada (Schaefer 1971; Bang and Dyerberg 1972). Of all the characteristics of Inuit
dietary habits that could possibly explain these findings, virtually all focus has been on omega-3 fatty
acids, while the absence of common Western foods has rarely been considered. For comprehensive
reviews of omega-3 fatty acids and cardiovascular prevention and treatment, see Chapter 10.
INSECTS AND LARVAE

The consumption of insects and larvae is thought to have been substantial in most African habitats
during the Paleolithic period, and they may have provided an important source of protein and fat
(DeFoliart 1999; Ungar 2007; Raubenheimer et al. 2014). Nonhuman primates regularly consume
them (Basabose 2002; Raubenheimer et al. 2014; Rothman et al. 2014). Yet, our knowledge of
their possible health effects is extremely limited.
ALCOHOL
The extent to which alcohol could have been a regular part of human’s original environment is
unknown (Kiple and Ornelas 2000). If storage vessels for alcohol were made of leather or plants
in earlier prehistoric times, they have long since disappeared without a trace. Even without delib-
erate production of alcoholic beverages, a low-level dietary exposure to ethanol via ingestion of
fermented fruit may have characterized our lineage of humans and human-like ancestors for about
40 million years (Smith 1999; Dudley 2002). For a comprehensive review of alcohol and cardiovas-
cular disease, see Chapter 31.
FOODS WITH NO OR MINIMAL CONTRIBUTION IN PALEOLITHIC DIETS

In the context of public health, it may hypothetically be more crucial to focus on foods that were
not staple items during human evolution, instead of trying to estimate accurately intake of foods that
were. On average, roughly 75% of the calories in Western countries are today provided by foods that
were practically unavailable during human evolution: wheat and other cereal grains, dairy foods,
refined fats and sugar (Cordain et al. 2005). In addition, the intake of sodium and chloride is now
considerably higher (Cordain et al. 2005).
PALEOLITHIC DIETS AND ABSENCE OF GRAINS

During our evolution, wild seeds were available from various plants, but not from the grass family
(Poaceae), which includes today’s wheat, rice, maize, and so on, and rarely or never from one plant
species every day. There is evidence for sporadic consumption of legume seeds during the latter
part of the Paleolithic period (Jones 2009). Nevertheless, seeds from legumes apparently became
staple foods only during the emergence of agriculture, as evidenced by gradual changes in their
form and quality as a consequence of domestication (Zohary and Hopf 1973; Berger et al. 2003).
Contemporary hunter–gatherers, in particular those living in arid, hot, marginal environments
(Australian Aborigines, Kalahari Bushmen), often include large, fatty seeds in their diet, but these
provide a relatively small amount of energy on an annual basis, and much less than is now provided
by wheat, rice, or maize (Lee 1968; O’Connell et al. 1983; Cordain et al. 2005).
When seeds from any one particular plant species are consumed in large quantities on a regular
basis, an interesting situation arises. The plant kingdom contains thousands of bioactive substances
and other natural chemicals, called phytochemicals, many of which are thought to be part of the
defense system against herbivores (Wynne-Edwards 2001; Herrera and Pellmyr 2002). The highest
concentrations of phytochemicals are generally found in the most vital parts of the plant, namely
sprouts, seeds, and beans and can make up 5%–10% of the plant’s dry weight (Perantoni 1998).
Prehistoric foragers were able to limit the intake of each of them by having access to a large number
of various plant species, by consciously avoiding the most poisonous ones (Ulijaszek and Strickland
1993), and by cooking, which destroys some of these plant chemicals (Wrangham and Conklin-
Brittain 2003). Most phytochemicals have not been properly studied with regard to their effects on
human health and only a few examples will be mentioned here.
One example is plant lectins, which are glycated sugar-binding proteins (Chrispeels and Raikhel
1991; Van Damme et al. 1998), and which are found in the highest concentration in cereal grains, beans,
potatoes, and peanuts. Notably, unrefined grain products have a higher lectin content than refined seed
products (van Damme et al. 1998). Lectins in wheat, rye, rice, and potatoes bind to GlcNAc-domains
(GlcNAc = N-acetylglucosamine) on receptors in the “host organism,” receptors of crucial importance
in metabolic programming mainly through the hexosamine biosynthetic pathway (Hardivillé and Hart
2014). Lectins are not completely destroyed during normal cooking (Freed 1999), although hydrat-
ing beans and then cooking them to 100°C for a minimum of 10 min significantly decreases its lectin
content (Grant et al. 1982). Nevertheless, these types of procedures are not normally applied to cereal
grains, so it is expected that these type of foods present a significant lectin content even after cooking
(Cordain 1999; Freed 1999). This may be clinically relevant since lectins are also relatively resistant
to enzymatic breakdown in the gastrointestinal tract, are thought to penetrate the intestinal mucous
membrane (Liener 1986; Pusztai et al. 1993), and thereby enter the bloodstream (Pusztai et al. 1989;
Wang et al. 1998; Cordain 1999). One of the most studied lectins in in vitro experiments and animal
models is the wheat lectin (wheat germ agglutinin, WGA). WGA may contribute to atherosclerosis
since it can bind to macrophages and smooth muscle cells of the vessel wall (Davis and Glagov 1986;
Kagami et al. 1991), activate the epidermal growth factor receptor (Wang et al. 2001) and the toll-like
receptor-2 (Unitt and Hornigold 2011), and increase the synthesis of IL-12, IL-2, INFγ, TNFI, and
IL-1 (Muraille et al. 1999; Sodhi and Kesherwani 2007). Activation of the epidermal growth factor
receptor and the toll-like receptor-2, hypothetically, can cause dysfunction of the glycocalyx, the pro-
tective barrier covering the endothelium of blood vessels, thereby allowing atherosclerosis to progress
(Henry and Duling 2000; Drake-Holland and Noble 2009; Becker et al. 2010; Liu et al. 2011; Reitsma
et al. 2011; Pahwa et al. 2016). Another lectin of potential relevance is peanut lectin, which has been
shown to enter the systemic circulation in humans (Wang et al. 1998) and to produce atherosclerosis in
rabbits (Kritchevsky et al. 1981, 1998). This may offer an explanation for the unexpected atherogenic
effect of peanut oil in various animal models (monkeys, rabbits, and rats) (Cordain 1998).
Wheat is also rich in gliadins, which are usually designated as prolamins with lectin-like properties.
Prolamins (together with glutelins that compose gluten) are present in the wheat endosperm, while
WGA is more abundant in the germ. There is ex vivo human evidence that gliadins can increase
intestinal permeability not only in individuals with celiac disease, but also in wheat-sensitive and normal
individuals (Hollon et al. 2015), although in vivo human studies are necessary before we can reach
a definitive conclusion. There is also in vitro and animal evidence that WGA may increase intestinal
permeability (Cordain et al. 2000b; Dalla Pellegrina et al. 2009). Increased intestinal permeability may
lead to endotoxemia, which may cause low-grade chronic inflammation (Carrera-Bastos 2011). Chronic
inflammation has been proposed as an important component in the pathogenesis of atherosclerosis and
insulin resistance (Frostegård 2013; Straub 2014). We need better data from randomized controlled
trials before we can draw any conclusions regarding cereal grains.
The leptin receptor, which is activated by the satiety hormone leptin, is glycosylated (Haniu et al.
1998) and, as such, is expected to bind lectins and lectin-like molecules like gliadin. In vitro, WGA has
been shown to bind to the leptin receptor (Kamikubo et al. 2007), whereas peptides derived from wheat
gluten digestion inhibited the ability of leptin to bind to its receptor (Jönsson et al. 2015). Reduced
leptin action and/or leptin resistance appears to be an early step in ectopic lipid deposition, lipotoxicity,
and the metabolic syndrome (Unger and Scherer 2010). Highly relevant in this context are findings in
C57BL/6 mice fed ad libitum isocaloric diets matched for macronutrient composition, where gluten
intake promoted weight gain and increased plasma levels of leptin, IL-6, and other pro-inflammatory
cytokines and adipokines independent of energy intake and diet macronutrient composition (Soares
et al. 2013; Freire et al. 2016). In one of these studies, radiolabeled gluten was detected both in the cir-
culation and in peripheral organs including visceral adipose tissue, showing once again that this protein
is not fully degraded into amino acids before absorption, and that direct action of gluten on these tis-
sues may be possible (Freire et al. 2016). In fact, nondegraded gliadin has been found in the breast milk
of healthy mothers (Chirdo et al. 1998). Another pertinent finding is cytotoxicity and accumulation
of intracellular lipid droplets in cell cultures exposed to gliadins (Dolfini et al. 2003). Together, these
findings, if replicated in humans, may be relevant to the lipid-laden foam cells of early atherosclerosis
(Beltowski 2006) as well as progressive beta cell failure and other aspects of the metabolic syndrome
and type 2 diabetes (Jönsson et al. 2005; Unger and Scherer 2010).
Cereals may also negatively affect glucose metabolism by means of their glycated proteins. Again, the
best studied and perhaps more relevant example considering the current dietary and cooking patterns is
the wheat germ agglutinin, which has been found to bind to several hormone receptors including insulin
receptors and other tyrosine kinase receptors (namely IGF-1 and EGF receptors) (Cuatrecasas and Tell
1973; Shechter 1983; Wang et al. 2001). The binding of WGA to the insulin receptor is strong and
long-lasting with high molecular efficiency, suggesting that it may hinder insulin effects for many hours
(Cuatrecasas and Tell 1973; Shechter 1983, #5701; Lindeberg 2010). Hence, it is theoretically capable of
causing insulin resistance. Furthermore, WGA increases glycolysis (Yevdokimova and Yefimov 2001) as
well as fat storage (Freed 1991). In contrast to insulin, which has the same effects, WGA does not seem to
stimulate protein synthesis (Pusztai et al. 1993), which is relevant since loss of muscle mass (sarcopenia)
has been suggested to impair insulin sensitivity (Cleasby 2016). Nevertheless, at this point, it remains a
theory and data from randomized controlled trials are necessary.
Another example of phytochemicals in legumes and seeds are the protease inhibitors, which
inhibit protein-degrading enzymes in the digestive tract such as trypsin, chymotrypsin, and amylase.
This very ancient defense mechanism of plants allows their seeds to pass through the gastrointestinal
system undamaged. The concentration of protease inhibitors in legumes and cereals is so high that the
digestion of dietary proteins other than those in the seed can be substantially reduced (Cordain 1999).
This increases the risk of undigested (and potentially bioactive) proteins and peptides entering the
circulation in susceptible individuals with increased intestinal permeability. Interestingly, an in vivo
study demonstrated that wheat alpha-amylase/trypsin inhibitors, which appear to resist proteolytic
digestion (Schuppan and Zevallos 2015), can activate the innate immune system both in individuals
with and without celiac disease, and therefore upregulate inflammation (Junker et al. 2012).
Our knowledge of the health effects of the many bioactive substances in grains and legumes
remains fragmentary. Nevertheless, one of the few controlled dietary intervention trials with hard
endpoints, DART (Diet And Reinfarction Trial), reported a tendency toward increased cardiovascu-
lar mortality in the group advised to eat more fiber, the majority of which was derived from cereal
grains (Burr et al. 1989). This nonsignificant effect became statistically significant after adjust-
ment for possible confounding factors, such as medication use and health status (Ness et al. 2002).
Furthermore, Cochrane systematic reviews concluded that the quality of evidence is poor and that
there is not enough evidence to recommend whole grain cereals for coronary heart disease (Kelly et al.
2007) and type 2 diabetes (Priebe et al. 2008).
Thus, although whole grains are increasingly recommended to increase fiber intake, it might be
prudent for patients at high cardiovascular disease risk to obtain most of their dietary fiber from
nuts, tubers, fruit and vegetables, until better and more definitive data from randomized controlled
trials in cardiovascular disease patients testing whole grains against other plant foods (as opposed to
refined grains as is often the case) are available.
PALEOLITHIC DIETS AND ABSENCE OF DAIRY

The impact of dairy foods on cardiovascular health has been debated for decades. A human autopsy
study in 1960 found myocardial infarction to be more common among patients who had undergone
the milk-based Sippy diet, than among those treated at hospitals where the Sippy diet had not been
prescribed (Briggs et al. 1960). Internationally, cardiovascular mortality has been positively associ-
ated with the intake of dairy products (Segall 1994), and secular trends in milk consumption have
correlated positively with changes in mortality rates of coronary heart disease in Europe (Moss and
Freed 2003). However, prospective cohort studies within Western populations have shown a slightly
lower risk with higher intake of dairy, or no association, suggesting that lifestyle factors other than
dairy foods are more important to explain the variation of coronary heart disease and stroke in such
populations (Elwood et al. 2005; Soedamah-Muthu et al. 2011; Rice 2014; Praagman et al. 2015).
Dairy may also improve cardiometabolic health, due to some of its numerous bioactive peptides, which
have been proposed to exert various beneficial effects, namely inducing satiety and hence contributing
to weight management, increasing insulin secretion, and decreasing blood pressure (Nongonierma and
FitzGerald 2015). Regarding the latter effect, there is evidence from randomized controlled trials that
milk-derived peptides present in both the whey and casein fractions of milk modestly decrease blood
pressure (Xu et al. 2008; Nongonierma and FitzGerald 2015). In accordance with this, following the
DASH diet, which includes dairy products, has been shown to lead to a reduction in blood pressure
(Appel et al. 1997; Chiu et al. 2015). However, the specific effect of dairy (as opposed to isolated dairy-
derived proteins or peptides) have not been properly investigated in randomized controlled trials against
the DASH diet, which minimize confounders by choosing adequate control foods and matching each
group for protein and calcium intake. Indeed, protein, and especially calcium intakes are normally high
in the dairy groups and low in the nondairy ones (Machin et al. 2014).
It has been known for decades that milk, despite presenting a low glycemic index, elicits a
high insulin response (Nilsson et al. 2004; Melnik 2015a). Theoretically, this could be beneficial
for glucose control in type 2 diabetic patients, but because milk has the ability to overactivate the
mechanistic target of rapamycin complex 1 (mTORC1), a high milk diet could increase insulin
resistance (Melnik 2015a,b). Indeed, randomized controlled trials in Danish boys have found that
high intake of cow’s milk, but not meat, markedly increased plasma levels of insulin, IGF-1, and
IGF-1/IGFBP-3 (Hoppe et al. 2004), and induced insulin resistance, assessed by calculation with
the homeostasis model assessment (HOMA) from fasting concentrations of glucose and insulin
(Hoppe et al. 2005). Moreover, animal studies have found that casein, the dominant milk protein,
promotes atherosclerosis (Wilson et al. 2000; Foo et al. 2009) as well as insulin resistance (Lavigne
et al. 2001) apparently by inducing lipotoxicity (Ascencio et al. 2004; Tovar et al. 2005; Tovar and
Torres 2010). In addition, in obese men whey protein increases glucagon (Hutchison et al. 2015),
a hormone increasingly suspected of playing a central role in the metabolic syndrome and type 2
diabetes (Unger and Scherer 2010; Lee et al. 2014).
Of interest, Allen and Cheer found a striking inverse association between diabetes and lactase per-
sistence rates with the latter explaining almost half of the worldwide variation in diabetes prevalence,
indicating cow’s milk as a potential contributing factor behind type 2 diabetes (Allen and Cheer 1996).
The uncertainty of the results of a recent systematic review of randomized controlled trials analyzing
the effect of dairy upon insulin sensitivity (Turner et al. 2015a) led the authors to themselves conduct a
trial in overweight and obese subjects, demonstrating that a diet high in primarily low-fat dairy (from
milk, yogurt, or custard) with no red meat reduced insulin sensitivity more than a diet high in lean red
meat with minimal dairy, and a control diet that contained neither red meat nor dairy (Turner et al. 2015b).
Given the conflicting studies mentioned, it might be prudent not to increase dairy intake in
patients with cardiometabolic diseases until more definitive data from randomized controlled trials
with adequate control meals and matched nutrient intakes between groups are available.
NUTRITIONAL CHARACTERISTICS OF PALEOLITHIC DIETS

In addition to being more or less devoid of grains and dairy, refined fats and sugar were obviously
not included in Paleolithic diets. Hence, total intake of fiber and most micronutrients was generally
higher (Cordain et al. 2005; Lindeberg 2010). There are no known nutrients in grains, milk, or
refined fats required by humans that are not provided by a mixture of meat, fish, shellfish, vegetables,
fruit, nuts, and eggs (Lindeberg 2010). One exception is calcium intake, which may not always have
achieved current recommendations, especially when the intake of green leafy vegetables was low
(Cordain et al. 2005; Lindeberg 2010).
However, Paleolithic-type diets, low in salt and high in fruits, vegetables, and tuber, are net base-
yielding diets, which can decrease calcium excretion and hence calcium requirements (Frassetto et al.
2001, 2007, 2008). This is because diets with high contents of fruits and vegetables are high in the pro-
portion of base-containing precursors to acid-containing precursors derived from biochemical break-
down of amino acids, fats, and organic anions in food (Lennon and Lemann 1968). While all foods
contain acid precursors, only fruits and vegetables contain base precursors (Frassetto 2008). Sebastian
et al., taking into account various geographies and climates, came up with acid-base estimates of
potential Paleolithic-type diets with varying food compositions, and found that the vast majority of
them were net base-producing (Sebastian et al. 2002). Diets low in acid precursors lower calcium
excretion, while diets high in acid precursors increase calcium excretion (Lemann et al. 1967).
As an example, a study in individuals with the metabolic syndrome showed that a Paleolithic diet
led to lower calcium and magnesium excretion compared to the control diet (an isoenergetic healthy
reference diet, based on the guidelines of the Dutch Health Council), which together with higher
magnesium intake would not compromise calcium homeostasis (Boers et al. 2014).
Another potential nutrient possibly consumed in insufficient amounts with a Paleolithic-type
diet is iodine. Since iodized salt and dairy products were not available, only those ancestors with
high regular access to fish or shellfish would be expected to have reached the currently recom-
mended intake of iodine (unless they were regularly consuming animal thyroids) (Lindeberg 2009).
This suggests that including moderate amounts of fish, shellfish, and minor amounts of algae in
“modern” Paleolithic-type diets might be needed to prevent possible iodine insufficiency.
Regarding macronutrients, Paleolithic diets were often high in protein, typically 15%–35% of
energy (E%), but not always low in carbohydrate (Cordain et al. 2000a; Kuipers et al. 2010).
Most available foods during the Paleolithic era were voluminous, with a high water and fiber con-
tent, and therefore had a low energy density. This is possibly one of the more important characteris-
tics of Paleolithic diets, since it may prevent excessive energy consumption, perhaps by increasing
satiety (Rolls et al. 2005; Jebb 2007). In animal experiments, restriction of dietary energy has been
found to increase life span in dogs, rats, mice, fish, worms, yeast, and fruit flies, but not in primates
(Heilbronn and Ravussin 2003; Fontana and Klein 2007; Colman et al. 2009). Although calorie
restriction has not been shown to retard atherosclerosis or prolong life, markedly beneficial effects
have been noted on cardiovascular risk factors in controlled trials in nonhuman primates (Bodkin
et al. 2003; Colman et al. 2009). Nevertheless, in humans, the effect of caloric restriction on hard
endpoints, such as premature death, has not been investigated systematically (Zhao et al. 2014).
CONTEMPORARY HUNTER–GATHERERS AND OTHER NON-WESTERN GROUPS

The rarity of ischemic heart disease has been noted in a number of clinical investigations and autopsy
studies in Melanesia, Malaysia, Africa, South America, and the Arctic. A British autopsy study in
Uganda, East Africa, at the beginning of the 1950s revealed only 1 in 1427 people (0.7%) above the
age of 40 years with histologic signs of previous myocardial infarction (Thomas et al. 1960). Among
age-matched subjects in the United States, a high occurrence of a previous myocardial infarction
was noted (Table 26.1).
Roughly, two dozen studies from Papua New Guinea paint the same clear picture; prior to urban-
ization, myocardial infarctions were unknown among the local population. These studies include
two systematic reviews of 2000 and 3999 hospital case records, respectively, completed during the
first half of the twentieth century (Campbell and Arthur 1964; Dewdney 1965).
Additional support comes from three systematic interviews in the original home environment
(Truswell and Hansen 1976; Sinnett 1977; Lindeberg 1994). Truswell found no evidence of sud-
den, spontaneous death when interviewing 96 adults among the San tribe, hunter–gatherers in the
Kalahari desert, Botswana, South Africa (Truswell and Hansen 1976). Sinnett noted no occurrence
of retrosternal chest pains matching angina pectoris among the Murapin in the highlands of Papua
New Guinea (Sinnett 1977). However, it is difficult to distinguish these pains from musculoskeletal
pain in the rib cage during an interview.
In addition to the rarity of ischemic heart disease, the medical records from Kenya and Uganda,
which became British protectorates in 1920, strongly indicate the absence of noninfectious stroke
in these countries before 1940 (Trowell and Burkitt 1981; Lindeberg 2010). British doctors in the
1920s, who worked in East Africa after receiving their training in Great Britain, have documented
this very convincingly. At the medical clinic in Kampala, Uganda, there was no case of noninfectious
stroke among 269 consecutive patients with neurological diseases (Muwazi 1944). Furthermore, in
an overview of 3000 careful autopsies in Uganda during the 1930s and 1940s, 4 cases of cerebral
hemorrhaging, but no cases of ischemic stroke were uncovered (Davies 1948). Hugh Trowell, in his
meticulous accounts of noninfectious diseases of East Africa, did not record any cases of ischemic
stroke among the aboriginal population, despite his almost 30 years as a doctor and researcher in
Uganda (Trowell 1960). The medical literature also highlights the absence of stroke in Papua New
Guinea before 1970 (for review, see Lindeberg 2010).
The prevalence of diabetes among hunter–gatherers has not been studied using current methods,
but indirect studies of glucose tolerance, blood sugar, serum insulin, waist circumference, and other
TABLE 26.1
Percentage of Deceased Men in the United States and Uganda
1951–1956 with Signs of Previous Myocardial Infarction at Autopsy
Age, Years United States Uganda
40–49 31 of 178 (17%) 0 of 178
50–59 51 of 199 (26%) 1 of 199
60–69 32 of 98 (33%) 0 of 98
70–79 8 of 24 (33%) 0 of 33
≥80 2 of 9 (22%) 0 of 9
Source: Thomas, W. et al., Am. J. Cardiol., 5, 41, 1960.

TABLE 26.2
Blood Pressure at Age 40–60 Years among Hunter–Gatherers,
in Kitava and in Sweden (mm Hg, Mean ± Standard Deviation)
Population Men Women Reference
Kung San 108/63 ± 11/7 118/71 ± 13/6 Kaminer and Lutz (1960)
Yanomamo 104/65 ± 8/8 102/63 ± 14/12 Oliver et al. (1975)
Xingu 107/68 ± 20/11 102/66 ± 23/11 Baruzzi and Franco (1981)
Kitava 113/71 ± 13/7 121/71 ± 16/8 Lindeberg (1994)
Sweden 134/92 ± 15/10 126/86 ± 16/11 Lindeberg (1994)
related variables provide evidence of a markedly low prevalence of type 2 diabetes and the meta-
bolic syndrome (Joffe et al. 1971; Merimee et al. 1972; Spielman et al. 1982; Lindeberg 2010).
Many studies have shown that being overweight is extremely rare among hunter–gatherers and
other traditional cultures (for review, see Lindeberg 2010). This simple fact is quickly apparent to
all foreign visitors. The average BMI at 40 years of age is typically around 20 kg/m2 for men and
19 kg/m2 for women. After the age of 40, BMI for both sexes drops, because both muscle mass
and water content decrease with age and because fat is not increasingly accumulated (Gallagher
et al. 2000).
Regarding hypertension, multiple studies have convincingly shown that it is very rare among
hunter–gatherer populations, and that the average blood pressure is low compared to the Western
world (for review, see Lindeberg 2010). Table 26.2 shows the average blood pressure in middle-aged
hunter–gatherers, traditional horticulturalists in Kitava, Trobriand Islands, and in Sweden. Blood
pressures increase with increasing BMI, which may be one reason why hunter–gatherers have lower
blood pressures (Droyvold et al. 2005).
THE KITAVA STUDY

Around 1990, our research group performed a survey in the island of Kitava, Trobriand Islands,
Papua New Guinea, where we noted an apparent absence of cardiovascular disease and associated
risk factors among Kitava’s 2,300 inhabitants (6% of which were 60–95 years old), as well as among
the remaining 23,000 people in the Trobriand Islands (Lindeberg and Lundh 1993; Lindeberg 1994,
2010; Lindeberg et al. 1997). The Kitavans are not hunter–gatherers but their staple foods resemble
what could have been available to preagricultural humans in Africa. Yam, sweet potato, taro, and
fruit were staple foods in Kitava, while grains, dairy, and refined fats and sugar were absent from
the diet.
In our systematic surveys, we noted a lack of sudden cardiac death, exertion-related chest pain
suggestive of angina pectoris, nor any cases of hemiplegia, aphasia, or sudden imbalance (Lindeberg
and Lundh 1993). The survey responses covered at least two past generations, probably more, due
to detailed knowledge of living and dead fellow residents. Clinical examinations did not reveal
any manifestations of stroke. The results were confirmed by physicians and medical scientists with
extensive knowledge of the Trobriand Islands and other parts of Melanesia. Child mortality from
malaria and other infections was relatively high, and the estimated average life span was around
45 years. The remaining life expectancy at 45 years of age is more difficult to determine, but may
be similar to Swedish figures. Our age estimates were based on known historical events. The main
causes of death after age 45 were infections, accidents, and senescence.
In Kitava, diabetes, being overweight, and hypertension were absent, and the average fasting
serum glucose and insulin levels were markedly lower than in Sweden (Lindeberg et al. 1999).
The average BMI at 40 years of age was approximately 20 kg/m2 for men and 19 kg/m2 for women
(Lindeberg 1994). No Kitavan person was larger around their waist than around their hips.
Blood pressure was measured in 272 Kitavans, aged 4–86. Compared to Westerners, the most marked
difference was seen for diastolic blood pressure, which was low in all age groups, with an average of
70.4 ± 6.7 mm Hg (range 51–89). Among adults, diastolic blood pressure did not increase with age.
In a European population, half of those over age 40 would fall above the highest measured value in
Kitavans (Lindeberg 1994). The extent to which blood pressure rises after middle age in traditional
populations is not clear. In Kitava, after the age of 40, there was a significant increase of systolic blood
pressure among men (r = 0.12, p = 0.0005, n = 92) and women (r = 0.12, p = 0.01, n = 46). On average,
the systolic pressure among the 15 men over the age of 75 was 133 mm Hg (range 100–162). This
was significantly (p = 0.002) higher than among the 31 men who were between the ages of 60 and 74,
whose average blood pressure was 115 mm Hg (range 84–166). No other previous study of traditional
populations had included so many different age groups as the Kitava study. In addition, the accuracy
of our age estimates was unusually high.
While the metabolic syndrome was apparently absent, serum lipids showed more of an overlap
with Western populations. Total and LDL cholesterol levels among Kitavan men were somewhat
lower than among Swedish men, and close to today’s Japanese, while Kitavan women had levels
comparable with Swedish women, especially for women below 60 years of age (Lindeberg 1994).
A possible contributing factor is a high intake of saturated fat from coconut, approximately the same
as in Sweden (Lindeberg and Vessby 1995). In fact, one could expect an even higher level of serum
cholesterol from high intake of saturated fat, and since the two major fatty acids in coconut, 12:0
lauric acid and 14:0 myristic acid, are considered to have a stronger cholesterol-raising effect than
16:0 palmitic acid, which is the dominant fatty acid in Western countries (Becker and Pearson 2002).
Lauric and myristic acid raise LDL and HDL, thereby contributing to a lower Total:HDL cholesterol
ratio than palmitic acid (which raises Total:HDL ratio) (Mensink et al. 2003).
In other surveys among aboriginal populations, the picture in terms of blood lipids is not as con-
sistent as it is in terms of their lack of being overweight, or having abdominal obesity and high blood
pressure. There are examples of populations with a very low prevalence of ischemic heart disease
despite blood lipid levels that would be considered high risk among Westerners. The Kitava study
adds to the notion that some of our most common diseases are fully preventable (Lindeberg 2010).
EFFECTS OF URBANIZATION
The emergence of ischemic heart disease has been well documented in a number of populations
after adopting Western dietary habits and lower levels of physical activity (for review, see Lindeberg
2010). The first case of a myocardial infarction in New Guineans, which was verified with an
autopsy, was reported in 1955 and individual cases were seen during the 1960s and 1970s (Conyers
1971; Somers 1974). After the 1970s, the number gradually increased (Kevau 1990). Among blacks
in Africa, the first case of classic angina pectoris was recorded in 1958 in an overweight housewife
with free access to Western food (Gelfand and Kaplan 1958). Later autopsy studies appeared to
indicate that myocardial infarctions were more common among those people with a higher income
(el Hassan and Wasfi 1972).
Table 26.3 shows the almost explosive growth of stroke, a previously unknown illness, among the
native population of Uganda. By 1955, stroke already made up 11% of the neurological cases (Hutton
1956), and by 1968, it became the most prevalent neurological diagnosis (Billinghurst 1970). At the
same time, hypertension, another previously unknown phenomenon, began to spread—a curious
development that was the topic of lively debates among British doctors in Uganda and Kenya at
the time (Trowell and Burkitt 1981). Parallel with the changes in the landscape of diseases, these
countries also underwent rapid social change from original traditional societies to Western-style
colonial societies.
The East African pattern is now being repeated in Papua New Guinea (Lindeberg 2003). The first
known case of stroke at Port Moresby General Hospital was seen in 1975 (Kevau I., personal com-
munication), a few years after the first documented case in Lae (Mathews 1974). Since then, cases
TABLE 26.3
The Proportion of Stroke among Patients with Acute Neurological
Disease in Kampala, Uganda, in Three Consecutive Series
Year N % Reference
1942 0 0 Muwazi (1944)
1954 11/100 11 Hutton (1956)
1968 17/50 34 Billinghurst (1970)
have become increasingly more common, and in 1998 there were almost two stroke cases per week
at the Port Moresby General Hospital.
As discussed earlier, many surveys among hunter–gatherers or similar non-Western populations
have found a striking absence of ischemic heart disease, stroke, diabetes, hypertension, and abdomi-
nal overweight. Not only did dietary habits differ in these populations compared to Western coun-
tries, but daily physical activity was more intense.
It has been argued that traditional populations may have been genetically protected against the
chronic degenerative diseases that occur in industrialized countries, yet when non-Westernized indi-
viduals adopt a more contemporary lifestyle, their risk for chronic degenerative diseases is similar
or even increased compared with modern populations (Yusuf et al. 2001; Lindeberg 2010; Carrera-
Bastos 2011).
Another common counterargument is the short average life expectancy at birth of hunter–
gatherers and other traditional populations. The problem with this marker is that it is influenced
by childhood mortality and fatal events (e.g., accidents, warfare, infections, exposure to “outdoor
dangers”) (Carrera-Bastos 2011). Today, average life expectancy is higher not because of a healthier
diet or lifestyle but because of less physical trauma, and improved sanitation, vaccination, medical
care, and social stability (Carrera-Bastos 2011). Although it is not possible to get a fair estimate of
life expectancy for Paleolithic cultures due to methodological limitations in osteology (Hoppa and
Vaupel 2002), among contemporary hunter–gatherers, the frequency distribution of ages at death
peaks around 70 years (Gurven and Kaplan 2007). Despite data that prehistoric hunter–gatherers
had a relatively high risk of death at young age, available evidence suggests that there were elderly
people and that they were important enough for the survival of grandchildren for age-related dis-
eases to be relevant in terms of nutritional adaptation (Henke and Tattersall 2007). Of more impor-
tance, these individuals reach age 60 years or beyond without the signs and symptoms of chronic
degenerative diseases that afflict the majority of the elderly in industrialized countries (Carrera-
Bastos 2011). Furthermore, in Western countries, various chronic degenerative diseases, including
cardiometabolic diseases, which are rare or virtually absent in hunter–gatherers, horticulturalists,
and traditional pastoralists, are now increasing in younger age groups (Carrera-Bastos 2011).
Nevertheless, it should be mentioned that while it is appropriate to provide information regard-
ing health status of hunter–gatherers and the effects of urbanization, the limitations of observational
studies, in particular effects due to residual confounding, preclude the inference that dietary prac-
tices are sufficient to account for differences in disease patterns.
CONTROLLED TRIALS OF “PALEOLITHIC DIETS”

There is a paucity of randomized controlled trials of “Paleolithic diets.” A systematic review
using the GRADE approach (Guyatt et al. 2008), as recommended by major Health Technology
Assessment organizations worldwide, found four randomized controlled trials of sufficient quality
where a Paleolithic diet was compared with another prudent diet in the treatment of the five compo-
nents of the metabolic syndrome (Manheimer et al. 2015). In total, 159 participants were included
in the meta-analysis. The control diets were based on distinct national nutrition guidelines but were
broadly similar. Food intake was ad libitum except for one trial where detailed menus were provided.
Adherence to the diets was assessed with food records. Paleolithic nutrition resulted in greater short-
term improvements (from 2 weeks to 6 months) than did the control diets (random-effects model)
for waist circumference (mean difference: −2.38 cm; 95% CI: −4.73, −0.04 cm), triglycerides
(−0.40 mmol/L; 95% CI: −0.76, −0.04 mmol/L), systolic blood pressure (−3.64 mm Hg; 95% CI:
−7.36, 0.08 mm Hg), diastolic blood pressure (−2.48 mm Hg; 95% CI: −4.98, 0.02 mm Hg), HDL
cholesterol (0.12 mmol/L; 95% CI: −0.03, 0.28 mmol/L), and fasting blood glucose (−0.16 mmol/L;
95% CI: −0.44, 0.11 mmol/L). The quality of the evidence for each of the five metabolic compo-
nents was moderate.
Two of these clinical trials were performed by our group (Lindeberg et al. 2007; Jönsson et al.
2009). In the studies, we noted beneficial effects of advice to eat a “Paleolithic” diet with regard to
waist circumference, glucose tolerance, blood sugar, blood pressure, and blood lipids (Lindeberg
et al. 2007; Jönsson et al. 2009). In both studies, we found evidence that the “Paleolithic” diet was
more satiating, such that the meals gave the same feeling of fullness at a lower level of energy intake
(Jönsson et al. 2010, 2013). In the first trial (Lindeberg et al. 2007), we randomly assigned 29 sub-
jects with type 2 diabetes or glucose intolerance to one of two prudent diets, with or without grains
and dairy products, a Mediterranean or Paleolithic diet, respectively. After 3 months, the Paleolithic
diet improved glucose tolerance more than the Mediterranean diet, independently of weight loss.
Also, after 3 months, all 14 subjects on the Paleolithic diet had normal plasma 2 h glucose compared
with 7 out of 15 in the Mediterranean group (p = 0.0007 for group difference). Regarding the second
study, a crossover study involving 13 patients, after 3 months, the Paleolithic diet improved glycated
hemoglobin, blood lipids, and blood pressure more than the recommended official diet for type 2
diabetes in Sweden (Jönsson et al. 2009). Another trial included in the foregoing meta-analysis tested
the long-term effects of a Paleolithic diet in 70 obese postmenopausal women (Mellberg et al. 2014).
The participants were randomized to either a Paleolithic diet or Nordic Nutrition Recommendations
(NNR) (Becker et al. 2004) for 2 years. Compared to the NNR, the Paleolithic diet resulted in greater
decreases in total fat loss, waist circumference, and abdominal sagittal diameter at 6 but not 24 months.
The effects were not maintained at 24 months, probably due to poor compliance with protein intake in
the Paleolithic diet. However, triglycerides levels were reduced more in the Paleolithic than the NNR
after 6 and 24 months. Liver fat also decreased more on the Paleolithic than the NRR after 6 but not
24 months (Otten et al. 2016). The fourth study included in the meta-analysis involved 32 subjects,
with at least two components of the metabolic syndrome, who were randomized to follow a 2-week
Paleolithic or an isoenergetic healthy reference diet according to the Dutch Health Council (Boers
et al. 2014). The Paleolithic diet improved blood pressure, blood lipids, and the number of character-
istics of the metabolic syndrome (1.07) more than the reference diet.
Although the control groups in these four trials were advised to follow a prudent diet and were
told that it was unknown if one diet was inferior to the other, it cannot be excluded that differences
in outcomes were due to the effect of residual confounding.
In a recent short-term (21 days) study in patients with type 2 diabetes, a Paleolithic diet induced
a significant reduction in HbA1c, fasting glucose, fructosamine, total and LDL cholesterol,
triglycerides and increased insulin sensitivity (as measured by euglycemic hyperinsulinemic
clamp), and increased HDL cholesterol (Masharani et al. 2015). However, the control diet based on
recommendations by the American Diabetes Association (Bantle et al. 2008), containing moderate
salt intake, low-fat dairy, whole grains and legumes, also improved HbA1c and worsened HDL
cholesterol compared to the baseline diets in both groups. No differences were observed between
the control and Paleolithic diets.
In a nonrandomized controlled study, after 4 months, a Paleolithic diet improved plasma lipids
more than the American Heart Association heart-healthy dietary recommendation (Eckel et al.
2014), in patients with hypercholesterolemia (Pastore et al. 2015). The macronutrient composition
of the AHA diet was 56%, 21%, and 23%, for carbohydrate, protein, and fat, respectively in men,
and 60%, 17%, and 23% in women. The macronutrient composition of the Paleolithic diet was
23%, 37%, and 40% for carbohydrate, protein, and fat, respectively in men and women. The lack of
randomization of this study represents a serious limitation.
In a metabolically controlled study, nine nonobese sedentary healthy volunteers consumed a
Paleolithic diet ensuring no weight loss (Frassetto et al. 2009). The participants consumed their
habitual diet for 3 days, then ate 7 days “ramp-up” diets to increase potassium and fiber intake,
and finally a Paleolithic diet for 10 days. All meals were prepared by the clinical research center
kitchen and provided to the participants to guarantee compliance with the dietary recommendations.
Frassetto et al. found improved levels of plasma insulin in the fasting state (by 68%) as well as after
a glucose load (by 39%), blood lipids, and blood pressure after the Paleolithic diet compared to their
usual diet. Similarly, marked improvements in body weight, blood glucose, blood lipids, and blood
pressure were noted in urbanized Aborigines with type 2 diabetes when they returned to a hunter–
gatherer lifestyle for 7 weeks (O’Dea 1984). Physical activity was also increased in that study.
There are few obvious risks associated with the consumption of a Paleolithic diet. The effect of
a high protein intake on kidney function is debatable in subjects with healthy kidneys and would be
expected to be outweighed by any beneficial effects on abdominal obesity and other health-related
variables (Lindeberg 2010). Even in patients with type 2 diabetes, but without chronic kidney dis-
ease, a low-carbohydrate diet (14% of total energy intake) high in fat and protein does not impair
renal function (Tay et al. 2015). Nevertheless, a Paleolithic type diet is not always high in protein.
People with genetic hemochromatosis, a hereditary disease that results in enhanced iron absorp-
tion, need to limit their intake of meat (especially red meat) and fish but should otherwise do well
on a Paleolithic diet. Heterozygous carriers are advised to monitor their iron status regularly after
middle age. Individuals treated with ACE-inhibitors, angiotensin II receptor blockers, or diuretics
should convert slowly to a salt-free Paleolithic diet in order to avoid a sharp drop in blood pressure
(Milan et al. 2002). Subjects with kidney failure cannot eat a high potassium diet, and should not
attempt a Paleolithic diet, unless under the care of a kidney specialist. Patients with type 2 diabetes
who are on sulfonylurea preparations (glipizid, glibenklamid, glimepirid) are at risk for hypoglyce-
mia when making a radical switch to a Paleolithic diet. People with a history of intestinal obstruc-
tion, intestinal dysmotility, or multiple intestinal surgeries can be at risk of recurrent obstruction
when eating a very high fiber diet, especially if not accompanied by an increase in fluid intake.
Converting to a Paleolithic diet during ongoing warfarin treatment should be done in consultation
with a physician or nurse.
CONCLUSION
A Paleolithic diet may serve as a model for healthy foods, in particular in clinical trials in compari-
son with other prudent diets. Hypothetically, food choice is more important than counting calories
or macronutrients in order to avoid common health problems in the Western world. Diets providing
wild meat, fish, shellfish, vegetables, tubers, fruit, berries, nuts, and eggs can be tested in the pre-
vention and treatment of disease. Hypothetically, dairy products, margarine, oils, and refined sugar
and cereal grains, which provide 70% or more of the dietary intake for modern humans, are not
optimal food choices for long-term health. However, much more research is needed to confirm the
benefits of Paleolithic diets for cardiometabolic health. Although this review has focused on what
most humans would have in common, studies on possible ethnic differences in susceptibility to diet-
induced disease are also needed.
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27 Fasting Intermittently or
Altering Meal Frequency
Effects on Plasma Lipids
John F. Trepanowski and Krista A. Varady
CONTENTS
Introduction.....................................................................................................................................517
Methods...........................................................................................................................................518
Results.............................................................................................................................................519
Effects of Intermittent Fasting on Plasma Lipids.......................................................................519
Mechanisms for Modulation of Plasma Lipids due to Intermittent Fasting...............................519
Effects of High Meal Frequency on Plasma Lipids...................................................................521
Mechanisms for Modulation of Plasma Lipids due to High Meal Frequency...........................522
Summary and Conclusion...............................................................................................................524
Acknowledgment............................................................................................................................524
References.......................................................................................................................................524
ABSTRACT
In this systematic review, we examined whether plasma lipids are improved by intermittent fasting
(5:2 diet, modified alternate day fasting) or high meal frequency dietary patterns. We found that
intermittent fasting is effective at reducing plasma triglycerides and that this effect may be second-
ary to weight loss. Intermittent fasting is not effective for modulating LDL-cholesterol or HDL-
cholesterol. High meal frequency (at a minimum of 9 meals/day) decreases LDL-cholesterol, but
plasma triglycerides and HDL-cholesterol are unaffected by this type of dietary pattern.
INTRODUCTION
A high LDL-cholesterol concentration is known to increase the risk of coronary heart disease
(Lewington et al. 2007). More controversially, a high triacylglycerol concentration is believed
by some scientists and clinicians to do the same (Nordestgaard and Varbo 2014). Treating these
lipid abnormalities with statins and fibrates has been shown to reduce coronary heart disease risk
(Jun et al. 2010, Mihaylova et al. 2012).
Intermittent fasting (IF) and high meal frequency (HMF) have recently emerged as two novel
dietary patterns for improving plasma lipids (Table 27.1). Intermittent fasting alternates between a
period of unrestricted food intake and a period of either substantial energy restriction (e.g., energy
intake at 25% of energy needs) or no food intake at all. For example, “strict” alternate day fasting
(ADSF) alternates between a “feast day” of unrestricted intake and a “fast day” in which no food
is consumed (Heilbronn et al. 2005b). Modified ADF (ADMF) which allows for the consumption
of one small meal equivalent to 25% of energy needs on the fast day, is an alternative to strict ADF
(Varady et al. 2011a). To date, plasma lipids have been measured in studies of ADMF but not in
studies of strict ADF. Another example of IF is the 5:2 diet, which consists of 5 consecutive days of
517
TABLE 27.1
Intermittent Fasting and High Meal Frequency Dietary Patterns
Dietary Pattern Description
Intermittent fasting (IF) Alternates between unrestricted food intake and substantial energy restriction
or no food intake
Intermittent fasting 2 out of 7 days (IF2-5) Alternates between 5 consecutive days of unrestricted intake and
2 consecutive days consuming 25% of energy needs
Modified alternate day fasting (ADMF) Alternates between a day of unrestricted intake, and consumption of
one small meal (25% of energy needs) on the fast day
Strict alternate day fasting (ADSF) Alternates between a day of unrestricted intake and a day in which no food
is consumed
High meal frequency 5 or 6 times/day
Very high meal frequency >10 times/day
unrestricted intake alternated with 2 consecutive days of consuming 25% of energy needs (Harvie
et al. 2011). With one exception (Soeters et al. 2009), each of the IF studies reviewed in this chapter
aimed for weight loss. The putative mechanisms by which IF may improve cardiometabolic health
have been reviewed recently (Horne et al. 2015). These include increased fat utilization for energy
(Duan et al. 2003), a hormetic response to the stress imposed by fasting (Anson et al. 2003), and
improved glucose homeostasis via activation of Forkhead Box A genes (Panowski et al. 2007).
While no universally agreed-upon definition of HMF currently exists, studies often have exam-
ined increasing meal frequency to 5 or 6 eating episodes per day although some investigations have
studied 12 or even 17 eating episodes per day (Jenkins et al. 1989, Murphy et al. 1996). Studies of
HMF have most commonly had a short duration, lasting anywhere between 12 hours and 4 weeks.
Each of these studies prescribed energy intake for weight maintenance. In contrast, a study lasting
24 weeks and another lasting 52 weeks prescribed energy intake for weight loss (Poston et al. 2005,
Bertéus et al. 2008). The main mechanism by which HMF may improve cardiometabolic health is
through decreased insulin secretion, both in response to each meal and overall (Heden et al. 2013).
However, HMF combined with positive energy balance has been shown to increase abdominal fat
and intrahepatic triglyceride content, and to impair insulin-mediated suppression of endogenous
glucose production and free fatty acids from adipose tissue (Koopman et al. 2014).
The purpose of this chapter is to review the effects of IF and HMF on plasma lipids. Additionally,
this chapter reviews the putative lipid-lowering mechanisms that have been examined in trials of
IF or HMF. These include weight loss, augmented insulin sensitivity, augmented fat oxidation, and
delayed gastric emptying.
METHODS
For a study to have been included in this review, it must have been a randomized controlled trial enroll-
ing human participants that measured at least one of the following as a primary outcome: body weight,
plasma lipids, energy intake, energy expenditure, appetitive hormones, glucose or insulin, macronutri-
ent oxidation, gastric emptying, or lipogenesis. These outcomes were chosen because they all relate
to body weight or plasma lipids. Investigations that enrolled participants with diabetes were excluded,
because diabetics experience unique plasma lipid responses when compared to nondiabetics (Rivellese
et al. 2004). For the studies of IF, prescribed energy intake during the feast period must have been
≥100% of needs, and prescribed energy intake during the fast period must have been ≤25% of needs.
For the studies of HMF, the difference in meal frequency between the intervention group and the con-
trol group must have been ≥2. No restrictions were placed on study duration or date of publication.
Fasting Intermittently or Altering Meal Frequency 519
On March 5, 2015, a Pubmed search was conducted. The following input was used for the studies
of IF: “alternate day fasting” or “caloric restriction” or “calorie restriction” or “intermittent calorie
restriction” or “intermittent fasting.” Limitations were set to include only randomized controlled
trials published in the English language that enrolled adults. There were 470 potentially relevant
publications identified through this search. Six publications were included after applying the inclu-
sion and exclusion criteria mentioned earlier. An additional seven publications were identified using
the references listed in these manuscripts. For the studies of HMF, the following input was used:
“eating frequency” or “eating pattern” or “feeding frequency” or “feeding pattern” or “gorging”
or “grazing” or “meal frequency” or “meal pattern” or “nibbling” or “snack.” The same limita-
tions as listed earlier were applied in this search. There were 365 potentially relevant publications
discovered through this search. Nine publications were included after applying the inclusion and
exclusion criteria. An additional eight publications were identified using the references listed in
these manuscripts.
RESULTS
Effects of Intermittent Fasting on Plasma Lipids
Consistent across studies, HDL-cholesterol did not change in response to IF (Table 27.2). The
findings regarding the effects of IF on other plasma lipids were heterogeneous. With the excep-
tion of one study that found a reduction in LDL-cholesterol (Varady et al. 2011a), the studies that
compared IF to a negative control group that did not receive any intervention (dietary or otherwise)
reported no change in any plasma lipid (Varady et al. 2011a, 2013, Bhutani et al. 2013, Hoddy
et al. 2014). In contrast, the studies that did not include a negative control group found reduc-
tions in LDL-cholesterol and/or plasma triglycerides, compared to baseline plasma lipids (Harvie
et al. 2011, 2013, Klempel et al. 2013b). Only one study to date has compared ADMF regimens
with different macronutrient compositions. This study found that a low-fat (25% of energy) and a
high-fat (45% of energy) ADMF regimen reduced LDL-cholesterol (25% and 15%, respectively)
and plasma triglycerides (14% in both groups) similarly (Klempel et al. 2013b). In the absence of
weight loss, fasting increases LDL-cholesterol and HDL-cholesterol, and decreases plasma tri-
glycerides (Sävendahl and Underwood 1999, Horne et al. 2013, 2014). Studies have consistently
found that ADMF improves LDL particle size (Varady et al. 2011a,b, 2013, Klempel et al. 2013a,
Hoddy et al. 2014).
Mechanisms for Modulation of Plasma Lipids due to Intermittent Fasting

Weight loss is known to lower plasma triglycerides and LDL-cholesterol while increasing HDL-
cholesterol (Dattilo and Kris-Etherton 1992). As shown in Table 27.2, IF consistently reduces body
weight in individuals seeking weight loss. It appears that individuals do not “binge” on feast days to
compensate for the large energy deficit incurred on the fast days, at least in the short term (Johnstone
et al. 2002, Klempel et al. 2010, Levitsky and DeRosimo 2010), and this may partially explain why
IF is successful at reducing body weight. Levels of the orexigenic hormone ghrelin do not change in
response to IF (Heilbronn et al. 2005a, Harvie et al. 2011), and no other appetite hormone has been
measured in IF studies to date.
Improved insulin sensitivity during weight loss is associated with improvement in plasma triglyc-
erides (McLaughlin et al. 2001), but recent evidence suggests that alterations in nutrient flux to the
liver play a more important role than alterations in insulin signaling (Otero et al. 2014) in mediat-
ing this effect. The effectiveness of IF for improving insulin sensitivity remains an open question.
Only one randomized controlled trial has investigated the effect of IF on insulin sensitivity under a
condition of weight maintenance (Soeters et al. 2009). This trial compared consuming 3 meals/day
to an IF regimen in which participants abstained from energy intake every second day for 20 hours.
520
TABLE 27.2
Intermittent Fasting: Effects on Body Weight and Plasma Lipids
Trial Body Weight Total Chol. LDL-Chol. HDL-Chol. Triglyceride
Length Selection of Comparison Change Change Change Change Change
Reference Subjects (Weeks) Dietary Regimen Meals Group (%) (%) (%) (%) (%)
Bhutani n = 16, MF 12 ADMF: lunch Fast-day meal No-intervention ↓6 0 0 0 0
et al. (2013) Age 42 ± 2 prepared control
BMI 35 ± 1
Harvie n = 42, F 24 5:2 diet Self-selected CR (isocaloric ↓7 ↓6 ↓10 0 ↓17
et al. (2011) Age 40 ± 4 comparison)
BMI 31 ± 5
Harvie n = 75, F 12 5:2 diet Self-selected CR (isocaloric ↓6 ↓5 ↓4 0 ↓14
et al. (2013) Age 47 ± 1 5:2 + PF comparison) ↓6 ↓4 ↓3 0 ↓12
BMI 30 ± 1
Hoddy n = 74, MF 8 ADMF: lunch Fast-day meal Each other ADF ↓4 0 0 0 0
et al. (2014) Age 45 ± 3 ADMF: dinner prepared group (isocaloric ↓4 0 0 0 0
BMI 34 ± 1 ADMF: small meals comparisons) ↓5 0 0 0 0
Klempel n = 32, F 8 ADMF: high fat All meals The other ADF ↓5 ↓13 ↓18 0 ↓14
et al. (2013b) Age 43 ± 3 ADMF: low fat prepared group (isocaloric ↓4 ↓16 ↓25 0 ↓14
BMI 35 ± 1 comparison)
Varady n = 13, MF 12 ADMF: lunch Fast-day meal No-intervention ↓5 0 ↓10 0 ↓17
et al. (2011a) Age 47 ± 2 prepared control
BMI 32 ± 2
Varady n = 15, MF 12 ADMF: lunch Fast-day meal No-intervention ↓7 0 0 0 ↓20
et al. (2013) Age 47 ± 3 prepared control
BMI 26 ± 1
Mean change: ↓6 ↓4 ↓6 0 ↓12
Notes: ADMF, modified alternate day fasting; BMI, body mass index (kg/m2); CR, daily calorie restriction (25%); F, female; M, male; PF, ad libitum protein and fat intake. Within-group
differences are reported so that across-study comparisons can be made.
Energy intake, macronutrient intake, and types of food consumed were matched between conditions,
and participants were provided amounts of food that were titrated to prevent weight change. Each
dietary assignment lasted 2 weeks in a crossover design. Glucose uptake as measured by the eugly-
cemic hyperinsulinemic clamp was not different between the dietary groups (Soeters et al. 2009). In
studies that examined IF with weight loss, ADF did not reduce the homeostasis model assessment
of insulin resistance (HOMA-IR) within group (Bhutani et al. 2013, Hoddy et al. 2014), while there
was a within-group reduction in HOMA-IR in response to the 5:2 diet (Harvie et al. 2011, 2013).
The studies of the 5:2 diet may have found a statistically significant reduction in HOMA-IR because
they had approximately twice the sample size compared to the ADF studies. Insulin sensitivity mea-
sured during an oral glucose tolerance test worsened in female participants but improved in male
participants in response to ADF (Heilbronn et al. 2005a). More favorable changes in insulin sensitiv-
ity in males versus females have also been observed in mice from the Balb/c, C57Bl/6J, and C3HJ
strains (Arum et al. 2009). The reason for this sexual dimorphism is presently unknown but may be
due to genotype-specific effects of IF (Goodrick et al. 1990).
Impaired fat oxidation is associated with an atherogenic lipoprotein phenotype (Faghihnia
et al. 2011). One mediator of this association may be insulin resistance, as impaired fat oxidation
may lead to increased intracellular (particularly intramyocellular) lipids that interfere with insulin
signaling (Shulman 2000). Weight loss increases resting fat oxidation in some (Corpeleijn et al.
2008) but not all studies (Kelley et al. 1999, Blaak et al. 2001). The magnitude of total fat mass
and visceral fat mass reduction may determine, in part, whether weight loss leads to increased
fat oxidation (Siri-Tarino et al. 2011). Fat oxidation does not appear to change in response to IF
without weight loss (Soeters et al. 2009), but does increase in response to IF with weight loss
(Heilbronn et al. 2005b).
Effects of High Meal Frequency on Plasma Lipids

HMF has been tested both in short-term (≤3 days) controlled dietary conditions where meals were
provided, as well as in longer-term (≥2 weeks) free-living conditions. The short-term studies have
generally found that HMF does not favorably modulate plasma lipids compared to consuming
3 meals/day (Table 27.3). Two studies found no difference in the plasma triglyceride area under the
curve between the HMF condition and the control condition (Schlierf and Raetzer 1972, Munsters
and Saris 2012), and a third study found that the plasma triglyceride area under the curve was
elevated in the HMF condition (Heden et al. 2013). Two studies found no difference in the total
cholesterol area under the curve between the HMF condition and the control condition (Jones et al.
1993, Heden et al. 2013), while a third study found a lower mean level of total cholesterol in the
HMF condition (Wolever 1990). The only study to measure postprandial LDL-cholesterol or HDL-
cholesterol found no difference for either parameter in the incremental area under the curve between
the HMF and control conditions (Heden et al. 2013). No study to our knowledge has examined the
effects of HMF on LDL particle size.
The longer duration studies have generally found that HMF does not favorably affect HDL-
cholesterol (Jenkins et al. 1989, Arnold et al. 1993, 1994, McGrath and Gibney 1994, Murphy
et al. 1996, King and Gibney 1999, Poston et al. 2005, Bertéus et al. 2008) or plasma triglycerides
in the absence of weight loss (Jenkins et al. 1989, Arnold et al. 1993, 1994, McGrath and Gibney
1994, Murphy et al. 1996, King and Gibney 1999, Poston et al. 2005) (Table 27.4). Some studies
have reported reductions in total cholesterol and LDL-cholesterol (Jenkins et al. 1989, Arnold et al.
1993, McGrath and Gibney 1994, Poston et al. 2005), while other studies have reported no change
in these parameters (Arnold et al. 1994, Murphy et al. 1996, King and Gibney 1999, Bertéus et al.
2008). A comparison of the studies that did or did not observe a reduction in LDL-cholesterol sug-
gests that meal frequency may need to be changed dramatically in order for LDL-cholesterol to be
reduced. Two of the studies that reported an LDL-cholesterol reduction in the absence of a change
in body weight compared either a 9 meals/day diet or a 17 meals/day diet to a 3 meals/day diet
TABLE 27.3
High Meal Frequency, Short-Term Feeding: Effects on Plasma Lipids
Dietary
Reference Subjects Trial Design Trial Length Regimen Effects on Blood Lipids
Heden n = 8, F Counterbalanced 12 hours 6 meals/day Plasma triglyceride incremental
et al. (2013) Age 39 ± 3 crossover area under the curve over
BMI 35 ± 1 12 hours was elevated in the
6 meals/day condition.
The incremental areas under the
curve for total cholesterol,
LDL-cholesterol, and HDL-
cholesterol were not different,
regardless of whether energy was
consumed over 3 or 6 meals.
Jones n = 6, M Parallel-arm 3 days 6 meals/day Total cholesterol decreased
et al. (1993) Age 31 ± 2 similarly, regardless of whether
BMI 23 energy was consumed over 3 or
6 meals.
Munsters and n = 12, M Counterbalanced 36 hours 14 meals/day Plasma triglyceride area under
Saris (2012) Age 23 ± 1 crossover the curve over 24 hours was
BMI 22 ± 1 similar, regardless of whether
energy was consumed over 3 or
14 meals.
Schlierf and n = 12, MF Counterbalanced 24 hours 6 meals/day In patients with
Raetzer (1972) Age 35 crossover normotriglyceridemia and in
BMI 25 patients with
hypertriglyceridemia, plasma
triglyceride area under the curve
was not different, regardless of
whether energy was consumed
over 3 or 6 meals.
Wolever (1990) n = 7, M Counterbalanced 12 hours Continuous Mean total cholesterol was lower
Age 23 ± 2 crossover feeding under the continuous feeding
BMI condition relative to the
unknown 3 meals/day condition.
Notes: BMI, Body mass index (kg/m2); F, Female; M, Male. Participants were provided all meals in each study. The control
regimen was 3 meals/day in each study.
(Jenkins et al. 1989, Arnold et al. 1993). The other study that reported an LDL-cholesterol reduc-
tion in the absence of a change in body weight examined a 6 meals/day diet, but the comparison
group decreased their meal frequency from a baseline of 6 to 3 meals/day during the intervention
(McGrath and Gibney 1994).
Mechanisms for Modulation of Plasma Lipids due to High Meal Frequency

Observational studies have often found that HMF is associated with lower body weight (Fabry
et al. 1964, Metzner et al. 1977, Ruidavets et al. 2002, Ma et al. 2003). However, a recent meta-
analysis of randomized controlled trials found that meal frequency was not associated with weight
loss (Schoenfeld, Aragon, and Krieger 2015). Therefore, the effects of HMF on plasma lipids are
unlikely to be related to changes in body weight.
TABLE 27.4
High Meal Frequency, Longer-Term Feeding: Effects on Body Weight and Plasma Lipids
Trial Dietary Regimen Selection of Body Weight Total Chol. LDL-Chol. HDL-Chol. Triglyceride
Reference Subjects Length (Meals/Day) Meals Change (%) Change (%) Change (%) Change (%) Change (%)
Arnold et al. n = 19, MF 2 weeks 9 Self-selected 0 ↓10 ↓14 ↓3 0
(1993) Age 32 ± 2
BMI 23 ± 1
Arnold et al. n = 16, MF 4 weeks 9 Self-selected 0 0 0 0 0
(1994) Age 50 ± 2
BMI 27 ± 1
Bertéus et al. n = 70, MF 52 weeks 6 Self-selected ↓5 0 0 0 ↓14
(2008) Age 40 ± 1
BMI 38 ± 1
Jenkins et al. n = 7, M 2 weeks 17 Prepared 0 ↓9 ↓14 0 0
(1989) Age 40
Fasting Intermittently or Altering Meal Frequency
Bodyweight 70 ± 3
King and n = 20, M 4 weeks 6 Self-selected 0 0 0 0 0
Gibney Age 48 ± 2
(1999) BMI 26 ± 1
McGrath and n = 23, M Not stated 6 Self-selected 0 ↓8 ↓12 0 0
Gibney Age 50 ± 2
(1994) BMI 24 ± 1
Murphy et al. n = 11, F 2 weeks 12 Prepared 0 0 0 ↓9 0
(1996) Age 22 ± 1
BMI 24
Poston et al. n = 50, MF 24 weeks 6 Self-selected ↓5 ↓4 ↓5 0 0
(2005) Age 40 ± 1
BMI 32 ± 1
Mean change: ↓1 ↓4 ↓6 ↓2 ↓2
Notes: BMI, Body mass index (kg/m2); F, Female; M, Male. The control regimen was 3 meals/day in each study. The experimental and control regimens were isocaloric in each
study, with one exception: Poston et al. compared 3 meal replacements/day with 3 meal replacements/day plus 3 snacks/day.
523
Robust measurements of insulin sensitivity in response to HMF are presently lacking. A few stud-
ies have found that fasting insulin, the major determinant of HOMA-IR (Abbasi et al. 2014), does
not change in response to HMF (Finkelstein and Fryer 1971, Poston et al. 2005, Bertéus et al. 2008).
On the other hand, HMF reduces the incremental area under the curve for insulin when assessed for
12 hours. This may explain why there is a decrease in cholesterol synthesis (measured by the
deuterium-uptake method) under HMF (Jones, Leitch, and Pederson 1993). Indeed, insulin secretion
is known to upregulate enzymes involved in cholesterol synthesis (Bhutani and Varady 2009).
Insulin also decreases the rate of fat oxidation in muscle and liver (Dimitriadis et al. 2011), and
therefore HMF has been hypothesized to increase fat oxidation by reducing day-long insulin secre-
tion (Allirot et al. 2013). However, both short-duration (≤36 hours) and long-duration (≥4 weeks)
studies have found that fat oxidation does not change with HMF (Bortz et al. 1966, Verboeket-Van
De Venne and Westerterp 1993, Munsters and Saris 2012, Allirot et al. 2013, 2014).
Delayed gastric emptying is known to improve plasma lipids (Meier et al. 2006). HMF has
been shown to delay gastric emptying (Jackson et al. 2007), but plasma lipids were not measured
in this trial.
SUMMARY AND CONCLUSION

Intermittent fasting appears to be effective at reducing plasma triglycerides, and this is likely a
consequence of body weight reduction. LDL-cholesterol and HDL-cholesterol do not appear to be
affected by IF. No study to date has examined the effect of IF on plasma lipids in the absence of
change in body weight. This is an interesting area for future research.
Most of the studies that have examined short-duration HMF have found no favorable changes
in plasma lipids. The long-duration studies suggest that LDL-cholesterol is reduced only when a
dramatic change in meal frequency is made (e.g., switching from 3 to 9 meals/day). Diets with high
meal frequency do not affect body weight, consistent with their lack of effect on plasma triglycer-
ides or HDL-cholesterol.
ACKNOWLEDGMENT
Departmental grant from Kinesiology and Nutrition, University of Illinois, Chicago.
The authors have no conflicts of interest to report.
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Section VI
Other Nutritional Influences
of Cardiometabolic Health
28 Early Life Nutrition,
Epigenetics, and Later
Mark H. Vickers, Clare M. Reynolds, and Clint Gray
CONTENTS
Background.....................................................................................................................................532
Evidence from Epidemiology and Clinical Cohorts.......................................................................533
Cardiometabolic Outcomes........................................................................................................533
Evidence from Animal Models.......................................................................................................534
Cardiometabolic Outcomes........................................................................................................535
Maternal Undernutrition............................................................................................................536
Maternal Overweight/Obesity....................................................................................................537
Inflammation and Programming.....................................................................................................538
Transgenerational Effects................................................................................................................541
Paternal Effects...............................................................................................................................542
Strategies for Intervention...............................................................................................................542
Discussion.......................................................................................................................................544
References.......................................................................................................................................545
ABSTRACT
Cardiometabolic disease arises from a complex interaction between many factors, including genetic,
physiologic, behavioral, and environmental influences. The increases in rates of metabolic disorders
over recent years suggest that genetics may play a relatively minor role, with increasing evidence for
environmental (e.g., epigenetic) and behavioral effects, underpinning the present disease epidemic.
In particular, alterations in the early-life nutritional environment (spanning the periconceptional
period through to infancy) are now well established to result in a “programmed” predisposition to
a range of metabolic and cardiovascular disorders in later life. In this context (preferentially termed
the “developmental origins of health and disease or “DOHaD”), it has been shown in both human
cohorts and experimental animal models that a range of altered early-life nutritional environments,
including both under- and overnutrition, can lead to cardiometabolic disorders in offspring. Further,
these effects can be amplified by the postnatal nutritional environment and also can be transmit-
ted across generations, thus leading to cycle of disease. Although the mechanisms are not fully
defined, this programming was initially considered an irreversible change in developmental trajec-
tory. However, it has now been shown that, at least in pre-clinical models, programmed metabolic
disorders are potentially reversible by nutritional or targeted therapeutic interventions during critical
periods of development. Given that fixed genomic variation may only explain a small proportion of
disease risk, there is an increasing interest in the role of epigenetics. As an example, epigenetic gene
promoter methylation at birth has been associated with later adiposity in childhood. These findings
suggest that a substantial component of disease risk may have a prenatal developmental basis and
perinatal epigenetic analysis may therefore have utility in identifying individual vulnerability to
later cardiometabolic disease.
531
BACKGROUND
Obesity and related cardiovascular and metabolic sequelae represent one of the greatest modern
threats to human health and lifestyle in the developed world. The incidence of cardiometabolic
disease has increased dramatically over the last two decades, driven primarily by an epidemic of
obesity in Western societies. These trends are also being mirrored in those developing nations that
are currently transitioning to First World lifestyles. Cardiometabolic disease arises from complex
interactions between genetic, physiologic, behavioral, and environmental factors. The rise in the
incidence of these metabolic disorders in recent years suggests that genetics plays a relatively minor
role, with increasing evidence for environmental (e.g., epigenetically mediated) and behavioral
effects, underpinning the present epidemic. Furthermore, intrauterine and early conditions are now
recognized as an influential factor in the progression or susceptibility of these pathologies.
The Developmental Origins of Health and Disease, or DOHaD, hypothesis has highlighted a
clear link between environmental exposures during early life development and the later risk for
obesity and related cardiovascular and metabolic disorders (preferentially termed “developmental
programming”). The DOHaD model speculates that during early life, adaptations occur in response
to cues from the environment. These predictive (mal)adaptations can result in adjustments in the set
points of homeostatic control systems in order to aid immediate survival and improve chances of
survival when placed in an expected adverse postnatal environment. However, if the interpretation
of prenatal cues is inappropriate or there are changes to the actual immediate environment, this can
result in a “mismatch” between prenatal predictions based on early life cues and postnatal reality.
As a result, these early adaptive processes, akin to the “thrifty phenotype” hypothesis developed
originally by Hales and colleagues (Hales and Barker 2001) and more recently the concept of pre-
dictive adaptive response (Gluckman et al. 2008), may actually be disadvantageous to offspring in
postnatal life, thus leading to an increased susceptibility for a range of diseases in adulthood and/or
the transmission of risk factors leading to a “feed-forward” cycle with inheritance of disease traits
across generations. Under the DOHaD framework, it is now well evidenced from epidemiological
observations, clinical cohorts, and a range of experimental animal models that alterations in the
nutritional environment during early development can manifest as an increased susceptibility for a
range of cardiometabolic disorders in later life (Fernandez-Twinn and Ozanne 2010).
Given the role of environmental influences, the role of epigenetic contributions to disease manifesta-
tion that arise due to developmental programming has become an increasing focus of recent studies.
Given the dynamic changes that occur during development, the epigenome is labile in nature, thus allow-
ing response and adaptations to environmental stressors, including early life nutritional modifications
(Jang and Serra 2014). It is understood that epigenetic switches control gene activity in a tissue-specific
manner. The observation that the level of imprinting in the agouti mutant gene can be modified via
changes in the maternal diet gave weight to the concept that alterations in maternal nutrition can affect
the epigenetic processes in the conceptus (Wolff et al. 1998, Cooney et al. 2002). Around the time of
blastocyst implantation, the majority of the genome is unmethylated and preimplantation blastocysts are
likely to be sensitive to environmental cues (Watkins et al. 2008, Eckert et al. 2012). The modification
of the maternal diet (i.e., supplementation or restriction) with dietary cofactors including methyl donors
(folate, methionine, or choline) can affect the establishment of DNA methylation patterns in offspring
(Lillycrop et al. 2005, Sinclair et al. 2007). Imprinted genes (whereby gene expression is in a parent-of-
origin-specific manner) are a class of genes that are considered a potential mediator of developmental
programming, because the normal expression of these genes is critically dependent upon epigenetic
modifications. Further, many such imprinted genes have key roles in the regulation of fetal growth
(including insulin-like growth factor-2 [IGF2]) as well as postnatal growth and metabolism. The meth-
ylation of DNA is also suggested as a mechanism underlying programming-induced changes in blood
pressure and glucose tolerance (Woods 2007), and the accumulation of methionine may predispose to
later development of cardiovascular disease (CVD), in both human studies and rodent models, primar-
ily through the demethylation of methionine and resultant production of homocysteine (Sutton-Tyrrell
Early Life Nutrition, Epigenetics, and Later Cardiometabolic Health 533
et al. 1997, Zhou et al. 2015). Kwong et al. reported that the persistent irreversible programming of
postnatal growth and physiology in rats was inducible during the preimplantation period of develop-
ment using a model of maternal protein restriction. A 30% reduction in the number of cells present
within preimplantation blastocysts was reported, which was associated with a reduced adult offspring
kidney weight. They postulated that maternal hyperglycemia and amino acid depletion may represent
early programming mechanisms that initiate “metabolic stressors,” thereby restricting early embryonic
cell proliferation and the subsequent generation of appropriately sized stem-cell lineages (Kwong et al.
2000). This work suggests that, in an environment that is nutrient restricted, the preimplantation embryo
is able to activate physiological mechanisms during this period of developmental plasticity in order to
stabilize the growth of the conceptus and enhance postnatal fitness. The activation of such acute
responses, however, may also be disadvantageous in the long term and predispose to a range of cardio-
metabolic disorders in later life as shown in a murine model (Watkins et al. 2008).
EVIDENCE FROM EPIDEMIOLOGY AND CLINICAL COHORTS

The basic concepts that underpin environmentally induced metabolic programming, and particularly
transgenerational transmission of acquired phenotypic characteristics, can be traced back to the French
biologist Lamarck (1809), who developed a theory around “inheritance of acquired characteristics.”
Later, Kermack reported a relationship between living conditions, socioeconomic status, and subse-
quent death rates (Kermack et al. 1934). Forsdahl and colleagues then described a correlation between
infant mortality and CVD-related deaths (Forsdahl 1977). The term “programming” was first used in
scientific literature by Dörner in the early 1970s following his observation that hormone, metabolite,
and neurotransmitter concentrations during sensitive periods of early development could program brain
development, reproductive parameters, and metabolism in human adults (Koletzko 2005). To encom-
pass the consequences of neonatal and early childhood nutrition, the term “nutritional programming”
was later proposed by Alan Lucas, and the concept went on to gain a broad and international acceptance
following the work of Hales and Barker (Hales and Barker 1992). Following these early observations,
a number of epidemiological studies and clinical cohorts have now accumulated a large amount of evi-
dence relating to the “fetal origins” or “developmental programming” of adult disease hypothesis. These
studies have highlighted that disease susceptibility is more than just a consequence of adult environ-
ment and lifestyle factors and that a range of nutritional stimuli or insults during the periconceptional,
gestational, or early postnatal environment can have a profound effect on developmental trajectory and
propensity for metabolic disorders in later life. Mechanistically, studies in the human that link epigen-
etic changes to later metabolic disease risk remain largely observational in nature, although there is
evidence that suggests the potential for tissue-specific inheritance of DNA methylation patterns (Silva
and White 1988). There are a number of limitations to human studies, including the accuracy and avail-
ability of methods, to measure discrete nutritional components involved in the one-carbon cycle and
whether disruptions to these processes exert tissue-specific effects. Different environmental exposures
can lead to differential patterns of epigenetic marks in the somatic tissues of individuals with evidence
for this provided in studies of twins whereby a divergence of DNA methylation and histone acetylation
patterns occurs more strongly in older twin pairs that are characterized by more marked life history
differences (Fraga et al. 2005). Work on human disorders, including Angelman (lack of maternally
contributed region 15q11) and Prader-Willi (imprinting disorder on paternally contributed regions of
chromosome 15) imprinting syndromes, further suggests that the inheritance of epigenetic marks may
play a role in the development of cardiometabolic disorders in the human (Kaminsky et al. 2006).
Cardiometabolic Outcomes
Human data are limited, and most evidence to date on epigenetic mechanisms underpinning develop-
mental programming has been derived from experimental animal models. Of the limited human data
available, the Dutch famine cohort (1944–1945) is one of the most cited historical cohorts used to
investigate early life nutritional deprivation on later health outcomes (Ravelli et al.1976, Roseboom
et al. 2000, 2001). The Dutch famine was a severe famine that occurred between November 1944 and
June 1945. The famine primarily affected the northwestern region of the Netherlands and resulted
from a blockade imposed by the German military occupation during the Second World War. The
Dutch famine cohort provides the most compelling evidence that the timing of nutritional adversity
in early life appears critical for changes in the adult methylome (methylated genome). Prenatal famine
exposure was linked to metabolic compromise in later life (impaired glucose handling, increased
adiposity and a higher body mass index [BMI], increases in total and low-density lipoprotein cho-
lesterol) and increased incidence of schizophrenia in later life (Lumey et al. 2011). The observations
of differential DNA methylation at promoters and imprinted regions for genes involved in metabolic
regulation following prenatal famine exposure indicated a role for epigenetic mechanisms in these
phenotypic associations. Moreover, data derived from the Dutch famine cohort have also shown that
differences in methylation after famine exposure are dependent upon the timing of exposure in addi-
tion to being sex specific (Heijmans et al. 2008, Tobi et al. 2009). As an example, a recent study in
this cohort has shown that early gestation, and not mid–late gestation, is a critical period for adult
DNA methylation changes, as assessed in blood, after prenatal famine exposure (Tobi et al. 2015). In
the Dutch famine cohort, individuals that had been prenatally exposed to famine had, 60 years later,
a reduction in the methylation of the IGF2 gene compared to same-sex siblings that were unexposed.
This association was specific to the timing of periconceptional famine exposure and reinforces that
very early developmental stages in mammals are crucial periods for the establishment and mainte-
nance of epigenetic marks (Heijmans et al. 2008, Tobi et al. 2009).
Although a role for macronutrient balance is clearly implicated in developmental programming,
for example, restricted or excessive protein and carbohydrate intake (McMillen et al. 2008), mater-
nal micronutrient concentrations are of interest due to their impacts on the one-carbon metabolism
involved in the regulation of DNA methylation and numerous experimental observations that an
imbalance in micronutrients can influence the patterns of DNA methylation in offspring. For exam-
ple, an increase in vitamin B12 concentrations during pregnancy is linked to a decrease in global
DNA methylation in the human newborn, while increases in the concentration of serum B12 in
newborns are associated with a reduction in IGFBP3 methylation, an IGF-binding protein involved
in intrauterine growth (McKay et al. 2012). More recent work examining parental obesity has shown
altered DNA methylation patterns at imprinted genes in newborns of obese parents (Soubry et al.
2015). Moreover, obesity in the father has been linked to the hypomethylation of IGF2 in newborns
(Soubry et al. 2013). Of note, the observation of significant associations between paternal obesity
and methylation status in offspring demonstrates that the developing sperm is susceptible to environ-
mentally mediated insults. The acquired “imprint instability” may be transmitted across generations
and increase the risk for chronic diseases in later life (Soubry et al. 2015).
EVIDENCE FROM ANIMAL MODELS

Given the short generation times and scope for transgenerational studies, tissue-specific analysis, and
ability to implement intervention strategies that allow the observation of outcomes over the life course
of the offspring, most data to date have been derived from a range of experimental animal models.
Developmental changes to epigenetic marks may induce lifelong alterations in gene expression, leav-
ing offspring metabolically disadvantaged and susceptible to later disease, particularly when exposed
to a “secondary trigger” such as a postnatal obesogenic environment. Numerous studies in animal mod-
els, using a diverse range of nutritional challenges across different experimental species, have exam-
ined changes in DNA methylation to investigate the impact of altered early life nutrition on epigenetic
regulation of both imprinted and nonimprinted genes (Gicquel et al. 2008). As described earlier, given
that one-carbon metabolism is dependent upon the availability of dietary methyl donors and cofactors
(including folic acid, choline, and vitamin B12), it is not unexpected that alterations in the early life
nutritional environment can influence DNA methylation (MacLennan et al. 2004, Gicquel et al. 2008,
Vanhees et al. 2014). Further, maternal nutritional restriction during the periconceptional period can
result in changes in the expression of microRNAs (miRNAs) in offspring that have been associated
with the development of insulin resistance in later life (Lie et al. 2014). Maternal dietary manipula-
tions including low-protein (LP) exposure can result in aberrant changes in DNA methylation in key
genes, changes that can be prevented by maternal dietary supplementation with cofactors (Lillycrop
et al. 2005, Cho et al. 2013). For example, in the rat, Lillycrop et al. have reported altered promoter
methylation and gene expression in LP-exposed offspring for both the hepatic glucocorticoid receptor
(GR) and the peroxisome proliferator–activated receptor α (PPAR-α) (Lillycrop et al. 2005, 2007),
influencing carbohydrate and lipid metabolism (Burdge et al. 2007a). In this study, a maternal low-
protein (MLP) diet resulted in PPAR-α and GR hypomethylation in offspring; these alterations were
normalized to that of the control group when the mother was supplemented with folate (Lillycrop et al.
2005). Further, alterations in the folate content of maternal or postweaning diets in the rat can result in
differential changes in phosphoenolpyruvate carboxykinase (PEPCK, an enzyme primarily involved in
gluconeogenesis) gene expression and promoter methylation (Hoile et al. 2012). In addition to folate,
there is emerging evidence around optimal dietary choline intake (a vitamin-like essential nutrient
also involved in methylation or one-carbon transfer) for normal fetal development (Zeisel 2009). In
the rat, maternal choline supply during pregnancy can impact upon fetal histone and DNA methyla-
tion, suggesting that a concerted epigenomic mechanism is in place that contributes to the long-term
developmental effects evident when choline intake is varied (Davison et al. 2009). In the rat, choline
is involved in the methylation of histone H3, expression of histone methyltransferases G9a (Kmt1c)
and Suv39h1 (Kmt1a), and methylation of their respective genes in fetal liver and brain (Davison et al.
2009). Data on vitamin B12 and altered methylation are less clear. As with folate, B12 is a requirement
for the synthesis of methionine and S-adenosyl methionine, the common methyl donor required for the
maintenance of methylation patterns in DNA; thus, a deficiency in B12 can result in hypomethylation.
One of the most epigenetically regulated loci that has been characterized, the paternally imprinted
IGF2 gene, has a labile pattern of methylation that is dependent on the nutritional stimuli or cues that
are received by the developing organism during early life (Murphy and Jirtle 2003, Waterland et al.
2006b). Of note, in postweaning animals, work by Waterland showed that the paternal allele of Igf2
differentially methylated region 2 was hypermethylated in the kidneys of mice fed a synthetic control
diet. These data, if translatable, suggest that compositional differences in the diet postweaning can
exert persistent effects on IGF2 expression, thus suggesting that diet in early childhood could poten-
tially contribute to a loss of IGF2 imprinting in humans (Waterland et al. 2006b).
Cardiometabolic Outcomes
Work across a range of experimental animal models of developmental programming has provided
clear evidence that a wide range of environmental challenges during the periconceptional period,
pregnancy, or early neonatal life can result in changes in gene promoter methylation. These changes
will subsequently either directly or indirectly affect gene expression in pathways associated with a
range of cardiometabolic disorders (Pham et al. 2003, Weaver et al. 2004, Bogdarina et al. 2007,
Jirtle and Skinner 2007, Dudley et al. 2011). A primary focus of work to date has been on changes
in DNA methylation. However, data are emerging on histone modifications arising due to aberrant
developmental programming. Alterations in histone methylation are key structural changes that can,
depending on the location, impact upon gene expression, leading to either promotion or repression
of gene expression. The methylation of either histone or DNA proteins requires methyl donor avail-
ability in addition to a range of methyltransferases (Wolffe 1998, Callinan and Feinberg 2006). In
addition to DNA methylation and histone modifications, epigenetics also integrates small noncod-
ing miRNAs with an emerging framework that integrates methylation, histone, and miRNAs. As
described earlier, although research to date has predominantly focused on changes in DNA methyla-
tion in response to altered nutrition (Delage and Dashwood 2008), an increasing number of reports
are highlighting the impact of early life nutritional challenges on miRNAs and histones. Work on
miRNAs has suggested a complex network of reciprocal interconnections with miRNAs not only
involved in the control of gene expression at a posttranscriptional level but also connected to meth-
ylation processes. miRNAs thus represent a new important class of regulatory molecules as via
regulatory loops they are also directly connected to other components of the “epigenetic machinery”
(Iorio et al. 2010). On one hand, DNA methylation and chromatin modifications can regulate the
expression of certain miRNAs. Reciprocally, miRNAs can impact upon the methylation machinery
and the expression of proteins involved in histone modifications (Iorio et al. 2010). As a combi-
natorial mechanism, this process may then ultimately determine gene expression and the ensuing
offspring phenotype. Epigenetic modifications are intimately linked with embryonic development,
differentiation, and stem-cell programming. These epigenetic mechanisms can be affected by envi-
ronmental stimuli (e.g., pregnancy complications, maternal smoking, drug use, alcohol, and diet)
and can increase predisposition to cardiometabolic disease. miRNAs lead to the posttranscriptional
degradation of target gene messenger RNA and translational inhibition of protein expression. As an
example of this, silencing of miRNAs targeting histone deacetylases has been linked to cancer pro-
gression, but the impact of such processes in the context of developmental programming are not well
described. Currently, the epigenetic regulation of miRNA-encoding gene expression following the
methylation of CpG dinucleotides within the promotor regions of these genes has not been described
in the context of CVD, but proof-of-principle studies in other areas (e.g., insulin sensitivity [Flowers
et al. 2015] and endothelial dysfunction [Prattichizzo et al. 2015]) suggest that miRNAs have utility
as biomarkers or potential therapeutic use in the setting of programming-related disorders.
Maternal Undernutrition
The most commonly used models in rodents have utilized the models of maternal undernutrition
(either global dietary restriction or isocaloric LP diets) or ligation of the uterine artery in order
to induce intrauterine growth restriction (IUGR). In response to alterations in early life nutrition,
gene expression can be fine-tuned by organisms to achieve environmental adaptation via epigen-
etic alterations of histone markers of gene accessibility (Zinkhan et al. 2012). Feeding a MLP diet
or inducing uteroplacental insufficiency during pregnancy can result in altered DNA methylation,
which later manifests as endothelial dysfunction, increased angiotensin II type 1 receptor expres-
sion in the kidney and adrenal gland, hypertension, and nephron deficits (Bogdarina et al. 2007,
Morgado et al. 2015). In the rat adrenal gland, Bogdarina and colleagues have shown that MLP
diet–induced hypertension in offspring is associated with hypomethylation and increased expression
of the angiotensin type 1b (AT1b) receptor gene (Bogdarina et al. 2007). Further examples are those
in the models of growth restriction whereby uteroplacental insufficiency can lead to a decrease in
postnatal IGF1 mRNA variants, H3 acetylation, and the gene elongation mark histone 3 trimethyl-
ation of lysine 36 of the IGF1 gene (H3Me3K36) (Fu et al. 2004, Zinkhan et al. 2012). Tosh et al.
have also shown in the rat that early patterns of body growth following early growth restriction (i.e.,
rapid vs. delayed catch-up growth) resulted in differential changes in hepatic IGF1 mRNA expres-
sion and histone H3K4 methylation (Tosh et al. 2010). A common molecular phenotype associated
with growth-restricted rats is a decrease in the expression of pancreatic and duodenal homeobox fac-
tor-1 (PDX1). PDX1 is a key transcription factor involved in the regulation of pancreatic develop-
ment, and a reduction in PDX1 activity has been associated with alterations in histone modifications
(Park et al. 2008). Similar findings have also been observed in the muscle of IUGR rats for the glu-
cose transporter GLUT4 (Raychaudhuri et al. 2008). There are also important associations between
phospholipid status in the mother and the one-carbon cycle (Khot et al. 2015). The inadequacy of
long-chain polyunsaturated fatty acid (LCPUFA)-containing phospholipids may cause the diversion
of methyl groups toward DNA and eventually result in aberrant DNA methylation patterns (Khaire
et al. 2015). Changes in these patterns of DNA methylation can lead to alterations in the expression
of key genes, including angiogenesis-related genes, and may thereby contribute to aberrations in
angiogenesis/vasculogenesis further affecting the development of the placenta (Khot et al. 2015).
The adipokine leptin has received significant attention in studies of developmental programming
with leptin treatment to neonatal rats shown to reverse the detrimental effects of global maternal
undernutrition (Vickers et al. 2005, 2008, Gluckman et al. 2007, Gibson et al. 2015) with similar
effects observed in a piglet model of IUGR (Attig et al. 2008). Mechanisms underlying epigenetic
modifications that result in an increased susceptibility to altered programming of leptin and insulin
signaling have been discussed previously (Holness and Sugden 2006). Embedded within a CpG
island, leptin has a 3kb promoter region and contains a number of putative binding sites for known
transcription factors including a glucocorticoid response element. In animal models, it has been
shown that the degree of variation observed in the DNA methylation of the leptin promoter varies
with the degree of obesity (Shen et al. 2014, Crujeiras et al. 2015) and the epigenetic regulation of
leptin signaling pathways can be modified by nutrients through altered histone modification and
binding of DNA methyltransferases at the level of the leptin promoter (Haggarty 2013). In addi-
tion to leptin, a further focus regarding the potential for epigenetic regulation is that of the fat mass
and obesity-associated (FTO) gene (Dina et al. 2007, Lawlor et al. 2008, Sebert et al. 2010, 2014,
Mayeur et al. 2013). An overview of the potential programming effects of the FTO gene in the devel-
opment of obesity has been undertaken previously by Sebert and colleagues (Sebert et al. 2014). As
evidenced by experimental work in both sheep and rat models (utilizing paradigms of both undernu-
trition and overnutrition), the FTO gene has been identified as a potential key target for nutritional
programming. As an example, leptin resistance associated with obesity in IUGR offspring and rapid
postnatal catch-up growth may arise due to altered FTO methylation and gene regulation (Sebert
et al. 2011). However, as is a limitation of many data around altered DNA methylation, although
significant associations between FTO and BMI are consistently replicated in humans, the precise
mechanisms by which FTO is involved in the regulation of weight gain remain to be defined (e.g.,
the impact of RNA demethylation on the control of energy balance). However, recent evidence
suggests that single nucleotide polymorphisms identified within the FTO gene display a functional
link to IRX3, a protein that regulates early neuronal development and is associated with obesity
onset. In addition, differences in the nutritional perturbations used can lead to differential effects on
the regulation of the FTO gene. For example, placental FTO expression is reduced by fetal growth
restriction but not by macrosomia in rodents and humans (Mayeur et al. 2013, Smemo et al. 2014).
A range of metabolic traits that result from low birth weight can be passed to subsequent genera-
tions, thus suggesting that epigenetic changes are maintained during meiosis (see “Transgenerational
Effects” section). An example of this, as noted earlier, is the variation in coat color in the agouti
mouse where color variation has been correlated to epigenetic marks that are established early
in development (Jirtle and Skinner 2007). This was evidenced by Dolinoy et al. using intrauter-
ine exposure to genistein via the maternal diet to heterozygous agouti viable yellow mice (Avy).
This resulted in alterations in coat color toward pseudoagouti and appeared to confer protection
in offspring against the development of obesity in later life, a characteristic feature of the agouti
phenotype (Dolinoy et al. 2006). Utilizing mouse models, work by Waterland et al. has shown that
some alleles are particularly sensitive to changes in methylation arising due to changes in maternal
nutrition (Waterland et al. 2006a, Waterland and Michels 2007). This work suggested that maternal
nutrition in the preconception period and during pregnancy can affect the establishment of CpG
methylation patterns and the lifelong expression of metastable epialleles (epigenetically modified
alleles) (Waterland et al. 2006a).
Maternal Overweight/Obesity
In addition to models of maternal dietary restriction, a number of studies have now shown that
an environment of maternal excess can elicit epigenetic changes in offspring. Maternal and neo-
natal overnutrition can result in epigenetic modifications in key genes involved in insulin signal-
ing in skeletal muscle and predispose to insulin resistance in later life (Liu et al. 2013). Work by
Marco et al. has shown that a maternal high-fat diet (HFD) resulted in the hypermethylation of the
hypothalamic pro-opiomelanocortin (POMC) promoter and postweaning obesity in the rat (Marco
et al. 2013), and maternal fat intake has been associated with altered epigenetic regulation of genes
related to fatty acid synthesis (Hoile et al. 2013). In a rat maternal HFD model, the inhibition of
the hepatic cell cycle and associated changes in gene expression and DNA methylation has been
reported in offspring, but these changes did not persist into adulthood (Dudley et al. 2011). A mater-
nal HFD can also result in altered methylation and gene expression of dopamine- and opioid-related
genes and represents a further potential mechanism for early life programming of appetite control
and an increased preference for energy-dense foods postnatally (Vucetic et al. 2010).
Work in the primate has shown that, in addition to DNA methylation changes, obesity induced by
a calorie-dense maternal diet can lead to alterations in fetal chromatin structure via covalent histone
modifications (Aagaard-Tillery et al. 2008). Hepatic metabolism in the neonate can be altered in
the setting of a maternal HFD, albeit in a sex-specific manner, and these differences, in association
with histone modifications, may contribute to the known sexual dimorphism in oxidative balance
(Strakovsky et al. 2014). Further work in the nonhuman primate has shown that a maternal HFD
can modulate sirtuin 1 (SIRT1) histone and protein deacetylase activity in the fetus. This implicates
SIRT1 as a likely mediator of the fetal epigenome and metabolome in the setting of maternal obe-
sity (Suter et al. 2012). Via regulatory loops, miRNAs can cause histone modifications and altered
DNA methylation of promoter sites, which affect the expression of target genes (Hawkins and
Morris 2008, Tan et al. 2009). Further, in an ovine model of maternal obesity, miRNA expression in
fetal muscle is altered and therefore may be a mechanism by which intramuscular adipogenesis is
enhanced during early muscle development (Yan et al. 2013).
As described earlier, offspring of rat mothers fed a moderate HFD during pregnancy display
hepatic cell cycle inhibition that is associated with short-term changes in gene expression and DNA
methylation (Dudley et al. 2011). In particular, the cell cycle inhibitor cyclin-dependent kinase
inhibitor (Cdkn1a, p21) is hypomethylated at specific CpG dinucleotides, and hepatic mRNA
expression is increased in the liver of offspring of HFD mothers. Since the upregulation of Cdkn1a
has been associated with hepatocyte growth in pathologic states, these data are suggestive of early
hepatic dysfunction in neonates born to mothers consuming a HFD. Exposure to a maternal obe-
sogenic environment therefore contributes to early perturbations in whole body and liver energy
metabolism in offspring mediated in part by epigenetic effects. Early programming of mitochon-
drial dysfunction and a reduction in fatty acid oxidation in the liver may precede the development
of obesity-associated comorbidities including insulin resistance and hepatic steatosis in later life
(Brumbaugh and Friedman 2014, Kjaergaard et al. 2014, Pereira et al. 2015). Further, nutritional
modulation of miRNAs may also underlie susceptibility to type 2 diabetes mellitus (T2DM) in off-
spring—for example, a maternal HFD has been shown to alter the levels of certain hepatic miRNAs
concomitant with alterations in the expression of genes including IGF2, which is important for islet
β-cell survival (Zhang et al. 2009).
Although studied less widely as an independent dietary cofactor, the effect of a high mater-
nal salt intake, common in most Western-style dietary environments, has also been examined in
relation to epigenetic changes. Work by Ding et al. examined the influence of a diet high in salt
during pregnancy on the development of the heart and DNA methylation in the rat fetal heart in
relation to the subtype of angiotensin receptors. A high maternal salt intake resulted in changes in
a number of CpG sites in the fetal heart that were linked to the AT1b promoter. Further, cardiac
AT1 receptor protein in adult offspring was also higher following exposure to a maternal high-salt
diet (Ding et al. 2010).
INFLAMMATION AND PROGRAMMING

Accumulating evidence has indicated that chronic low-grade inflammation is a key factor in the pro-
gression of metabolic disease. Obesity was first linked to inflammation in 1993 when Hotamisligil
et al. demonstrated an increase in the pro-inflammatory cytokine TNFα in obese, insulin-resistant
mice (Hotamisligil et al. 1993). Subsequent studies have demonstrated that infiltration of innate
immune cells (macrophages, dendritic cells, and T cells [Lumeng et al. 2007a,b, 2009, Reynolds
et al. 2012]) and local production of pro-inflammatory mediators interrupt insulin signaling, repre-
senting an integral step in the pathogenesis of metabolic disease. While TNFα represents the first
association between inflammation and obesity, there are now numerous cytokines and adipokines
known to disrupt insulin responsiveness, instigating a drive toward anti-inflammatory (both phar-
macological and nutritional) treatments for obesity and its comorbidities. There is increasing evi-
dence that these inflammatory processes are governed, at least in part, by environmentally induced
epigenetic changes. Peripheral blood monocytes (PBMCs) represent a useful noninvasive tool for
the assessment of epigenetic alterations in the immune cells of obese subjects. A study in PBMCs
from monozygotic twins indicated that obesity and metabolic dysfunction are associated with dif-
ferentially methylated genes in a range of metabolically relevant targets (Ollikainen et al. 2015).
Indeed, the methylation of specific inflammatory genes has been linked to metabolic dysregula-
tion. Campion et al. determined that TNFα promoter methylation was reduced following weight
loss in obese men (Campion et al. 2009). There is also evidence that obese women with calorie
restriction–induced weight loss have lower adipose tissue methylation levels in both the TNFα and
leptin promoter regions (Cordero et al. 2011). Finally, there is evidence from human adipocytes
and THP-1 (human macrophage cell line) cells that inflammatory stimuli alter the expression of
miRNA (miR-221/222, miR-155), which may be responsible for inflammation-related metabolic
dysfunction (Ortega et al. 2015).
Despite the relative importance of meta-inflammation (the chronic low-grade inflammatory
response to obesity) in the pathogenesis of insulin resistance and T2DM, the role of maternal pro-
gramming of metabolic inflammation has not been clearly defined. However, there are indicators
that give reason to speculate that epigenetic alteration of inflammatory mediators may influence
metabolic disease in the offspring of mothers subjected to adverse environments during pregnancy.
Evidence from human studies has indicated that the development of the immune system begins at an
early embryonic stage (Naito 1993, Palmer 2011). While a rudimentary immune system is present
at birth, this system does not fully mature until later in life and is therefore susceptible to program-
ming stimuli from gestation to the postweaning period (Cedar and Bergman 2011, Palmer 2011).
The immune system is highly plastic, and a series of epigenetic marks govern the differentiation
of hematopoietic stem cells to functional immune mediators (Attema et al. 2007). There is signifi-
cant evidence that exposure to inflammatory stimuli during pregnancy can influence the offspring’s
immune system in later life. Further, maternal inflammation and infection represent a major cause
of preterm birth, which in itself is associated with significant effects on offspring’s long-term health
outcomes. This appears to be linked to epigenetic cues. Sanders et al. demonstrated that specific
miRNAs present in the cervix in a cohort of 53 pregnant women were associated with several factors
including inflammation and predicted gestational length (Sanders et al. 2015), while Hillman et al.
indicate that distinct DNA methylation profiles associated with autophagy, oxidative stress, and hor-
monal regulation in preterm placentas may predispose to future disease risk (Hillman et al. 2015).
Maternal infection and inflammation also represent the sources of epigenetic regulation, which may
affect future disease risk in adults. Infection of pregnant mice with gram-negative bacteria (A Iwoffii
F78) protected offspring from inflammatory lung disease through the alteration of histone acetyla-
tion in the promoter region of the anti-inflammatory cytokine IL-4 (Brand et al. 2011). Given that
HFDs, particularly those rich in saturated fats, activate the immune system both in humans and in
animal models (Fernandez-Real and Ricart 1999, Ferrante 2013), it is reasonable to speculate that
this nutrient-derived inflammation may play an important role in programming immune function
and indeed subsequent meta-inflammation in offspring (Li et al. 2013).
While the evidence for the role of epigenetic processes in developmentally programmed meta-
bolic inflammatory disease is limited, there are several studies that implicate poor maternal nutrition
in the development of chronic low-grade inflammation and subsequent metabolic disease. Reynolds
et al. demonstrated that moderate maternal global undernutrition resulted in increased adipose tissue
inflammation, which is linked to insulin resistance in Sprague-Dawley rats (Reynolds et al. 2013a).
This study was followed up with evidence that bone marrow macrophages from undernourished
offspring have an increased inflammatory profile upon immune stimulation and demonstrate signs
of polarization from the anti-inflammatory M2 phenotype to the pro-inflammatory M1 phenotype
(Reynolds et al. 2013b). Interestingly, macrophage polarization is a process that is heavily gov-
erned by epigenetics, whereby demethylases act on histones that bind to the promoters of genes
that characterize the M2 phenotype. Therefore, it is plausible that the effects observed in offspring
from undernourished mothers may, in part, be mediated by epigenetic alterations (Satoh et al. 2010).
Indeed, inflammatory changes are also observed in relation to maternal obesity. Li et al. demonstrate
that maternal obesogenic diets (rich in sugars and fat) program hepatic inflammation in rat neonates,
prior to the onset on overt metabolic disease (Li et al. 2013). However, whether these effects are
due to direct exposure in utero or via epigenetic changes remains to be examined. A recent study
examining transgenerational HFD-induced programming observed progressive increases in infiltrat-
ing macrophages across generations accompanied by increased gene expression of critical innate
immune mediators such as NLRP4 and TLR2/4 in adipose tissue. These findings were associated
with the hypomethylation of inflammatory gene promoter regions, thus demonstrating evidence of
transgenerational epigenetic transmission of meta-inflammation (Ding et al. 2014).
While direct evidence linking epigenetic changes in inflammatory genes to developmentally pro-
grammed obesity and metabolic dysfunction is limited, it is clear that epigenetics plays a key role
in the pathogenesis of obesity-mediated comorbidities. There is however evidence from early life
interventions with anti-inflammatory nutrients (including n-3 polyunsaturated fatty acids [PUFA]
and conjugated linoleic acid [CLA]) demonstrating beneficial effects on adult onset metabolic dis-
ease. There is increasing evidence that maternal dietary supplementation with n-3 LCPUFAs ame-
liorates the metabolic disturbances caused by a HFD in hamsters and rats (Guermouche et al. 2004,
Kasbi-Chadli et al. 2014), although the outcomes can be dependent upon the source of lipid used.
Furthermore, these beneficial changes have been linked to epigenetic alterations. Casas-Agustench
et al. demonstrated that pregnant rats supplemented with fish oil during early pregnancy decreased
the expression of hepatic miRNAs (miR192, miR21, miR26, miR10b, and miR377) involved in
glucose and insulin metabolism in offspring (Casas-Agustench et al. 2015). Supplementation with
n-3 PUFA during pregnancy was also seen to alter methylation profiles in CD4+ T cells derived
from cord blood (Amarasekera et al. 2014). Another anti-inflammatory PUFA, CLA (found primar-
ily in ruminant meat and dairy products), has been demonstrated to have beneficial effects in terms
of developmental programming of metabolic dysfunction. The supplementation of the maternal
HFD with CLA during pregnancy and lactation prevented early onset puberty and hyperlipidemia
in female offspring (Reynolds et al. 2015). In normal mice, however, maternal CLA consumption
induced the hypermethylation of hypothalamic POMC promoter regions in adult offspring and led
to metabolic dysfunction (Zhang et al. 2014), thus providing a potential epigenetic mechanism for
the differential effects of this anti-inflammatory nutrient. While there is a shortfall in the evidence
regarding specific epigenetic alterations in inflammatory genes as a direct result of maternal diet–
induced developmental programming, there is certainly a strong case for further investigations.
A combination of obesity-induced inflammation and adipose tissue hypertrophy promotes
the release of reactive oxygen species (ROS), thus promoting a state of oxidative stress. There
is evidence to suggest that this process can inhibit histone deacetylase, activating previously
silenced genes (Adler et al. 1999). Increased ROS production in insulin responsive tissues has
been suggested as a potential mechanism for the onset of obesity-related insulin resistance
(Nishikawa et al. 2007). Indeed, oxidative stress has been observed in several models of mater-
nal diet–induced developmental programming of metabolic dysfunction (Alfaradhi et al. 2014,
Preidis et al. 2014, Rodriguez-Gonzalez et al. 2015). Furthermore, programmed oxidative stress
responses have been linked to epigenetic changes in offspring. Strakovsky et al. demonstrate that
a maternal HFD induces hepatic promoter histone modifications in offspring that influence oxida-
tive balance (Strakovsky et al. 2014).
TRANSGENERATIONAL EFFECTS
Extensive evidence from experimental and human studies suggests that the process of developmen-
tal programming should be regarded as a transgenerational phenomenon with evidence for both
germline and somatic inheritance of epigenetic modifications that may underlie phenotypic changes
across generations. As such, programming should be viewed as a form of epigenetic inheritance,
either via the maternal or paternal line (Aiken and Ozanne 2014). It has been proposed that transgen-
erational epigenetic transmission of phenotype allows future generations to be maximally competi-
tive in their environment (Dunn and Bale 2011). Under this assumption, adaptations acquired during
the lifespan of the parent(s) persist into the next generation, thereby enabling future generations to
better survive in a potential environment of adversity. However, evidence suggests that environmen-
tal exposures including poor early life nutrition can result in maladaptive parental traits being passed
to offspring. The transmission of such epigenetic traits can therefore lead to the manifestation of
a phenotype at a population-wide level that occurs over several generations and can exacerbate
the rapid onset of phenotypes including obesity and noncommunicable diseases (NCDs) currently
observed in human populations (Dunn and Bale 2011).
A number of nutritional challenges have been reported to induce transgenerational phenotypic
changes in mammals (Kaminsky et al. 2006). Following undernutrition in the F0 generation, changes
in the hepatic methylation of the GR promoter are reflected in the F2 generation without any further
dietary manipulation of F1 female offspring (Benyshek et al. 2006). A MLP diet during F0 pregnancy
in the rat induces transgenerational changes in the hepatic transcriptome in offspring and includes
altered fasting glucose homeostasis and changes in PEPCK promoter methylation and expression
across three generations (Burdge et al. 2007b, Hoile et al. 2011). Using a model of a sustained envi-
ronmental dietary challenge (25% increase in energy compared to control diet), the expression of
DNA methyltransferase (Dnmt) 3a2, but not Dnmt1 or Dnmt3b, increased, and the methylation of its
promoter decreased from F1 to F3 generations (Burdge et al. 2011). These data suggest that, within a
generation, the regulation of energy balance during pregnancy and lactation can be influenced by the
maternal phenotype in the preceding generation and the environment during the current pregnancy.
The transgenerational effects on phenotype were associated with altered DNA methylation patterns
of specific genes in a manner consistent with de novo induction of epigenetic marks in each genera-
tion (Burdge et al. 2011). Dietary methyl donor supplementation has been shown by Waterland et al.
to prevent transgenerational amplification of obesity (Waterland et al. 2008). Although a number
of studies, primarily in the rodent, have reported phenotype transmission to the F2 lineage, trans-
mission to F3 or beyond (representing true transgenerational effects as it avoids the confounding
contributions of the initial maternal environmental insult) is less clear with some studies reporting
a resolution or amelioration of the programmed phenotype by the F3 generation. In a meta-analysis
by Aiken and Ozanne, of nine transgenerational studies carried through to F3, five failed to show any
effect at F3 (Aiken and Ozanne 2014).
Transgenerational inheritance via paternal influences has also been reported (Fullston et al.
2013), with obesity in fathers linked to metabolic disturbances across two generations of mice. Diet-
induced obesity in males resulted in alterations in miRNA content in sperm and germ methylation
status, thus impacting potential signals that program offspring health and initiate the transmission of
obesity to future generations. Studies in sperm in the F1 generation have also suggested a potential
role for alterations in IGF2 and H19 expression in the transmission of a phenotype to the F2 off-
spring (Ding et al. 2012). However, epigenetic alterations in the F1 sperm have not been reported
in all studies reporting a paternal line transmission (Drake et al. 2011). In work by Radford et al.,
there was no evidence that changes in the nutritional environment altered susceptibility to epigen-
etic reprogramming of imprinting control regions in the germline, thus suggesting that mechanisms
other than direct germline transmission may be responsible (Radford et al. 2012).
Although transgenerational transmission of phenotypic traits is often viewed as a form of epi-
genetic inheritance, there is also evidence for the effects of nongenomic components. This includes
the interaction between the developing fetus and the intrauterine environment in the propagation of
programmed phenotypes. These factors include a suboptimal reproductive tract and maternal con-
straint and altered maternal adaptations to pregnancy. The propagation of developmental program-
ming effects may therefore occur de novo through the maternal lineage in generations beyond F2 via
development in a suboptimal intrauterine tract and not necessarily directly transmitted via epigen-
etic mechanisms. Further, as aging can exacerbate the programmed metabolic phenotype, increases
in maternal age may also be a factor in increasing the likelihood of developmental programming
effects being transmitted to future generations.
PATERNAL EFFECTS
The paternal influence on epigenetic alterations in offspring cannot be neglected. As the father is
the sole transmitter of genetic and epigenetic factors to the oocyte, it has been argued that the father
may serve as a better model to explore epigenetic involvement in the setting of developmental
programming (Vanhees et al. 2014). Ng et al. reported a chronic paternal HFD programmed β-cell
dysfunction in female rat offspring paralleled by changes in DNA methylation profiles including
the hypomethylation of the II13ra2 gene. This was the first time that nongenetic, intergenerational
transmission of metabolic sequelae of a HFD from father to offspring was reported in mammals
(Ng et al. 2010, 2014). Offspring of male mice fed a protein-depleted diet and control-fed females
display an increased expression of genes involved in fat and cholesterol biosynthesis with increases
in methylation in an enhancer for PPAR-α, which could therefore regulate hepatic gene expression
(Carone et al. 2010). As noted earlier, the significant association between obesity in males and meth-
ylation status in offspring suggests that the developing sperm is susceptible to environmental insults
(Soubry et al. 2015). Further, studies in sperm in the F1 generation have suggested a role for altered
IGF2 and H19 expression in the transmission of phenotype to the F2 offspring (Ding et al. 2012),
but, as shown by Drake et al., not all studies examining paternal line transmission have reported
epigenetic alterations in F1 sperm (Drake et al. 2011).
STRATEGIES FOR INTERVENTION

Animal models have provided great utility in allowing the investigation of intervention strategies
aimed at ameliorating or reversing the effects of adverse early life developmental programming
including those described earlier regarding neonatal leptin treatment or maternal methyl donor sup-
plementation. Leptin was an early focus in nutritional programming studies with leptin treatment to
rat neonates shown to reverse the effects of IUGR on later cardiometabolic disorders in offspring in
rodents (Vickers et al. 2005, Gluckman et al. 2007) and piglets (Attig et al. 2008). The leptin gene
promoter is subject to epigenetic programming, and leptin expression can be modulated by DNA
methylation (Melzner et al. 2002, Stoger 2006, Iliopoulos et al. 2007). In the rat, leptin exposure
during lactation confers protective effects against the development of later obesity and related meta-
bolic disorders and may be associated with changes in promoter methylation of the hypothalamic
POMC gene (Palou et al. 2011). Further, leptin receptor activation can also induce the expression
of the suppressor of cytokine signaling-3 (SOCS-3) gene. SOCS-3 inhibits further leptin signaling
and can also inhibit insulin signaling. Alterations in SOCS-3 methylation may therefore have per-
sistent effects on the feedback loop that exists between leptin and insulin (the adipoinsular axis) and
negatively impact on phenotype development (Holness and Sugden 2006). In 3T3-L1 cells, specific
CpG site methylation and a methylation-sensitive protein may contribute to the regulation of leptin
gene expression during adipocyte differentiation (Yokomori et al. 2002). During preadipocyte to
adipocyte differentiation, it has also been demonstrated that both methylation of specific CpG sites
and a methylation-sensitive transcription factor contribute to the regulation of the GLUT4 gene
(Yokomori et al. 1999). In addition to the work on leptin and insulin signaling, differential DNA
methylation has been observed in promoters of genes known to be involved in glucose metabolism
including GLUT4 (Yokomori et al. 1999) and uncoupling protein 2 (UCP-2) (Carretero et al. 1998).
In addition to leptin, the GLP-1 analog exendin-4 can increase histone acetylase activity and reverse
epigenetic modifications that silence PDX1 in the growth-restricted rat (Pinney et al. 2011). As noted
previously, hypertension in offspring of LP-fed rat mothers is associated with changes in methyla-
tion and gene expression of the AT1b receptor (Bogdarina et al. 2007)—supplementation to LP-fed
mothers with metyrapone, an 11β-hydroxylase inhibitor, during the first 14 days of pregnancy, nor-
malized blood pressure, DNA methylation, and gene expression profiles in offspring (Bogdarina
et al. 2010). In a model of restricted maternal nutrition in the mouse to induce a preeclampsia-type
phenotype, pulmonary vascular dysfunction is associated with alterations in DNA methylation pat-
terns in the lungs of offspring. The administration of histone deacetylase inhibitors (butyrate and
trichostatin A) to offspring of these diet-restricted mice normalized pulmonary DNA methylation
and pulmonary vascular function. Further, nitroxide administration (Tempol) to the mother during
dietary restriction prevented vascular dysfunction and dysmethylation in the offspring, thus further
demonstrating the importance of epigenetic alterations in the programming of later vascular func-
tion (Rexhaj et al. 2011, Scherrer et al. 2015). In line with lifestyle modifications as a potential ave-
nue for the prevention of mitochondrial dysfunction and related cardiometabolic disorders, exercise
as an intervention modality has also been shown to lead to alterations in the DNA methylation of
the peroxisome proliferator–activated receptor gamma coactivator 1-alpha (PGC1α) promoter that
favors gene expression responsible for mitochondrial biogenesis and function (Cheng and Almeida
2014). In the MLP rat model, hypomethylation in offspring of PPAR-α and GR can be normalized
following maternal supplementation with folic acid (Lillycrop et al. 2005). As shown by Hoile et al.,
increasing the folic acid content of either the maternal or postweaning diets in the rat leads to differ-
ential effects on PEPCK expression and promoter methylation (Hoile et al. 2012). Maternal methyl
donor supplementation can reduce fatty liver and has been shown to modify the fatty acid synthase
DNA methylation profile in rats fed an obesogenic diet (Cordero et al. 2013a). Similarly, hyperho-
mocysteinemia induced by a maternal diet high in fat and sucrose intake can be prevented via the
supplementation of methyl donors during lactation, possibly via altered hepatic DNA methylation
and changes in the methionine–homocysteine cycle (Cordero et al. 2013b).
Imbalances in maternal micronutrients can influence LCPUFA metabolism and global methylation
in the rat placenta, mediated in part by a reduction in mRNA expression of methylene tetrahydrofo-
late reductase (MTHFR) and methionine synthase and increased cystathionine β-synthase (CBS) (Khot
et al. 2014). Supplementation with adequate concentrations of selenium and folate in female offspring
of mothers fed a HFD deficient in these factors during gestation and lactation can alter global hepatic
DNA methylation (Bermingham et al. 2013). Supplementation with omega-3s can ameliorate many of
these observed changes arising from maternal micronutrient imbalance through normalizing MTHFR
and CBS levels. Maternal supplementation with CLA has also been shown to improve maternal and
offspring outcomes, particularly as regards inflammatory profile and endothelial function in the set-
ting of maternal obesity (Gray et al. 2015, Reynolds et al. 2015), but the experimental data on CLA
are conflicting and the role of epigenetic processes is not well defined. In normal rodent pregnancies,
CLA can reduce adipose tissue mass via apoptosis (Tsuboyama-Kasaoka et al. 2000), and it has also
been shown that CLA supplementation can lead to the hypermethylation of the proximal specificity
protein (Sp1) binding site that suppresses hypothalamic POMC in neonates and therefore may contrib-
ute to metabolic disorders in adults (Zhang et al. 2014). In addition to folic acid and B vitamins, other
micronutrients including vitamins A and C, iron, chromium, zinc, taurine, and flavonoids play a role
in developmental programming. A maternal diet high in zinc can attenuate intestinal inflammation by
reducing DNA methylation and elevating H3K9 acetylation in the promoter of the anti-inflammatory
protein A20 (Li et al. 2015a). The efficacy of the methyl donors glycine and choline and the sulfonic
acid taurine in ameliorating the adverse effects of both maternal undernutrition and maternal obesity
across a range of experimental animal models has also been reported (Boujendar et al. 2003, Brawley
et al. 2004, Bai et al. 2012, Li et al. 2013, 2015b), although the epigenetic basis of these observations is
not well defined. It is known that taurine depletion in the perinatal period can increase oxidative stress
and mediate blood pressure control throughout life and that taurine depletion during early life leads to
epigenetic programming that can impact on later physiological function (Lerdweeraphon et al. 2013,
Roysommuti and Wyss 2014). As detailed earlier, maternal choline status modifies fetal histone and
DNA methylation and is involved in histone H3 methylation, expression of histone methyltransferases,
and DNA methylation of their genes in rat fetal liver and brain (Davison et al. 2009). Further, the impor-
tance of dietary methionine in the programming of hypertension in the setting of a MLP diet has been
highlighted. While one MLP diet preparation containing methionine consistently produces hypertension
in offspring, a further MLP diet without methionine supplementation resulted in either no change or a
slight reduction in blood pressure in offspring (Langley and Jackson 1994, Langley-Evans 2000).
As regards paternal transmission, it has been shown that targeted lifestyle interventions in the
obese father can normalize the transmission of disease traits to female offspring. In mice, diet or
exercise interventions for 8 weeks (covering two full cycles of spermatogenesis) in obese males led
to a restoration of insulin sensitivity and normalization of fat mass in female offspring (McPherson
et al. 2015). The diet and/or exercise regime also normalized the abundance of X-linked sperm miR-
NAs that are target genes involved in cell cycle regulation and apoptosis, pathways that are central
to oocyte development, and early embryogenesis. In addition, comorbidities associated with obesity,
including inflammation, glucose intolerance, stress, and hypercholesterolemia, served to be good
predictors for sperm miRNA abundance and offspring phenotypes.
Aims currently being pursued relate to the identification of epigenetic biomarkers in early life
to assess an individual’s disease susceptibility and the development of protocols for tailored dietary
treatments/advice to counterbalance adverse epigenomic events. As there is no “one size fits all,”
such approaches will allow early diagnosis and the potential for the facilitation of targeted therapeu-
tic strategies in a personalized “epigenomically modeled” manner to combat obesity and metabolic
disorders (Martinez et al. 2012).
DISCUSSION
Developmental programming during critical windows of plasticity has shown us how later life
health or an individual’s susceptibility to disease may be influenced by suboptimal early life nutri-
tion. To date, comprehensive genome-scale views of differential methylation following alterations in
early life nutrition are lacking in humans. The characterization of the genomic regions and biologi-
cal pathways involved are key in order to understand the environmentally induced plasticity of the
epigenome and its role in disease development (Tobi et al. 2014). To date, a range of experimental
models have elucidated distinct phenotypes showing marked effects of early life events on later
cardiometabolic status. In addition, many studies have demonstrated how these effects are verti-
cally passed to the offspring and increase the risk factors of cardiometabolic disorders including
obesity, hypertension, and metabolic syndrome. Furthermore, continued exposure to such refined,
poor-quality Western-style diets has the potential to exacerbate the effects of adverse early life pro-
gramming and lead to a feed-forward cycle of adverse health outcomes through future generations.
This may, in part, explain the exponential increase in NCDs observed throughout developed societ-
ies and those societies that are currently transitioning to First World economies.
Suboptimal early life nutrition can induce epigenetic alterations including altered DNA methyla-
tion, histone modifications, chromatin remodeling, and/or regulatory feedback by miRNAs, all of
which can modulate gene expression and promote cardiometabolic phenotype (Desai et al. 2015).
Such epigenetic mechanisms, given their importance in processes around orchestrating and stabiliz-
ing cellular differentiation, likely play a major role in controlling the physiological set points that
act to promote obesity and related disorders in our current obesogenic environment (Waterland
2014). Suboptimal nutrition in the periconceptional period, pregnancy, and/or lactation can lead to
a range of metabolic and cardiovascular disorders in offspring. Such programming effects are medi-
ated in part by epigenetic processes that represent an integrated network encompassing information
encrypted by DNA methylation patterns, histone modifications, and miRNAs (Martinez et al. 2012).
The extent of the windows of developmental plasticity in which epigenetic changes are induced in
key physiologic systems remains to be well defined, but the period of plasticity appears to extend
from the periconceptional period into early postnatal life (Weaver et al. 2004, Sinclair et al. 2007).
Given the transgenerational impacts, understanding the mechanisms by which developmental pro-
gramming effects can be transmitted across generations is an urgent area of research and is of
particular relevance to those populations transitioning between traditional and Western lifestyles
(including both changes in the types of nutritional exposures and potential for decreased physi-
cal activity). Of note, some disease traits appear to be resolved in subsequent generations where
others persist suggesting that there are divergent mechanisms of transmission involved. Moreover,
evidence to date suggests that those metabolic traits that do persist are capable of being transmitted
via the male germline (Dunn and Bale 2011). However, human evidence remains largely unsubstan-
tiated with most studies either observational or purely associative in nature. As such, data derived
primarily from the rodent provide the strongest argument for the potential of transgenerational epi-
genetic inheritance in humans (Morgan and Whitelaw 2008). As most work in human cohorts to date
on the epigenetic basis of disease is associative in nature, the extent to which such modifications
may mediate the effects of developmental programming of cardiometabolic disorders remains to be
defined. As an example, quantification of DNA methylation in blood may provide little linkage with
phenotype and therefore cannot be easily justified on a functional basis (Ojha et al. 2015). However,
these epigenetic markers may have utility as predictive biomarkers of later disease risk (e.g., the
link between cord blood promoter methylation of retinoid X receptor alpha and adiposity and bone
mineral content in childhood) (Godfrey et al. 2011, Harvey et al. 2014).
Understanding the role of the early life nutritional environment and mechanisms of epigenetic
inheritance across generations is essential in order to develop and validate effective intervention
strategies targeted at disease prevention to modulate not only that of the immediate adult phenotype
but also that of offspring, grand-offspring, and beyond. Given that around 80% of pregnant women
in the United States, for example, take supplements, the evidence surrounding maternal methyl
donor and vitamin supplements, one-carbon metabolism, and altered DNA methylation patterns
also raises the issue that more attention should be given to the safety and potential long-term effects
of such supplements on offspring (Vanhees et al. 2014). Importantly, epigenetic modifications that
occur during early development may not have any effect on phenotype development until later in
life, especially if they affect genes that modulate responses to later environmental challenges or sec-
ondary triggers, such as postweaning dietary challenges with energy-dense diets (i.e., the so-called
second hit). The impact of paternal effects can also not be underestimated as interventions aimed
at improving paternal metabolic health during specific windows prior to conception can normalize
aberrant epigenetic signals in sperm and improve the metabolic health of female offspring. In addi-
tion to parent-of-origin effects, it is also important to note that many studies to date have not con-
sidered the importance of sex-specific effects in offspring programming. Such sexually dimorphic
responses to programming stimuli need to be incorporated into future experimental studies as they
add translational value and aid in the mechanistic understanding of how cardiometabolic phenotypes
evolve. Further, adopting a life course perspective allows earlier identification of markers of risk
(Godfrey et al. 2010), with the possibility of implementing nutritional and other lifestyle interven-
tions early in life that have obvious implications for the prevention of NCDs across generations.
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29 Gene–Diet Interactions
Silvia Berciano and Jose M. Ordovas
CONTENTS
Abbreviations................................................................................................................................. 555
Introduction.................................................................................................................................... 556
Genetics of CVD and CVD Risk Factors...................................................................................... 558
Gene–Diet Interactions and CVD Risk..................................................................................... 558
Gene–Diet Interactions and Energy Balance............................................................................ 558
Gene–Diet Interactions and Glucose-Related Risk Factors...................................................... 563
Gene–Diet Interactions and Lipid Metabolism......................................................................... 565
CVD Risk Has Rhythm............................................................................................................. 566
Translation of Knowledge.............................................................................................................. 566
References...................................................................................................................................... 567
ABSTRACT
Cardiometabolic diseases are the principal cause of death globally, and are strongly driven by both
genetic and environmental factors, mainly nutritional. Nutrigenetic studies investigate the relation-
ship between genetic variants and diet in modulating cardiometabolic risk. Numerous studies have
reported statistically significant gene–diet interactions related to cardiovascular risk factors (obesity,
insulin resistance, and dyslipidemias) and more recently some evidence is emerging related to car-
diovascular events. However, current advances in this field are hampered by the lack of reproduc-
ibility across studies. Here, we describe the current state of the field of nutrigenetics with respect
to cardiometabolic disease research using some of the best characterized loci, and outline direc-
tions for the translation of this and future findings into new preventive and therapeutic options for
cardiometabolic disease.
ABBREVIATIONS
APOA1 Apolipoprotein A-1
APOC3 Apolipoprotein C-3
APOE Apolipoprotein E
ARIC Atherosclerosis risk in communities
BDNF Brain-derived neurotrophic factor
BMI Body mass index
BPRHS Boston Puerto Rican Health Study
CETP Cholesteryl ester transfer protein
CHARGE Cohorts for Heart and Aging Research in Genomic Epidemiology
CHO Carbohydrate
CLOCK Clock Circadian Regulator
EPIC European Prospective Investigation into Cancer and Nutrition
555
FTO Fat mass and obesity-associated

GI Glycemic index
GIPR Gastric inhibitory polypeptide receptor
GL Glycemic load
GOLDN Genetics of Lipid Lowering Drugs and Diet Network
GRS Genetic risk score
GWAS Genome-wide association studies
HDL High density lipoproteins
HOMA Homeostatic model assessment
IR Insulin resistance
IRS1 Insulin Receptor Substrate 1
LEP Leptin
LIPC Hepatic lipase
LOC Loss of control
MC4R Melanocortin 4 Receptor
MDCS Malmö Diet and Cancer Study
MedDiet Mediterranean diet
MESA Multi-Ethnic Study of Atherosclerosis
miRNAs MicroRNAs
MRE miRNA recognition elements
MRESS MRE seed sites
PCSK7 Proprotein Convertase Subtilisin/Kexin Type 7
PLIN1 Perilipin 1
PPM1K Protein Phosphatase, Mg2+/Mn2+ Dependent, 1K
PREDIMED PREvención con DIeta MEDiterránea
SNP Single nucleotide polymorphism
T2D Type 2 diabetes
TG Triglycerides
TCF7L2 Transcription Factor 7-Like 2
VLDL Very low density lipoprotein
INTRODUCTION
“The controversy concerning heredity versus environment is largely an argument of the past. It is now
generally accepted that genes act within the environments in which they find themselves, and that the
genetic potentialities of an individual may or may not find full expression because of the limitations
of the environment in which they exist. One aspect of the environment, which may affect the expres-
sion of genes, is nutrition.” Whereas these statements are totally appropriate and timely, we cannot
take ownership as they belong to the introduction of a review published in 1969 by Lucille S. Hurley
(1969), well before the advent of contemporary genetics and the upsurge of personalized nutrition.
The same author went on to emphasize this last concept: “Individual differences in reactions to food
are, it is believed by the writer, an expression, in part, of innate genetic individuality.” However,
this is not by any means the first explicit description of gene–diet interactions. We can go back more
than eight decades and find a similar discourse in the work by W. Franklin Dove (1935), describing
differences in bodily demands between genotypes: “Different genotypes or phenotypes controlling
form or function must be closely allied to the ability to choose food wisely. In the same way that one
individual is able to alter its choice of food in accordance with the requirements of each stage of its
Gene–Diet Interactions 557
growth or life cycle, so are individuals with differing genetic make-up able to make a selection of
food in accordance with their differences in form and function.” These nuggets are not unique in the
quarry of the scientific literature. Rather, they represent a minimal sample to show the oldness of
many research topics and how, sometimes, we forget their existence for decades just to bring them
up again to the limelight with contemporary names such as Nutrigenetics or Nutrigenomics.
The purpose of this chapter is not to review the ancient literature but rather to bring to the reader
the current status of the field in relation to gene–diet interactions and cardiometabolic health and
to project a vision of the future in this most relevant health area that, if properly used, may change
the future of preventive medicine and how we can achieve a healthier aging. Conscientious of this
potential and armed with the new weapons of genetics and molecular biology, an increased num-
ber of basic, nutritional, and clinical scientists have launched an offensive to conquer the essential
knowledge needed to materialize personalized nutrition. However, it is important to warn about the
overstatements that have been made in relation to personalized nutrition. The scientific objectives
are far from being accomplished and what we have in our hands is a large amount of promising and,
in most cases, unconnected findings that still need to crystalize into solid knowledge before the field
can move into clinical practice applications.
Beginning in the 1990s, there was an extensive and growing literature related to the contempo-
rary approach to gene–environment and particularly gene–diet interactions concerning cardiometa-
bolic health. Nevertheless, about one-third of the articles found in PubMed on this topic are review
articles. Moreover, the quantitative richness may not be paralleled by the qualitative wealth, con-
sidering that there is an appalling lack of replication among the findings that hampers the ultimate
objective of translating this knowledge into better cardiovascular prevention and therapy for every
member of society. The lack of replication is partially due to the limitations associated with previous
study designs:
• Mostly observational and retrospective, providing a low level of evidence

• Inadequate sample size to properly address the statistical power related with the complex-
ity inherent to the analysis of gene–environment interactions
• Uncertainty and subjectivity associated with gathering environmental information, includ-
ing diet
Progress to overcome some of these caveats, specifically regarding sample size, has been made
possible, thanks to the assembly of the Cohorts for Heart and Aging Research in Genomic
Epidemiology (CHARGE) consortium, created to enable meta-analyses of genome-wide associa-
tion studies (GWAS) and replication among many large population-based cohort studies (Bis et al.,
2009). However, improved methods that allow direct measurement of individual dietary intake are
urgently needed (Tucker et al., 2013).
In order to compose a bird’s-eye view of the current status of this research, we have recently cata-
loged the literature related to cardiometabolic gene–environment interactions (Parnell et al., 2014).
From this inclusive list of 386 publications, including blood lipids, glycemic traits, anthropometrics,
blood pressure, and inflammation, we concluded that the gene–environment single nucleotide poly-
morphisms (SNPs) listed in the catalog showed little overlap with variants identified for those same
traits in GWAS. These observations highlight the incomplete description of contribution to pheno-
typic variance by main effect associations such as those in GWAS, and strengthen the importance
of gene–environment interactions as contributors to that variance. This then implies that genetic
contributors alone are insufficient diagnostic tools for assessing disease risk, and that those calcula-
tions also must include the gene–environment term. Besides, the publications were enriched with
SNPs related to adaptation to environmental factors, such as climate. This may have had evolution-
ary implications on energy homeostasis and response to physical activity. Overall, the current body
of literature suggests that SNPs involved in cardiometabolic gene–environment interactions often
exhibit transcriptional effects or are under positive selection.
Withstanding the relevance of other environmental factors, our focus will be on gene–diet
interactions. After all, food is an essential component of our daily lives that, in addition, we can
individually modify to fit our needs and our health (Ordovas and Corella, 2004).
GENETICS OF CVD AND CVD RISK FACTORS

It has long been known that CVD has a strong genetic component, thanks to evidence gathered ini-
tially from twin studies and from inborn errors of metabolism (i.e., familial hypercholesterolemia).
Moreover, the knowledge of the physiology and biochemistry associated with specific metabolic
pathways involved in the development of the disease (i.e., plasma lipid metabolism) facilitated the
early adoption of the “candidate gene approach,” well before the advent of the GWAS, thus provid-
ing a “heads-up” to the field. However, a more precise and complete identification of the genetic
factors associated with CVD and its risk factors has been hampered by the significant influence of
environmental variables and the intrinsic complexity of the disease (Corella and Ordovas, 2009a).
Gene–Diet Interactions and CVD Risk

We are not aiming to provide a complete list and evaluation of the knowledge accumulated since
the 1990s; rather, we will focus on some representative and more impactful loci related to gene–diet
interactions and cardiometabolic traits. In some cases, the choice may be clear: fat mass and obesity-
associated (FTO) and energy metabolism; TCF7L2 and dysglycemia. For other CVD risk factors,
the choice may not be so evident and we will use our own discretion to bring examples that represent
the progress made on the field.
Gene–Diet Interactions and Energy Balance

The initial focus of gene–diet interactions in relation to cardiometabolic health fell in the area of
lipid metabolism; however, the interest has been increasingly shifting toward the pressing topic of
energy balance and obesity.
Obesity has become, in the mind of many, the number one public health enemy. Its prevention
and therapy have proved to be extremely difficult and the mindset has been shifting from a simple
balance equation based on energy in and energy out (and a conceptually simple solution: “eat less
and move more”) to a much more complex formula with an ever-increasing number of variables.
Some of them, like the microbiota and chronobiology, are gaining increased interest and relevance
alongside the more traditional factors (i.e., diet, physical activity). Even within the diet itself, we
have evolved from calorie-counting to a more elaborate scenario in which the different combinations
of macro (and micro) nutrients may play a key role. Finally, the complexity increases exponentially
when we add genetic individuality.
In practical terms, there are two sides to the obesity conundrum. One involves the prevention of
unhealthy weight gain and the other relates to successfully losing the excess of weight. Whereas
both have a significant genetic component, it is becoming evident that the overlap among the
genes involved is far from complete. Attaining a healthy body mass index (BMI) becomes harder
at increased adiposity levels, and this is due to both genetic and environmental factors. A recent
study (Fildes et al., 2015) that analyzed data for 76,704 obese men and 99,791 obese women has
shown that obese individuals have low chances (1.7% for men and 2.2% for women) of recover-
ing a normal weight or even reducing it by a modest 5% during a maximum of 9 years’ follow-up,
refueling disbelief in the current obesity management strategies. Lifestyle modification is a central
part of these interventions, and relies on the ability of the patient to adopt new, healthier habits. This
may be notoriously difficult for some individuals, and we are currently studying how the interplay
between genetics, epigenetics, and environment affects cognitive inhibition—that is, one’s ability to
process stimuli before eliciting a response—a key function in this process. It also needs to be noted
that, while the follow-up duration is limited to a few months in most studies, successful short-term
adherence may not imply long-term habit formation, and further research needs to be carried out on
this subject to better understand how healthy eating behaviors can become stable habits to support
weight maintenance in the long term.
Regarding weight gain, the state of the art of GWAS meta-analyses has led to the identification of
over 100 loci for common, polygenic anthropometric traits (i.e., BMI; waist circumference and body
fat distribution) (Locke et al., 2015; Shungin et al., 2015). These variants have modest effect and for
most of them their functionality remains unknown. In fact, for the most significant of these obesity-
predisposing loci, the FTO gene, the increase in BMI associated with the presence of each risk allele
is ~0.40 kg/m2. To put this in practical terms, this figure is similar to the average seasonal variability
in BMI found in humans through the year (van Ooijen et al., 2004), or the variability associated with
BMI changes resulting from dietary habits during certain holidays (Yanovski et al., 2000).
FTO was the first gene to be strongly associated with obesity using the GWAS approach and,
unsurprisingly, it has been one of the most investigated genes in terms of obesity-related associa-
tions and interactions. Nevertheless, the mechanism by which the FTO might contribute to obesity
remains unsolved. In fact, despite the metabolic phenotypes found in FTO rodent models and the
location of SNPs in intron 1 of the human gene, the implication of other neighboring genes in obe-
sity cannot be disregarded. Based on the high FTO expression levels in the hypothalamus, it has
been suggested that the mechanism of action could be mediated by a potential role in food intake
(Fredriksson et al., 2008), which is consistent with growing evidence since its discovery and with
the role proposed for many of the other newly identified obesity-predisposing genes.
Interactions between the FTO gene and environmental factors were initially reported for physi-
cal activity and the most comprehensive of these studies (Ahmad et al., 2013) was carried out in
111,421 individuals of European ancestry, with the FTO being part of a 12-loci genetic risk score
(GRS). Historically, the analysis and presentation of genetic findings has focused on individual
genes; however, we have to keep in mind the polygenic nature of CVD and its risk factors, and that
the practical translation of this research will not rely on individual loci but in more complex algo-
rithms incorporated in GRS. The use of GRS for prediction of risk or dietary response has two major
benefits. First, it takes into account the already mentioned complex polygenic nature of common
diseases. Moreover, it partially overcomes the problem of multiple comparisons associated with the
statistical analysis of many individual SNPs.
Returning to the FTO, secondary analyses of the rs1121980 SNP demonstrated a significant gene
by physical activity interaction (p = 0.003). The results emerging from the analysis of FTO by diet
interactions are more complex. Our group has found a significant interaction between saturated fat
(SFA) intake and FTO on BMI, in two independent American populations, the Genetics of Lipid
Lowering Drugs and Diet Network (GOLDN, n ~ 1100) and the Boston Puerto Rican Health Study
(BPRHS, n ~ 1300) (Corella et al., 2011a). We examined rs9939609 (in the GOLDN) and rs1121980
(in the GOLDN and BPRHS) SNPs. Our results show that subjects homozygous for the FTO-risk
alleles had a higher mean BMI than those with the other genotypes only when they had a high-SFA
intake, whereas no associations with BMI were found at lower SFA intakes in models adjusted for
energy intake. We also examined the potential interaction with carbohydrate (CHO) intake but our
analysis did not reveal any statistically significant findings. Therefore, from these data we can con-
clude that SFA intake modulates the association between FTO and BMI in these two populations of
White and Hispanic Americans.
A second study also based on three American populations (9,623 women from the Nurses’ Health
Study, 6,379 men from the Health Professionals Follow-up Study, and a replication cohort of 21,421
women from the Women’s Genome Health Study) posed a different, although related, question
and analyzed the interactions between genetic predisposition to obesity and consumption of fried
food using a GRS based on 32 BMI-associated gene variants (Qi et al., 2014a). The data revealed a
significant interaction between fried food consumption and the GRS by which the genetic associa-
tion with adiposity was strengthened with higher consumption of fried foods, with the FTO locus
showing the strongest result. Conversely, a meta-analysis performed based on data from 177,330
adults (154,439 Whites, 5,776 African Americans, and 17,115 Asians) from 40 studies did not find
significant interactions between the FTO gene and dietary intake of total energy, protein, carbohy-
drate, or fat on BMI (Qi et al., 2014b).
Given the potential role of FTO in food intake, this locus has been investigated in relation to
emotional eating and food consumption (Cornelis et al., 2014). For this purpose, information on
eating behavior and BMI was collected by questionnaires for 1471 men and 2381 women from the
two U.S. cohorts indicated earlier (Nurses and Health Professionals). The same GRS used earlier
was applied to this study and it was positively associated with emotional and uncontrolled eating.
Another study (Harbron et al., 2014) investigated associations between polymorphisms including
the FTO rs1421085 and rs17817449 haplotypes and dietary intake and eating behavior. The popu-
lation was much smaller (n = 133) than in the previous study and consisted of overweight/obese
Caucasian adults seeking treatment to lose weight. Weight and height were measured and eating
behavior was assessed by the Three Factor Eating questionnaire, which measures dietary restraint,
disinhibition, and hunger (Stunkard and Messick, 1985). The risk alleles of the FTO SNPs were
associated with poorer eating behaviors (higher hunger, internal locus for hunger, and emotional
disinhibition scores), a higher intake of high fat foods and refined starches supporting an effect of
the FTO locus on eating behavior.
The topic of FTO and eating behavior has been also examined in children, specifically in rela-
tion to loss of control (LOC) eating, a behavior associated with weight gain (Tanofsky-Kraff et al.,
2009). For this purpose, 289 youth aged 6–19 years were studied by genotyping the FTO-rs9939609
SNP and determining their level of LOC eating, a behavior assessed by interview and by participa-
tion in a buffet meal modeling an LOC episode. Subjects carrying the A risk allele had significantly
greater BMI and fat mass. Of these, ~35% reported LOC compared with ~18% of the TT subjects
(p = 0.002). Despite consuming the same amount of total energy, carriers of the A allele consumed
a significantly greater percentage of energy from fat than did the TT subjects. Therefore, this study
suggests that the youth carrying the FTO risk allele reported more often LOC eating and selected
more often the consumption of foods higher in fat. FTO is highly expressed in regions of the hypo-
thalamus that are considered key for appetite regulation and eating behavior. The preference for
fat and energy-dense foods has been proposed as a potential mechanism to explain the association
between the FTO risk allele A and obesity. Both findings (LOC eating and high-density food prefer-
ence) support that hypothesis. However, the authors selected a population enriched for overweight.
We know that there is an interaction between this FTO risk allele and saturated fat intake that results
in effective development of obesity only when the daily saturated fat intake exceeds 22 g per day.
The fact that the FTO carriers in this study had a significantly higher BMI than noncarriers could
have biased the results as selecting for children that are already on the road to obesity could mean
that carriers that have not displayed a preference for fat are left out. Therefore, this combination of
effects may be driving the increased BMI associated with this risk allele. Additional support for the
hypothesis involving FTO and eating behavior comes from the LOOK-AHEAD study, where the
risk allele at the FTO rs1421085 SNP predicted more eating episodes per day even after adjustment
for body weight (McCaffery et al., 2012). FTO rs1421085 was also associated with an increased
percentage of energy intake from fat (p = 0.019), although this effect was relatively small (0.52%
per risk allele copy).
The previous findings suggest certain consistency in terms of the association between the FTO
locus and eating behavior; however, the evidence is still very limited. Moreover, this question can
be addressed from a different perspective by examining macronutrient intake in relation to FTO
and BMI. This was examined in the Atherosclerosis Risk in Communities (ARIC) study including
10,176 Whites and 3,641 African Americans aged 45–64 years (Hardy et al., 2014). As expected, the
FTO SNPs (rs17817449, rs8050136) were significantly associated with higher BMI; and in media-
tion analysis, the FTO high-risk alleles were associated, in Whites, with higher BMI in part through
small effects on CHO and protein intake. In another meta-analysis, the FTO BMI-increasing allele
at the rs1421085 SNP was associated with higher protein intake, independent of BMI (Tanaka
et al., 2013). These findings and their magnitude suggest that the relationship between FTO variants
and BMI could be mediated, although moderately, through food intake. On one hand, there is an
interaction between saturated fat and FTO driving the BMI up. On the other hand, people with the
FTO variant eat more protein and carbohydrates, suggesting two independent mechanisms associ-
ated with this gene. One is driving food intake and the other one fat storage.
Analogous findings were reported in larger populations (n = 33,533 for discovery and n = 38,360
for replication) by Chu et al. (2013). In this study, the FTO rs10163409 was among the top associa-
tions for percent of total energy intake from protein and carbohydrate, suggesting preference for
these macronutrients with this SNP. Some early studies in children also showed that the energy
density of the food ingested as part of the experimental design was higher among carriers of the
risk allele as compared with the control genotype, which could explain some of their predisposition
to obesity (Cecil et al., 2008). Similar findings were observed in adult subjects participating in a
lifestyle intervention program to prevent diabetes (Haupt et al., 2009). In this study, the FTO risk
allele was significantly associated with higher energy intake even during dietary restriction, adding
more evidence in support of the role of the FTO gene influencing food intake (Haupt et al., 2009).
However, other earlier studies, despite showing the association between FTO and BMI, did not find
any nutrient-specific food preference associated with the risk allele (Bauer et al., 2009).
Gene–diet interactions related to weight loss have also been identified and have recently been
reviewed by Qi (2014). This excellent review highlights both the substantial progress made toward
revealing relevant gene–diet interactions as well as the many shortcomings that still need to be addressed.
In brief, the following loci were found to modulate the individual success of weight loss approaches
such as energy restriction and change in dietary macronutrient distribution: IRS1, TCF7L2, FTO, GIPR,
PPM1K, BDNF, and MC4R. In addition, loci including LEP, FTO, and BDNF were related also to dif-
ferences in weight regain following intervention. On the positive side, the availability of randomized
clinical trials is allowing the identification of loci that modulate the individual response to diet interven-
tions aimed at weight loss, as well as weight maintenance. On the negative side, the field still needs more
replication and identification of the functional variants and their mechanisms before translation of this
research into personalized diet interventions can be made a common clinical practice.
Since the publication of this review, other studies have been reported, but for the most part, they
focused on the interaction between obesity-related genes and weight-loss diets in relation to other
CVD risk factors such as plasma lipid profiles (Qi et al., 2015; Xu et al., 2015) and glucose-related
variables (Huang et al., 2015; Zheng et al., 2015). These studies support the notion that dietary fat
intake modifies the association between common genetic variants and plasma lipids (LIPC, CETP)
as well as insulin sensitivity (FTO); whereas dietary carbohydrate was a modulator of the associa-
tion between PCSK7 and insulin sensitivity.
We analyzed the association between a weighted obesity GRS, calculated on the basis of
63 obesity-associated variants, and BMI in the GOLDN study (n = 783) (Casas-Agustench et al.,
2014), focusing on gene–diet interactions with total fat and SFA intake. Moreover, we tested for rep-
lication of findings in the Multi-Ethnic Study of Atherosclerosis (MESA) (n = 2035). As expected,
a higher GRS was associated with increased BMI in both populations. More importantly, we found
significant interactions between total fat intake and the obesity GRS for the discovery and the rep-
lication population that reached a p-level for the interaction of 0.002 in the meta-analysis. The
interaction terms became even more significant for SFA intake. Thus, a high consumption of SFA
in combination with an elevated GRS magnifies the allele-raising effect as compared to those that
despite having the high GRS consume a low-fat diet and more specifically a low-SFA diet. A prac-
tical translation of these findings would be the strong recommendation to reduce total fat intake,
mainly by limiting SFAs, among those individuals with high obesity GRS. However, we need to
keep in mind that for “the whole to be greater than the sum of its parts,” these parts need to be well
characterized. Therefore, a detailed knowledge of each of the SNPs and their specific modes of
action is needed before their incorporation into any GRS.
We already mentioned that some of the candidate genes investigated in the past for associations
with CVD risk factors and related gene–diet interactions did not materialize in the corresponding
GWAS for main effects. One interpretation of this lack of concordance may be that the previous
associations were not real. A more positive twist is that the expression of the phenotypes associated
with those genes may be subject to strong gene–environment interactions that prevented their emer-
gence in the traditional GWAS. This may be the case for two of our best-studied candidate genes for
obesity: PLIN1 and APOA2.
Perilipin proteins were identified in the adipocyte, where they regulate lipid storage and lipolysis.
Perilipin 1 (PLIN1) is the most abundant of the adipocyte proteins, and over a decade ago we began
to investigate associations of the PLIN1 locus with obesity and related phenotypes focusing on six
SNPs (rs2289487, rs1561726, rs2304794, rs894160, rs2304795, rs1052700) (Qi et al., 2004a,b, 2015).
These studies revealed relatively consistent and gender-specific (women only) associations between
the SNPs and anthropometric and metabolic traits in different ethnic groups and geographical
locations.
Despite this compelling evidence, some studies failed to detect associations between PLIN1
and obesity-related phenotypes. Considering that dietary intake is one of the strongest determi-
nants of obesity, we launched a series of studies examining gene–diet interactions. The first of
them involved patients attending an obesity clinic (Corella et al., 2005). Subjects were exposed
to an energy-restricted intervention, and after 1-year follow-up, carriers of the minor allele at the
rs894160 SNP were much more resistant to weight loss compared with major allele homozygotes.
This could be related to the macronutrient composition of the diet (~40% of total energy from
fat, ~20% from protein, and ~40% from CHO). This hypothesis was tested in a subsequent study
based on Puerto Ricans living in the United States. In this population, dietary CHO interacted with
PLIN1 rs894160 SNP, in that carriers of the minor allele were protected from increased adiposity
in the context of high complex CHO intake, but were at risk of increased adiposity when complex
CHO intake was low (Smith et al., 2008). Findings from other studies can be interpreted in the
same light (Smith and Ordovás, 2012). The current evidence is consistent with the hypothesis that
a low-fat, high complex CHO diet may be protective against obesity for individuals with the minor
allele for rs894160.
Moreover, in one energy-restricted intervention (n = 177 Koreans), Jang et al. (2006) demon-
strated that carriers of the minor allele for either rs894160 or rs1052700, which are in strong linkage
disequilibrium in Asians, showed greater waist circumference and fat mass reduction as well as a
greater change in free fatty acids following weight loss. These two observations may be physiologi-
cally linked, since free fatty acids may reflect increased lipolysis accompanying weight loss, and
rs894160 is the same SNP for which Mottagui-Tabar et al. (2003) reported greater rates of lipolysis
in obese women.
The well-known connection between obesity and impaired glucose metabolism led us to investi-
gate PLIN1 in the context of glucose- and insulin-related traits. Thus, in a cohort of Spanish women
(n = 801) in whom linked PLIN1 SNPs rs2289487 and rs894160 were protective against adiposity,
an association between these SNPs and lower plasma glucose was also found, and adjustment by
BMI did not eliminate statistical significance of the association (Qi et al., 2004a). Another exam-
ple in support of taking the analysis beyond the traditional association studies and bringing diet
into the equation can be found for PLIN1 and insulin. Thus, PLIN1 genotype was not associated
with plasma insulin independently of nutritional factors in Singaporean Asian women (Malays,
Indians, and Chinese; n = 2198) (Qi et al., 2005); however, in these women, we detected interac-
tions between PLIN1 rs894160 and PLIN1 rs1052700 and saturated fat and CHO, which were
strongest when considered as a ratio (Corella et al., 2006). For homozygous minor allele carriers of
either SNP, the SFA:CHO ratio was associated with increased plasma insulin and HOMA-IR, and
this relationship was replicated in U.S. White women. In both Asian and White populations, inclu-
sion of a measure of adiposity (BMI or waist) did not attenuate the significance of the interaction,
suggesting that obesity was not the primary mediator of the SNP-related insulin resistance.
Another example of replicated evidence without support from GWAS findings comes from the
apolipoprotein A2 (APOA2) gene and its relation to obesity. We have identified and replicated a sig-
nificant interaction between the APOA2 gene variant [APOA2-265T > C (rs5082)] (which in vitro
has been shown to modulate APOA2 gene expression), dietary SFAs, and BMI. We analyzed
gene–diet interactions between the rs5082 SNP and SFA intake on BMI and obesity in subjects
from three American populations: (1) Framingham Heart Study (1454 Whites); (2) GOLDN (1078
Whites); and (3) BPRHS (930 Hispanics of Caribbean origin). We found that the magnitude of the
difference in BMI between the homozygotes for the risk allele C and carriers of the major allele
was dependent on SFA intake. We observed a difference of 6.2% in BMI between the two genotype
groups when SFA consumption was high (>22 g/d), but no difference when SFA consumption was
low. Moreover, the CC genotype was significantly associated with higher obesity prevalence in
these populations only when SFA intake was high (Corella et al., 2009b). These findings in three
independent populations were further replicated in other geographical areas and ethnic groups
(Corella et al., 2011b), and more recently, we replicated these interactions using specific foods
(i.e., dairy) (Smith et al., 2013).
Gene–Diet Interactions and Glucose-Related Risk Factors

Similar to obesity, most of the solidly established candidate genes involved in glucose metabolism
emerged from GWAS. This is the case of the Transcription Factor 7-Like 2 (TCF7L2) gene, the first
one to be consistently implicated in type 2 diabetes (T2D) (Grant et al., 2006; Voight et al., 2010),
especially in nonobese subjects (Bouhaha et al., 2010; Kalnina et al., 2012). Similarly to FTO and
obesity, the mechanistic basis for TCF7L2 and T2D is not well understood.
Several gene–diet interactions related to the TCF7L2 locus have been reported. Some of them
are related to glycemic traits; however, it is important to highlight that many genes cross the
boundaries of different CVD risk factors. Thus, we have already indicated that the TCF7L2 is
also involved in gene–diet interactions related to obesity (Fisher et al., 2012; Mattei et al., 2012;
Roswall et al., 2014). Moreover, one of the first TCF7L2 by diet interactions was reported in rela-
tion to postprandial lipemia (Warodomwichit et al., 2009). In that study, we reported that high
habitual intake (above the population median of 6.6% of total daily energy intake) of n-6 polyun-
saturated fatty acids (PUFA) was associated with atherogenic dyslipidemia (fasting plasma VLDL
concentrations and postprandial TG-rich lipoproteins) in carriers of the high-risk T-allele at the
TCF7L2 rs7903146 SNP as compared with CC subjects or T-carriers consuming low levels (below
the median) of n-6 PUFA.
In terms of glycemic traits, Fisher et al. (2009) investigated whether the protective effect of
whole grains related to postprandial glucose response and insulin demand might be attenuated in
the presence of the rs7903146 risk-conferring T-allele. These investigators tested their hypothesis
using a case-cohort approach that included 2318 randomized individuals and 724 incident T2D
cases from the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam
cohort. As expected, whole-grain intake was protective against T2D risk among rs7903146 CC
carriers, C being the low-risk allele. Conversely, carriers of the T high-risk allele did not benefit
from the protective action of whole-grain intake. A similar question was posed in the context of
a prospective population-based study (Wirström et al., 2013). The investigators examined the
8–10-year incidence of prediabetes and T2D in relation to the intake of whole grains in 2297 men
and 3180 women. Overall, a higher intake of whole grains was protective, with the exception of
men carrying the high-risk alleles at the TCF7L2 gene. Therefore, both studies support the notion
that the beneficial effect of whole-grain intake on T2D risk is modified by the common TCF7L2
rs7903146 SNP. These findings are further supported by another cohort of 24,799 nondiabetic
individuals from the Malmö Diet and Cancer Study (MDCS), who were followed for 12 years
and in whom high dietary fiber intake was associated with protection from T2D only among car-
riers of the low-risk allele for rs7903146. During this time, 1649 subjects developed incident T2D
(Hindy et al., 2012). Once again, this study demonstrates that the protective effect of a dietary
component—in this case, dietary fiber—may work preferably in those subjects who are nonrisk
allele carriers.
The interaction between this locus and dietary carbohydrates, with modulation of the effect of
the high-risk T-allele on T2D, was also investigated in the Nurses’ Health Study after stratifying
this population according to glycemic load and glycemic index (Cornelis et al., 2009). Using the
GG genotype as a reference, the multivariate-adjusted ORs (95% CI) of T2D associated with the TT
genotype were 2.71 (1.64, 4.46) and 2.69 (1.64, 4.43) in those subjects in the highest tertile of GL
and GI, respectively. The ORs (95% CIs) for TT subjects in the lowest tertile of GL and GI were
1.66 (0.95, 2.88) and 1.82 (1.11, 3.01). A more recent study in an Algerian population (Ouhaibi-
Djellouli et al., 2014) observed gene–diet interactions related to dessert and milk intake and T2D
risk. This risk was greater in T-allele carriers with high dessert and milk intakes as compared with
CC subjects or T-allele carriers with low dessert and milk consumption. Overall, similar to studies
in other populations, the T-allele at the TCF7L2 rs7903146 SNP was associated with a higher risk
of T2D and this association was magnified by high dessert and milk intakes; however, the specific
nutrients involved in these interactions were not reported in this study.
Although the experimental designs and the questions differ considerably across these studies,
there seems to be an agreement regarding the significant interaction between the TCF7L2 locus,
carbohydrate intake, and T2D risk.
Perhaps the most relevant of all the gene–diet interactions described so far relates to the risk
of stroke in the PREvención con DIeta MEDiterránea (PREDIMED) study (Corella et al., 2013).
The PREDIMED study was a randomized dietary intervention trial (two MedDiet intervention
groups and a control group) with 7018 participants. In this context, we investigated whether the
associations of the TCF7L2-rs7903146 SNP with T2D, glucose, lipids, and CVD incidence were
modulated by a Mediterranean diet (MedDiet). Data were analyzed at baseline and after a median
follow-up of 4.8 years. Consistent with current knowledge, the TCF7L2-rs7903146 SNP was
associated with T2D. Moreover, the MedDiet interacted significantly with rs7903146 on fasting
glucose at baseline. Thus, when adherence to the MedDiet was low, TT subjects had higher fast-
ing glucose concentrations than C-allele carriers (CC+CT). Conversely, when adherence to the
MedDiet was high, this increase in fasting glucose was not observed in TT subjects. Similar statis-
tically significant interactions were noted for total cholesterol, LDL cholesterol, and triglycerides
(p interaction < 0.05 for all). Moreover, during the randomized trial, TT subjects had a higher
stroke incidence in the control group compared with CC carriers (p = 0.006), whereas in the groups
consuming the MedDiet no differences in stroke incidence were observed between TT and CC
homozygotes (p = 0.892). These results support the concept that MedDiet reduces the otherwise
increased fasting glucose and lipids in TT subjects, but most importantly also lowers the incidence
of stroke to a level similar to that observed in CC subjects in both arms of the MedDiet intervention
(extra-virgin olive oil and nuts).
Additional gene variants associated with increased T2D risk and insulin resistance found in the
gene that encodes the Insulin Receptor Substrate 1 (IRS1)—a key player in insulin signaling path-
ways—were identified by GWAS. In particular, rs2943641 T-allele carriers and rs757826 G-allele
carriers have been shown to be less susceptible to insulin resistance, T2D, and metabolic syndrome
(MetS) than noncarriers. Over the last few years, several research groups have found interactions
between these SNPs and different macronutrient intakes. Our results from GOLDN and BPRHS
(Zheng et al., 2013) showed that carriers of both protective alleles (G-T haplotype) may only experi-
ence lower insulin resistance and MetS risk when their saturated fatty acid-to-carbohydrate ratio is
low (SFA:CHO ≤ 0.24). Similarly, we also found that the rs757826 G-allele was only associated
with a reduced risk of developing MetS when MUFA intake was lower than the median of each
population.
Additional evidence comes from the POUNDS LOST trial (Qi et al., 2011) (n = 738, predomi-
nantly White Americans, 2-year follow-up) regarding the fairly well-studied rs2943641 SNP: carriers
of two risk alleles (CC) randomized into the highest-carbohydrate (lower fat) diet group were found
to experience a greater decrease in plasma insulin concentrations, BMI, and insulin resistance
(HOMA-IR). Gender effects have also been shown to affect variables associated with this genotype,
particularly adiposity for which the interaction only held significance in men. The aforementioned
MDCS (Ericson et al., 2013) results provided further insight into gender-specific gene–environment
interactions for this locus by suggesting that the minor T-allele interacted with diet in a sex-depen-
dent manner affecting T2D risk (significantly decreased T2D incidence only in women in the lower
tertiles of carbohydrate intake, and men in the lowest tertile of fat intake). The latter results, despite
coming from an observational study and thus not providing the highest level of evidence to sup-
port this effect, may help us recognize the potential complexity of gene–environment interactions, in
which parameters like gender could completely shift the optimal dietary approach needed to neutral-
ize a genetic susceptibility.
Gene–Diet Interactions and Lipid Metabolism

Some pioneering studies in the field of gene–diet interactions and cardiometabolic health relate to
candidate genes in the path of lipoprotein metabolism, specifically associated with the APOE and
the APOA1/APOC3/APOA4/APOA5 gene cluster. However, whereas there are clear and consistent
associations between some of these loci and plasma lipid concentrations, the results from gene–diet
interactions suffer from the similar lack of replication found for other metabolic pathways.
In this section, we will focus on the lipoprotein lipase (LPL) gene, a key player in plasma lipo-
protein metabolism and more specifically in the catabolism of triglyceride-rich lipoproteins. Similar
to other candidate genes (i.e., APOE, APOA5), there are clear and consistent associations between
common LPL variants and plasma lipid levels. However, the functionality and mechanisms involved
in these associations are, for the most part, unknown. We will use this locus to illustrate the cross
talk between genetics (LPL), epigenetics (microRNAs), and diet (PUFA) and to demonstrate how
this new knowledge is helping to assign functionality to SNPs identified through candidate gene or
GWAS approaches.
MicroRNAs (miRNAs) are small, 20–24 nucleotide, noncoding RNAs that function as posttran-
scriptional inhibitors of gene expression by binding to miRNA recognition elements (MRE) within
the 3′UTR of their target mRNAs (Bartel, 2009). The most critical region for binding and repression
of mRNA by a miRNAs are positions 2–7 of the MRE, called the “seed site.” SNPs in MRE seed
sites (MRESS) have been shown to decrease or eliminate miRNA-mediated repression (Brennecke
et al., 2005). Moreover, some epidemiological evidence is mounting supporting that SNPs within
an MRE or MRESS are associated with phenotypic variation (Saunders et al., 2007; Sethupathy and
Collins, 2008; Richardson et al., 2011).
MicroRNAs have emerged as important epigenetic regulators in CVD. Therefore, based on previ-
ous findings related to gene–diet interactions for obesity and the results of a genome-wide search
for SNPs in MREs and MRESS, we decided to explore the rs13702T>C SNP (rs13702) in the
3′ untranslated region of the LPL gene for functionality based on miRNA-related mechanisms. This
SNP has an extensive literature over the past decade and a solid track record of associations with
plasma triglyceride and HDL-C concentrations. Furthermore, the rs13702 is in linkage disequilib-
rium with several SNPs identified by GWAS associated with HDL-C and TG. In our in silico pre-
diction, the rs13702 minor allele had been found to disrupt an MRESS for the human miRNA-410.
We used the CHARGE consortium to perform a meta-analysis and, consistent with the literature,
we found a highly statistically significant association of the rs13702 SNP with low triglyceride
(p = 3.18 × 10(−42)) and high-HDL-C (p = 1.35 × 10(−32)) with each copy of the minor allele
associated with 0.060 mmol/L lower triglyceride and 0.041 mmol/L higher HDL-C (Richardson
et al., 2013). Then, we carried out in vitro functionality tests and demonstrated that the expression
of an LPL 3′ UTR luciferase reporter carrying the rs13702 major T-allele was reduced by 40%
in response to a miR-410 mimic. Finally, we examined the interaction between intake of dietary
fatty acids and the LPL-rs13702 SNP. Our meta-analysis involving 10 of the CHARGE cohorts
demonstrated a highly significant interaction between the rs13702 SNP and dietary PUFA with
respect to plasma triglyceride concentrations (p = 0.00153). Thus, the protective effect of this SNP
on plasma triglyceride levels was further enhanced by an additional reduction of −0.007 mmol/L in
the presence of high PUFA intake. The same applied to the HDL-C raising effect that was further
increased by dietary PUFA. Our results demonstrate that the rs13702 SNP induces the allele-specific
regulation of LPL by miR-410 in humans and these effects (lowering TGs and increasing HDL-C)
were enhanced as the habitual intake of PUFA increased in the participants.
In a follow-up study, we intended to extend and solidify these findings by assessing the interac-
tion between the rs13702 SNP and fat intake on triglycerides at baseline and longitudinally by using
the PREDIMED dietary intervention design (Corella et al., 2014). We also examined as a primary
outcome the association of this variant with CVD incidence and its modulation by the MedDiet.
Gene–diet interactions for triglyceride were analyzed at baseline (n = 6880) and after a 3-year inter-
vention (n = 4131). As in previous populations, the rs13702 SNP was significantly associated with
lower plasma triglycerides in C-allele carriers. Moreover, we found a significant dietary interaction
at baseline with unsaturated fat similar to the one reported in the CHARGE cohorts. After 3 years
of intervention with MedDiet, high in unsaturated fat, the C-allele was associated with an even
greater reduction in triglyceride concentrations. Consistent with the protective lipoprotein profile
associated with the C-allele, we found an association with lower stroke risk that reached sta-
tistical significance only in the combined MedDiet intervention groups but not in the control group
(p interaction = 0.044).
Therefore, in addition to validating our previous study in terms of lipid associations and gene–
diet interactions, our findings in PREDIMED revealed a new association between rs13702 and
stroke incidence, which is modulated by diet in terms of decreasing stroke risk in rs13702 C-allele
carriers following a high-unsaturated fat MedDiet intervention.
CVD Risk Has Rhythm

The interest in chronobiology is experiencing a dramatic increase. This is in part due to the realiza-
tion that chronodisruption is associated with most chronic diseases including CVD. Moreover, we
should not forget that our metabolism and vital signs such as body temperature, respiratory rate,
and blood pressure present circadian variations that may affect the risk of suffering cardiovascular
events. For instance, myocardial infarctions do not occur evenly throughout the day, but rather they
concentrate during specific time frames (i.e., early morning). Therefore, when it comes to gene–diet
interactions—and particularly, personalized nutrition—time needs to be included in the equation.
Our own research shows that CLOCK SNPs (i.e., rs4580704 and rs1801260) are associated with
BMI and glucose-metabolism-related traits. Moreover, we found a modulation of the associations
of these SNPs with plasma glucose, insulin resistance, and anthropometric traits by MUFA and SFA
intakes (Garaulet et al., 2009). Furthermore, variation at the CLOCK locus was also associated with
energy intake (Garaulet et al., 2010).
TRANSLATION OF KNOWLEDGE
Further research into gene–diet interactions is crucial to generate solid knowledge that will allow
us to launch clinical applications aimed at early prediction of CV risk. The conceptual process is
straightforward. Once genetic variants associated with CVD or an intermediate phenotype (i.e.,
dyslipidemia, dysglycemia, obesity) are found and we know that a certain diet can counteract that
genetic risk, then one can forecast that CVD risk could be effectively reduced through recommenda-
tion of a personalized diet. To date, studies like PREDIMED have focused on primary prevention,
but there is also great interest in discovering gene–diet interactions in secondary CVD prevention in
order to provide appropriate dietary recommendations for individuals who have already had a non-
fatal CVD event, as will be possible with studies like CORDIOPREV (Garcia-Rios et al., 2014). The
results of this research can provide us with the much needed knowledge to achieve more successful
prevention and therapy of CVD.
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30 Its Relationship to Health and
Gut Microbiome
Its Modulation by Diet

Brian J. Bennett and Katie A. Meyer
CONTENTS
Introduction.................................................................................................................................... 572
Gut Microbiome............................................................................................................................. 572
Inflammation and the Microbiome................................................................................................. 575
The Gut as a Metabolically Active Tissue..................................................................................... 576
Microbiota and Their Metabolites Can Affect CVD Risk............................................................. 577
Microbiota and Cardiometabolic Risk Factors.............................................................................. 578
Functional Studies of the Microbiome and Cardio-metabolic Health: Study Designs to
Understand and Test Mechanisms.................................................................................................. 578
Gnotobiotics to Test Microbiota................................................................................................ 579
Human Fecal Transplants.......................................................................................................... 579
Effects of Diet on Microbiota........................................................................................................ 580
Specific Effects of Diet and Potential Interaction with Cardiometabolic Health.......................... 581
Fiber.......................................................................................................................................... 581
Polyphenols............................................................................................................................... 582
Pre-/Probiotics........................................................................................................................... 582
Food Additives.......................................................................................................................... 582
Interactions between Host Genetics and Microbiota..................................................................... 582
Host Genetics and Microbial Composition............................................................................... 583
Altered Microbiota across Life Span............................................................................................. 584
Newborns and Early Childhood................................................................................................ 584
Maternal Diet............................................................................................................................ 585
Microbiota and Elderly Populations......................................................................................... 585
Summary........................................................................................................................................ 585
Glossary......................................................................................................................................... 585
References...................................................................................................................................... 586
ABSTRACT
Recent advances in sequencing technologies and analytics have dramatically improved our
understanding of the gut microbiota. We now have a solid foundation for studying the microbiota
and thus recent efforts have focused on identifying how perturbations to the microbiota composition
and function affect disease risk. This chapter focuses on the relationship between the microbiota and
cardiometabolic risk with an emphasis on how diet modulates these effects.
571
INTRODUCTION
Alterations in the composition of the gut microbiome have been identified to increase suscep-
tibility to several chronic metabolic diseases including diabetes (Larsen et al. 2010), obesity
(Turnbaugh et al. 2008), and cardiovascular disease (Karlsson et al. 2012, Robles Alonso and
Guarner 2013). These findings have generated enthusiasm since they point to a largely unstudied
contributor to biologic variability that may have large effects on human health. Furthermore, the
microbiome appears to be modifiable through diet, pre-/probiotics, and transplantation, open-
ing avenues for strategic intervention. At the same time, genetics appears important, as does
early-life establishment of the gut community. We are still in the infancy of the science, and
much remains to be understood/discovered, including the causal importance of the microbiome.
Clearly, a better understanding of the microbiome’s role in disease, and modifiability by dietary
nutrients, could be useful as we begin to design nutritional interventions that target an indi-
vidual’s personal risk.
A developing body of literature indicates that microbial dysbiosis, an altered or unhealthy micro-
biome, including low microbial diversity, low abundance of bacteria considered beneficial, and
increased presence of pathobionts, resident microbes with pathogenic potential (Chow et al. 2011),
in the digestive tract (Vijay-Kumar et al. 2010, Ridaura et al. 2013) may influence systemic inflam-
mation by altering gut permeability and thus increasing circulating lipopolysaccharide (Ostos et al.
2002, Cani et al. 2008, 2009)—a powerful trigger for immune response. Another line of literature
reveals the microbiome to be a metabolically active, complex organ, producing many metabolites
that can directly influence disease susceptibility, including those metabolites derived from nutrients.
The hypothesized mechanisms of action relating the microbiome to disease are not exclusive, nor
necessarily independent of each other, and there is evidence that diet and perhaps specific food
components can affect disease risk directly or indirectly.
GUT MICROBIOME
The potential role for gut microbiota in our biology has long been recognized and studied via
culture-based methods (Falk et al. 1998), but recent developments in next-generation sequenc-
ing (NGS) technology and bioinformatics methods have accelerated discoveries and expanded
understanding of the health relevance of our microbial symbionts. These “culture-free” methods
do not rely on the ability to grow bacteria in a laboratory and the microbial species identified
through NGS have not been successfully cultured. These technological advances have promoted
an explosion of research into the microbiome over the past decade, including the creation of
major cooperative projects such as the NIH Common Fund’s Human Microbiome Project (HMP)
(Human Microbiome Project, Consortium 2012) and the European Metagenomics of the Human
Intestinal Tract (MetaHIT) (Arumugam et al. 2011), designed to delineate the composition and
function of human microbial communities across multiple sites in representative samples of
healthy human adults. These studies have revealed 10–100 trillions of symbiotic microbial cells
residing across and within the human body. Microbial ecosystems have coevolved to provide
specific local functions, with distinct microbial communities populating body compartments
such as the gut, oral cavity, and vagina, and contributing unique functions that enhance our biol-
ogy. The digestive system is one of the largest reservoirs of these microbes, and the microbial
community of the lower intestine is particularly relevant with respect to diet, nutrition, and car-
diometabolic disease.
As with many new fields, there is some confusion regarding the terminology used to describe the
gut community. Microbiota and microbiome are often used interchangeably; for the purposes of this
chapter, we use microbiota to specifically refer to the taxonomic membership of the gut community,
and microbiome to refer to the genomic information embodied in that community. We reserve the
use of the term metagenomics for shotgun sequencing of total DNA to distinguish it from analysis
Gut Microbiome 573
of microbial community composition by sequencing marker genes (e.g., 16S ribosomal RNA).
We focus specifically on the gut microbiome due to its close relation to nutrition and its associa-
tion with cardiometabolic risk, but note that there are multiple microbial communities throughout
the body, each with distinct ecologic characteristics. Most studies in humans that we will discuss
use stool samples as a proxy for the lower-gut microbiome, while studies in model organisms may
profile cecal contents or specific regions of the intestinal tract. Furthermore, we will largely restrict
our discussion to bacterial members of the gut community, but increasing research includes viral and
archaeal components (Hoffmann et al. 2013).
Much study of the gut microbiome to date has focused on the composition of the gut micro-
bial community, while there has been less work employing measures of genetic potential or
functional activity. The emphasis on community composition has been driven by both technical
and analytical practicalities, as many of the individual bacteria comprising the microbiota have
not yet been successfully cultured in the laboratory. The inability to culture these bacteria limits
the functional characterization of specific microbiota because they cannot be specifically tested
in vitro or in vivo.
Gut community composition is typically estimated by sequencing regions of the 16S ribo-
somal RNA (rRNA) marker gene, which is sufficiently conserved to identify bacteria, yet
includes hypervariable regions to distinguish taxonomic groups (Doi and Igarashi 1966, Colli
and Oishi 1969). In these analyses, 16S rRNA sequences are assigned to taxonomic groups
(called operational taxonomic units, OTUs) (Schloss and Westcott 2011), from which statistical
metrics of the ecologic community are derived, including clusters of taxonomic groups, and
measures of diversity and richness (Legendre and Legendre 2012). Several analytical tools have
been developed for 16S analysis and have been reviewed elsewhere (Kuczynski et al. 2012,
Goodrich et al. 2014a).
One goal of the HMP and MetaHIT was to serve as reference samples for describing a
core or optimal microbiome among healthy individuals. Based on results from these and other
studies, we now know that members of phyla Firmicutes and Bacteroidetes dominate the gut
microbiota with lesser representation from Actinobacteria, Proteobacteria, Verrucomicrobia,
Fusobacteria, and other phyla (Mahowald et al. 2009). In both HMP and MetaHIT, Bacteroides
was the dominant genus (Arumugam et al. 2011). These characterizations of the core composi-
tion of the microbiota are robust. In one comparative analysis, taxa with an OTU prevalence
of 0.50 or greater in the HMP sample were confirmed in at least 90% of non-HMP samples
(Fodor et al. 2012). Still, in spite of these relatively consistent taxa in humans, studies have
revealed large variability within and across populations in the composition of the gut microbi-
ome (Human Microbiome Project, Consortium 2012), and very few OTUs occurred in 95% of
the HMP sample (Huse et al. 2012). Thus, there is a core of microbes found in the microbiota
of humans alongside considerable variation in the overall makeup of the microbial community.
This individual variability in the microbiome is particularly apparent with respect to the relative
abundance of taxonomic groups. For example, the most abundant OTU in HMP gut microbi-
ome samples accounted for an average of 23% of total sequences across cohort members, but
ranged from 0.02% to 84% across individuals (Huse et al. 2012). Some studies have proposed
that individuals may be distinguished through enterotypes, loosely defined as similar clusters of
taxonomic groups (Turnbaugh et al. 2009, Arumugam et al. 2011). However, these clusters have
not been consistently observed across studies (Wu et al. 2011, Yatsunenko et al. 2012, Human
Microbiome Project, Consortium 2012), and it now appears more likely that a multitude of pos-
sible distinct clusters exist and that microbial composition may be better conceptualized as a
gradient or through continuous measures of compositional pattern (Huse et al. 2012, Knights
et al. 2014). Studies of age- (Biagi et al. 2010, 2012, Yatsunenko et al. 2012) and geographically
diverse (Yatsunenko et al. 2012) populations have revealed large between-person differences in
microbial composition, further illustrating the enormous natural variability of microbial com-
munities. Together, these findings do not provide strong support for a single core group of
microbiota across healthy populations. These studies also point to current limitations, despite
detailed studies in hundreds of humans, in creating a definitive classification scheme of the
microbial community among healthy humans.
The classification schemes described earlier focus on 16S rRNA characterizations of
the microbiota. An alternative approach is to focus on the overall functional characteristics
of the microbiota. Although relatively fewer in number, studies that have conducted whole-
genome metagenomics have shown that the large variation observed in compositional measures
of the microbiota does not imply large variability in the functional potential or activity of the
microbiota. In fact, these studies point to clear redundancy in gene presence and expression,
suggesting a core set of activities that can be fulfilled by different microbiota (Turnbaugh et al.
2009, Qin et al. 2010, Human Microbiome Project, Consortium 2012). We know from shotgun
metagenomics studies that the total microbial community DNA encompasses a rich set of genes
involved with carbohydrate and amino acid metabolism, illustrating a core functional role of
the gut microbiome in digestion and metabolism. The presence of specific microbial genes only
reflects functional potential, and to truly characterize functional activity of the microbiome,
it will be necessary to employ microbial measures of messenger RNA (metatranscriptomics),
proteins (metaproteomics), and metabolites (metametabolomics) (Integrative 2014). These
approaches may reveal variability related to health and disease not captured through 16S rRNA
characterization of the microbiota and contribute to understanding a healthy core microbiome
from a functional perspective. For example, a study utilizing metagenomics and metatranscrip-
tomics analysis revealed that there is greater variability in gene expression than in gene presence
(Franzosa et al. 2014). Similarly, proteins related to carbohydrate metabolism have been shown
to be expressed at a level greater than expected from metagenomics profiles (Verberkmoes et al.
2009). Furthermore, there is an indication that copy-number variation may also impact the func-
tional capacity of the microbiota (Greenblum, Carr, and Borenstein 2015). Understanding the
factors that regulate differences in microbiota gene expression and their relationship to disease
is still a critical gap in our knowledge.
Despite the lack of a clear compositional definition of a healthy microbiome, with respect to
specific patterns of community membership, certain objective features are often cited as consis-
tent with dysbiosis. Diversity is generally considered a component of healthy ecology, contribut-
ing to an ecosystem that is more stable and less susceptible to experiencing long-term altered
responses to external perturbations (such as—in the case of the gut community—treatment with
antibiotics), and it is possible to consider diversity in terms of microbial composition or func-
tional activity. Thus, in the absence of specific patterns of community membership fitting into a
notion of a core healthy microbiome, diversity—of either microbial composition or functional
activity—may be an important feature. It is important to note that there are different approaches
to defining dysbiosis. For example, dysbiosis is often defined relative to health, using features
that distinguish unhealthy individuals from healthy individuals as the measure of dysbiosis.
Applying such a definition, characteristics of dysbiosis will likely vary depending on the out-
come considered.
The gut microbiome of healthy adults is remarkably stable in the absence of large perturba-
tions—such as dramatic weight loss or gain, dietary changes, or antibiotic use (Costello et al.
2009, Jakobsson et al. 2010, Caporaso et al. 2011, Dethlefsen and Relman 2011, Faith et al.
2013). In a U.S. study of 27 healthy adults, sampled between 2 and 13 times over 5 years, 60%
of microbial strains were consistently observed over the study period (Faith et al. 2013). Samples
collected over time reveal greater between-person than within-person variability, and support
a relatively stable individual profile. While it is true that the resilience of diverse ecosystems
reflects a healthy stability, it is also true that microbial communities can degrade into unhealthy,
but stable, patterns. Furthermore, an unstable microbiota does not always reflect poor health,
as in infancy or if rebounding from a perturbation, such as antibiotic use or dietary changes. In
addition to returning to a previous steady state following a perturbation, the microbiome can
Gut Microbiome 575
rebound to a new steady state, which may differ from the original state. The time course of acute
changes and the return to a steady state is still currently under investigation. For example, in a
dietary crossover study, gut microbial changes were observed within 24 h after administration of
a vegetable- or meat-based diet, and reverted to the prestudy microbiome after 2 days of return-
ing to the original diet (David et al. 2014). Gut microbial changes have also been observed within
1 week of antibiotic use; in some instances, antibiotic-related changes remain after 4 years of
observation (Jakobsson et al. 2010). Much work is needed to delineate gut microbial changes in
response to exposure changes. In addition, these patterns of change vary by individual and we
do not as yet understand the predictors of response patterns (Jakobsson et al. 2010, Dethlefsen
and Relman 2011).
INFLAMMATION AND THE MICROBIOME

Inflammation plays a unifying role in cardiometabolic disease (Gorjao et al. 2012), affecting ath-
erosclerosis (Libby 2002), insulin resistance (Glass and Olefsky 2012, Romeo, Lee, and Shoelson
2012), and obesity (Cancello and Clement 2006). In addition, there is growing recognition for the
importance of the gut in immune system regulation, with subsequent metabolic effects. Details of
this system remain to be delineated, but evidence supports a complex interplay between immune
and gut microbial systems. For example, it has been shown that mutations in single genes related
to host immunity can have dramatic effects on the microbial community structure (Wen et al. 2008,
Thompson et al. 2010). Thus, specific deletion of gut mucosal expression of TLR-5 (toll-like recep-
tor 5), an innate immune system modulator, results in insulin resistance (Vijay-Kumar et al. 2010).
In addition to genetic manipulation of immune-related genes in mice, specific tests are used to iden-
tify inflammation in the gut. There is evidence that high fecal levels of calprotectin, an S100-related
protein found in neutrophils and granulocytes, is indicative of an inflammatory colonic environment
(Hildebrand et al. 2013). In addition to the identification of secreted proteins (such as calprotectin),
several taxa, such as members of the Clostridiales order, are known to be decreased in intestinal
inflammatory environments (Schwab et al. 2014). These findings suggest that differences in immune
response and chronic inflammatory disease susceptibility may result from differences in microbiota
composition.
Barrier function is crucial to guard against bacterial translocation from the gut into the circula-
tion. Impaired barrier function with subsequent endotoxemia has been linked to risk factors for
cardiometabolic disease, notably increased fasting glycemia, decreased glucose tolerance, increased
body and liver weight, increased liver triglyceride content, and increased energy intake (Cani et al.
2007). Studies utilizing knockout mice for specific antigen recognition receptors on immune cells
residing in the intestine, such as Cd14 and Nod1, demonstrate a critical link between phagocytosis
of bacteria in the microbiota and increased bacteria in the plasma and tissues of high-fat-fed mice
(Amar et al. 2011).
Specific bacteria may impact barrier function of the intestine and physiological traits.
Bifidobacterium longum, known to promote tight junction integrity, was associated with
decreased plasma triglycerides suggesting a protective role (Ulluwishewa et al. 2011). On the
other hand, Rumminococcus gnavus, a mucin-degrading species associated with reduced barrier
integrity and bile acid metabolism, was positively related to plasma triglycerides and plasma
glucose. Reduced bacterial translocation results in improved glycemic control and reduced adi-
posity, indicating perhaps a causal role for the microbiota in cardiometabolic disease through
effects on the intestinal barrier integrity. Specific comparisons of the microbiota of the oral
cavity, fecal samples, and carotid endarterectomy samples indicate that there are notable differ-
ences in the composition of each of these sites, but also highlight the possibility that some of
the specific bacteria present in the atherosclerotic plaque could also be derived from the distal
gut or the oral cavity (Koren et al. 2011). Interestingly, altered Proteobacteria:Firmicutes ratios
have been observed in circulating bacteria among patients with CVD as compared to “healthy”
Food
Generation of microbial-
derived metabolites
Local effects Microbiota

on intestine
Direct/indirect
effects on CVD risk
Systemic inflammation
Dyslipidemia LPS
Insulin resistance
Obesity
FIGURE 30.1 (See color insert.) Potential mechanisms by which the gut microbiota affects cardiovascular
risk. Changes in microbial community structure can change gut permeability and allow microbes to enter the
bloodstream (leaky gut syndrome). Lps or other bacterial products can induce low-grade systemic inflamma-
tion. Alternatively, the microbiota can metabolize nutrients in food to specific metabolites that either increase
or decrease susceptibility to cardiometabolic disease.
controls with detectable bacteria in their blood (Rajendhran et al. 2013). We have outlined the
inflammatory and gut permeability pathways in Figure 30.1.
THE GUT AS A METABOLICALLY ACTIVE TISSUE

The gut is a primary site of digestion and we now recognize the complex role the microbiome has in
the metabolism of many individual foods and nutrients that compose our diet. Part of this renewed
appreciation is driven by technological advances in metabolomics, which now allow the detection
of many small compounds that were previously undetectable.
For example, the colonic metabolome of patients with nonalcoholic fatty liver disease (NAFLD),
as compared to healthy controls, contains increased fecal ester volatile organic compounds, such
as butanoic acid, and altered levels of certain Lactobacillus and Firmicutes bacteria (Raman et al.
2013). This presents the possibility that altered microbiota may affect the levels of endogenously
produced toxins that increase susceptibility to metabolic disease. These results are supported by stud-
ies comparing germ-free (GF) mice with conventionalized mice. Dozens of circulating molecules
were detected only in conventionalized mice, and ~10% of commonly observed molecules differed
significantly in concentration between GF and conventionalized mice (Wikoff et al. 2009). A high-
fat diet has also been shown to affect both the global metabolome and microbiota community as
well as perturb specific taxa and metabolic pathways such as bile acid and hormone metabolism
(Daniel et al. 2014). Consistent with these findings are those from a 2-week dietary intervention
among African-Americans and Africans in which African-Americans were allocated a high-fiber,
low-fat diet, more typical of a standard native African diet, and Africans were allocated a high-fat,
low-fiber diet, more typical of an African-American diet. Changes in microbiota were apparent, with
Gut Microbiome 577
increased pathogenic proteobacteria among Africans after consuming the high-fat/low-fiber diet, but
compositional changes in microbiota were less pronounced than changes in microbial metabolites
following the dietary intervention, with increased butyrate production associated with the high-fiber,
low-fat diet and proinflammatory metabolites associated with the low-fiber, high-fat diet (O’Keefe
et al. 2015). The approach of linking health-related metabolites to the gut microbial community in
diseased and healthy states allows us to delineate mechanisms through which the microbiome influ-
ences disease and identify potential clinical targets for intervention.
This meta-organismal approach was recently applied to cardiovascular disease and led to the
discovery of an association between a specific bacterial metabolite, trimethylamine N-oxide, and
cardiovascular risk in humans and mice (Brown and Hazen 2014). We describe this pathway and its
relationship to cardiometabolic disease in detail later in this chapter.
MICROBIOTA AND THEIR METABOLITES CAN AFFECT CVD RISK

It is important to note that in order to design novel therapies for CVD based on microbiota, it is
important to identify specific microbes and their biologic products that can affect disease. In 2011,
Trimethylamine N-oxide (TMAO) was identified as a novel risk factor for CVD in humans and a series
of animal and human studies have subsequently characterized several genetic polymorphisms, dietary
components, and microbial factors affecting TMAO levels (Wang et al. 2011, Bennett et al. 2013,
Koeth et al. 2013, Tang et al. 2013, Hartiala et al. 2014). Plasma TMAO levels show a dose-dependent
association with the severity of coronary atherosclerosis in cardiac patients. Subjects in the upper
quartile for TMAO levels (compared with the lowest quartile) had a significant, approximately three-
fold increased risk of experiencing a major adverse cardiac event (MACE), such as death, myocardial
infarction (MI), and stroke, as well as overall poorer event-free survival, over a 3-year period (Tang
et al. 2013). Notably, this relationship was independent of traditional CVD risk factors, renal function,
and medication use. Atherosclerosis-susceptible ApoE−/− mice fed a diet supplemented with choline,
l-carnitine, or TMAO had increased lesion size (Wang et al. 2011, Koeth et al. 2013). Additionally,
studies with multiple inbred strains of mice suggest that TMAO explains about 11% of the total varia-
tion of atherosclerosis (Bennett et al. 2013). Since the initial reports, several studies have replicated the
association of TMAO with CVD, including studies of patients with chronic heart failure (Troseid et al.
2014), diabetes mellitus (Lever et al. 2014), and renal disease (Tang et al. 2015).
Thus, this meta-organismal pathway may be an important new paradigm to consider for an
improved understanding of atherosclerotic heart disease and perhaps other cardiometabolic dis-
ease processes (Tang and Hazen 2014). We give a brief overview of this pathway with a focus on
the metabolism of dietary nutrients. One route for the initial catabolism of dietary choline and
l-carnitine (a nutrient important for fat metabolism) is mediated by intestinal microbes and leads to
the formation of trimethylamine (TMA). Foods rich in choline and l-carnitine, such as eggs, milk,
and red meat, can thus lead to increased TMA production (Zeisel et al. 2003). TMA is efficiently
absorbed from the gastrointestinal tract and oxidized in the liver by the flavin-containing monooxy-
genase (FMO) enzymes to form trimethylamine N-oxide (TMAO) (Bennett et al. 2013). Studies
have shown that Fmo3 is indeed the primary FMO responsible for hepatic metabolism of TMA
to TMAO through a series of experiments that modulated Fmo3 mRNA levels using adenoviral
overexpression, transgenic overexpression, and in vivo antisense oligonucleotides and examined the
effect on circulating levels of TMAO (Bennett et al. 2013).
Recent efforts have focused on the link between the microbiome and the atherogenic metabolite
TMAO. The microbiome plays an obligate role in the formation of TMA (from trimethylamine-
containing nutrients choline and carnitine), and antibiotic knockdown studies show clearly that
TMAO is not formed in the absence of the microbiome (Tang et al. 2013). Bacterial species harbor-
ing putative choline utilization gene clusters (cut-c) have been suggested to play a central role in
enteric TMA formation(Craciun and Balskus 2012) (and therefore down-stream TMAO produc-
tion); however, the specific microbiota have not been fully ascertained.
Mice receiving cecal microbes from atherosclerosis-prone donors demonstrated dietary choline-
dependent enhancement in atherosclerotic plaque burden compared to atherosclerosis-resistant donors.
Using adoptive transfer approaches, cecal contents from distinct inbred donor strains with differing ath-
erosclerosis potential and TMAO production capacity were identified and then introduced via gastric
gavage into recipient mice in which endogenous gut microbes were initially suppressed through use of
an oral antibiotic cocktail. A global 16S analysis revealed successful transplantation of donor microbiota,
which tended to show coincident proportions with plasma TMAO levels (Gregory et al. 2015).
MICROBIOTA AND CARDIOMETABOLIC RISK FACTORS

In addition to TMA and cardiovascular risk, it is important to note that the microbiota’s influence
on other cardiometabolic risk factors such as dyslipidemia, insulin resistance, diabetes, and obesity
has been extensively investigated.
Several elegant studies have clearly established a role for the microbiome in regulation of body
weight and adiposity (Backhed et al. 2004, Ridaura et al. 2013). A shared genetic regulation has
also been reported, with loci regulating complex traits of body composition coinciding with loci
regulating microbial abundances (McKnite et al. 2012, Parks et al. 2013). The negative relation-
ship of Roseburia, Blautia, and other unclassified genera of the Lachnospiraceae family with body
weight and fat mass, and their positive relationship with lean mass suggest a relationship between
butyrate production in the intestine and adiposity. In addition to providing a nutrient source for the
enteric epithelium (De Vadder et al. 2014), increased butyrate levels may also increase host energy
expenditure (Gao et al. 2009). Additionally, butyrate can influence gut peptide secretion, such as
glucagon-like peptide 1 (GLP-1) and peptide YY (PYY), with the potential secondary effect of
increased satiety (Hosseini et al. 2011). These effects may be mediated via short-chain fatty acid
(SCFA) receptors such as GPR43 (Kimura et al. 2013).
Studies of humans identified a bimodal distribution of the microbiota composition with individu-
als having either low gene counts or high gene counts. Interestingly, there was increased adiposity
in the low gene count group, and this was associated with increased serum leptin, decreased serum
adiponectin, insulin resistance, increased levels of plasma triglycerides, and increased inflammation
as determined by highly sensitive C-reactive protein (hsCRP) levels and higher white blood cell
counts (Le Chatelier et al. 2013).
Blood profiling of patients identified that an increase in the proportion of Proteobacteria phylum
was associated with long-term cardiovascular risk; from initial analysis, CVD risk appears to be
independent of plasma lipoprotein levels (Amar et al. 2013).
FUNCTIONAL STUDIES OF THE MICROBIOME AND CARDIO-METABOLIC

HEALTH: STUDY DESIGNS TO UNDERSTAND AND TEST MECHANISMS
One challenge in understanding the role of the microbiome in health is the difficulty establish-
ing causality between actual microbes within the microbiota and disease. The basic framework for
establishing causality of a microbe and disease was laid forth by Robert Koch in the late 1880s and
they are called “Koch’s postulates” (Fredricks and Relman 1996):
1. The parasite occurs in every case of the disease in question and under circumstances that
can account for the pathological changes and clinical course of the disease.
2. The parasite occurs in no other disease as a fortuitous and nonpathogenic parasite.
3. After being fully isolated from the body and repeatedly grown in pure culture, the parasite
can induce the disease anew.
This framework states that if the disease-causing entity is a microbe, differences in disease
susceptibility should follow transplantation of the microbe into a new host. Using adoptive transfer
Gut Microbiome 579
approaches, it has been possible to identify pathogenic microbes that cause disease. Obviously, as
medicine and technology have evolved, these principles have been refined (Falkow 2004). In the
case of the microbiota, establishing causality is more difficult as these bacteria may not be uniformly
associated with the disease. In spite of this limitation, the basic concept of experimental validation of
the relationship between a specific entity in the microbiota and cardiometabolic disease is important.
There are several additional challenges in order to apply these principles to the microbiota. The
first difficulty is that microbiota are a complex community and the effects observed on human health
may be the result of the interactions among multiple taxa. Secondly, the association between the
gut and cardiovascular health is spatially separated, and thus changes in the microbiota most likely
affect disease risk through complex pathways with potentially many susceptibility factors that vary
among people. Lastly, many of the bacteria in the microbiota have not yet been cultured successfully,
and thus functional tests to establish causality remain technologically challenging. Obviously, estab-
lishment of causality is a high threshold of scientific work and thus many of the studies described
throughout this chapter are associative in nature. What is clear from the vast number of associative
studies is that there are strong impacts of the gut microbiome on disease susceptibility, but the
underlying bacterial species, or gene sets from multiple species, causing disease remain elusive.
Gnotobiotics to Test Microbiota

One tool that has been particularly useful in demonstrating specific effects of the microbiome are
gnotobiotic animals, defined as an animal stock or strain derived by aseptic cesarean section (or
sterile hatching of eggs) that is reared and continuously maintained with germ-free techniques under
isolator conditions (Gordon and Pesti 1971). Gnotobiotic techniques have been used for well over
50 years in microbial research, but recent advances have facilitated creation of several large facili-
ties for using gnotobiotic mice. By using adoptive transfer approaches, various microbes can be
transferred from humans, mice, or specific cultured microbes into gnotobiotic mice. Most often
the adoptive transfer is performed using gastric gavage. These tools have been extremely useful to
demonstrate that a phenomenon observed in an associative study is due to specific differences in the
microbiome.
There are significant resource barriers to the development and use of gnotobiotic mice as this
approach requires specialized techniques, facilities, and equipment. These challenges have spurred
researchers to develop other methods to address causality in model organisms. A primary approach
is to use broad-spectrum antibiotic treatment to dramatically and effectively reduce the microbial
population of the gastrointestinal tract in rodents and then to perform an adoptive transfer experi-
ment using gastric gavage. These experiments are partially confounded by the nonspecific effects of
antibiotic treatment, but when utilized with appropriate controls, they have been used to demonstrate
the effects of microbiota on a number of clinically relevant phenotypes.
As development of high-throughput and cost-effective sequencing technologies expands, it is
possible to consider other experimental designs to demonstrate causality of the microbiota. For
example, mice are coprophagic or feces-consuming rodents, and thus cohoused animals may adopt
a similar microbiome. Therefore, one study design is to combine mice of differing genotypes, either
different inbred strains or gene targeted knockout mice, to test if one “microbiota” is dominant
amongst them. These studies can identify the invasive nature of microorganisms and the stability of
changes over time among mice with variable genetic makeup.
Human Fecal Transplants

In addition to the use of model organisms, there is evidence that perturbing, and even transplanting,
the microbiome between humans may have therapeutic benefit. For example, individuals with per-
sistent and recurrent Clostridium difficile infection have been treated with fecal transplants. Several
clinical studies have been performed and a recent meta-analysis of these data identified that 245 of
the 273 patients receiving fecal transplants resolved their Clostridium difficile infection (Kassam
et al. 2013). This initial success has prompted the design of studies investigating treatment of a vari-
ety of diseases, including cardiometabolic disease (Smits et al. 2013). One report demonstrated that
transplant of microbiota from lean individuals improved insulin sensitivity of patients with meta-
bolic syndrome (Vrieze et al. 2012). It is important to note that the clinical efficacy and potentially
unintended adverse effects of such approaches needs to be fully evaluated before these therapies are
moved into mainstream clinical practice.
EFFECTS OF DIET ON MICROBIOTA

The human microbiome utilizes both dietary and host-derived nutrients for survival. Thus, changes
in host diet can have a profound impact on the microbiome (Cotillard et al. 2013, Spor, Koren,
and Ley 2011), including altering the overall bacterial composition, influencing gene expression,
or promoting a bloom or inhibition of certain taxa. These dynamic changes have been shown in
both mouse and human microbial populations in response to dietary intervention (Spor, Koren, and
Ley 2011). As noted earlier, changes in the diet generally yielded changes in the adult microbiome
within 24 h, and a return to the baseline microbiome can be observed within 2 days following an
individual’s resumption of their usual diet (David et al. 2014). Interestingly, alterations beginning
early in life may have long-lasting effects on multiple phenotypes (Cox et al. 2014).
National comparisons have revealed significant differences in gut microbial composition with
distinct microbial patterns by country (Yatsunenko et al. 2012). These differences are apparent
across all age groups (0–70 years) (Yatsunenko et al. 2012) and appear as early as 6 months of
age (Grzeskowiak et al. 2012). Broadly, data indicate that Western adult populations may have
on average lower microbial diversity as compared to African or South American populations. In
addition, taxonomic differences are apparent, with higher Prevotella abundance among Africans
(De Filippo et al. 2010, Yatsunenko et al. 2012, Ou et al. 2013, Schnorr et al. 2014) and South
American (Yatsunenko et al. 2012) samples, as compared to Americans (Yatsunenko et al. 2012, Ou
et al. 2013) or Europeans (De Filippo et al. 2010, Schnorr et al. 2014). These studies support diet-
related differences, such as the enrichment of fiber-degrading microbiota in non-Western popula-
tions, but likely reflect a multitude of nondiet differences as well.
Observational and intervention studies support differences in the gut microbiome related to
macronutrient consumption. Among 178 elderly adults, a healthy food diversity index was sig-
nificantly associated with measures of microbial diversity, with higher microbial diversity among
individuals consuming a diet high in fruits, vegetables, and whole grains, and low in red meat and
snacks/sweets (Claesson et al. 2012). In another study, gut microbiota clustered into 3 taxonomic
groups among 98 adults based on their habitual dietary consumption patterns of predominantly
carbohydrate or predominantly high protein and fat (Wu et al. 2011). Cyclical changes in diet
may affect microbiota composition fairly quickly; a randomized control trial of high-fat/low-fiber
or low-fat/high-fiber diets found there were changes in the microbial composition within 24 h.
However, these changes within individuals are insufficient to overcome the taxonomic differences
between subjects observed at baseline (Wu et al. 2011). Similarly, in an intervention study of three
diets high in resistant starch, high in nonstarch polysaccharides (wheat bran), or high in protein/
low in carbohydrate, gut microbial changes were observed within days, but failed to overcome
initial between-person differences (Walker et al. 2011). These findings illustrate the strength of an
individual microbial pattern, but do not necessarily support a lack of large dietary effects on the gut
microbiome, particularly given data indicating clear functional redundancies among microbiota.
For example, a crossover study in 10 adults allocated to exclusively plant- or animal-based diets
revealed significant differences in functional measures, even in the absence of large compositional
changes in microbial pattern. After 24 h, bacterial gene expression changes distinguished subjects
based on their assigned diet, rather than their individual baseline microbiome, with enrichment
of microbial enzymes reflecting the needs of the specific diet (e.g., bile-acid enzymes increased
Gut Microbiome 581
with animal-based diet) (David et al. 2014). Studies in gnotobiotic mice reconstituted with known
human microbiota and fed a variety of diets demonstrate significant plasticity in gene expression
that is rapid, reproducible, and reversible in response to changes in diet (McNulty et al. 2013).
These studies further illustrate the need for functional measures in studies of diet, the gut micro-
biome, and health.
SPECIFIC EFFECTS OF DIET AND POTENTIAL INTERACTION

WITH CARDIOMETABOLIC HEALTH
Much of the efforts to understand the interactions of diet and the microbiome have focused on the
effects of a high-fat, calorie-rich diet. In model organisms, there has been a focus on identifying
the changes to the microbiota when a high-fat diet is consumed, and how altered composition of
the microbiota relates to susceptibility or resistance to disease. Clinical studies in humans have
focused on microbiota changes during weight loss interventions (Cotillard et al. 2013). In addition
to increased energy density or alterations in macronutrient composition, specific nutrients or dietary
components may have effects on the microbiota that are associated with cardiometabolic health.
Fiber
A large literature supports a protective role for fiber in various aspects of cardiometabolic dis-
eases, including insulin sensitivity, glucose homeostasis, circulating lipids, and body weight.
Fiber includes nondigestible carbohydrates or lignin that may be fermented by microbiota in
the large intestine. Fiber encompasses many compounds—including resistant starch and oligo-
saccharides—that differ in their potential for microbial fermentation, as well as their ability to
alter the composition and function of the gut microbial community. Extensive epidemiological
evidence has led to the broad recommendation to consume a diet high in fiber to reduce risk of
cardiovascular disease (American Heart Association Nutrition et al. 2006). The impact of fiber
on cardiometabolic disease may be partially mediated by products of gut microbial fermentation
of fiber and through fiber-induced (“prebiotic”) microbial changes to a more health-promoting
microbial profile.
Interestingly, the use of germ-free mice with a humanized microbiota determined that gastroin-
testinal transit time was faster when mice were fed a high-fiber (cellulose) diet, compared to mice
fed a polysaccharide-deficient diet. Additional tests with a diet containing a fermentable fructo-
oligosaccharide resulted in increased gastrointestinal transit time. These data suggest that a fiber-
enriched diet improves gastrointestinal motility due to the presence of insoluble, nonfermentable
fiber that reduces microbe-mediated carbohydrate fermentation (Kashyap et al. 2013a).
Microbe-mediated carbohydrate fermentation may be of particular interest. SCFAs (butyr-
ate, propionate, and acetate) are products of microbial fermentation of fiber that may influence
cardiometabolic health through local colonic as well as systemic pathways. Locally, SCFAs, in
particular butyrate, enhance colonic membrane integrity, limiting inflammatory responses due to
lipopolysaccharide movement from the gut to the circulation. In the periphery, SCFAs act as sig-
naling molecules for glucose homeostasis and insulin sensitivity in the liver, skeletal muscle, and
adipose tissues; influence concentrations of appetite-related hormones (peptide YY, glucagon-like
peptide 1, and leptin); and suppress the release of proinflammatory mediators. A clinical study
investigating diet alterations in obese subjects showed that reduced carbohydrate intake increases
butyrate-producing bacteria in the feces (Duncan et al. 2007).
The production of SCFAs varies according to the type, amount, and compositional form of avail-
able fiber substrate, and the composition and function of the gut microbial community (Walker et al.
2011). There is evidence for some patterns of enzymatic specificity within taxonomic groups, such
as greater acetate-producing bacteria within the Firmicutes phylum and greater butyrate-producing
bacteria within the Bacteroidetes phylum, but there is also much overlap in function across phyla.
Furthermore, SCFAs that are produced by some microbiota are further degraded by other microbiota,
highlighting the enormous complexity of the gut microbiome as an ecologic system.
Polyphenols
Polyphenols have been widely investigated due to their antioxidant and anti-inflammatory proper-
ties. Several risk factors associated with cardiometabolic disease are influenced by inflammation or
oxidative stress and therefore polyphenols have been hypothesized to potentially reduce cardiovas-
cular disease (CVD) risk. One area that is often overlooked is that an estimated 90%–95% of dietary
polyphenols can be metabolized by microbiota in the lower gut prior to entering the bloodstream
(Clifford 2004). Thus, the microbial community can directly influence the extent to which bioactive
polyphenol metabolites are available to the host, potentially explaining observed individual vari-
ability in metabolite production (Atkinson et al. 2008).
In addition, the health benefits of polyphenols may partly be mediated through polyphenol-induced
changes on the gut community. In fact, several studies have demonstrated microbial changes follow-
ing feeding interventions with polyphenol-rich foods. For example, cranberry extracts enriched for
polyphenols affected the levels of Akkermansia spp., a taxa associated with insulin sensitivity and
intestinal inflammation (Anhe et al. 2015). Increases in butyrate-producing bacteria Bifidobacterium
or Lactobacillus have been observed in interventions of isoflavones (Clavel et al. 2005), cocoa fla-
vanol (Tzounis et al. 2011), almonds (Liu et al. 2014), wild blueberries (Vendrame et al. 2011),
coffee (Jaquet et al. 2009), red wine (Queipo-Ortuno et al. 2012), green tea (Jin et al. 2012), apples
(Shinohara et al. 2010), and bananas (Mitsou et al. 2011).
Pre-/Probiotics
There has been tremendous interest in the role that pre- and probiotics have in modulating the
effects of the microbiome. For example, prebiotic feeding with oligofructose in ob/ob mice, a
genetically obese strain, had broad effects on the microbiota affecting >100 distinct taxa and result-
ing in improved glucose tolerance and reduced fat-mass (Everard et al. 2011). Prebiotic feeding
normalized abundance of A. muciniphila, a taxa increased in obese and diabetic mice, and the
change correlated with an improved metabolic profile (Everard et al. 2013). Further analysis of
metagenomic sequencing data revealed that high-fat diets and prebiotics affected the microbiota at
different taxonomic levels and that prebiotics counteracted high-fat-diet-induced metabolic disor-
ders (Everard et al. 2014). Similar effects of improved adiposity and plasma lipid levels have been
seen with administration of wheat arabinoxylans (Neyrinck et al. 2011).
Food Additives
A less frequently studied area of research is the effects of processed foods and specific food prod-
ucts on the microbiome. A recent study identified that noncaloric artificial sweeteners had negative
effects on insulin sensitivity that were nullified by antibiotic treatment (Suez et al. 2014), indicating
that specific alterations in the microbiota induced by sweeteners may cause this effect. The robust
nature of this specific interaction remains to be corroborated, but it does point to a limitation in our
current understanding of the health effects of food additives.
INTERACTIONS BETWEEN HOST GENETICS AND MICROBIOTA

Genetic factors are clearly important to determine the risk of cardiometabolic disease. What
is not clear is how the microbiota can buffer the risk of cardiometabolic disease or increase
risk independently of the underlying genetic composition of the individual and if these del-
eterious or positive effects of the microbiota work in conjunction with the consumption of
Gut Microbiome 583
a specific diet. An interesting possibility is that specific genetic loci in the host may affect
microbial composition and thereby modulate risk of cardiometabolic disease. We outline some
of these concepts here.
Host Genetics and Microbial Composition

Considering the interindividual variability at the level of the microbiome (Eckburg et al. 2005, Qin
et al. 2010), detailed studies integrating the intestinal microbiome with disease risk complement
genome-wide association studies (GWAS), and other efforts seeking to assess the basis for hetero-
geneity in health and disease status. Importantly, understanding how microbial diversity and specific
microbial species affect clinical phenotypes and risk of CVD will be beneficial as we begin to focus
on personalized approaches to nutrition and medicine. As our interest in the microbiome’s role in
chronic disease has expanded, so has interest in how host genetics influences microbial diversity.
Several groups have reported that enteric microbial composition is a heritable trait (Turnbaugh et al.
2010, Tims et al. 2013), although results from twin studies have shown evidence for discordance of
heritability (Turnbaugh et al. 2009). One limitation of the initial characterization of the heritability
of the composition of the microbiota is the limited population sample sizes available. Recently, 1000
fecal samples from the TwinsUK population were characterized. This cohort of ~416 twin pairs
demonstrated that specific members of the microbiota are influenced, at least in part, by underly-
ing host genetic factors. Interestingly, one highly heritable taxon, Christensenllaceae, is enriched in
individuals with low BMI and potential effects of this taxa on adiposity were confirmed in germ-free
gnotobiotic mice (Goodrich et al. 2014b).
Studies using naturally occurring genetic variation among panels of inbred mouse strains, in
addition to single gene mutations in genetically modified mice, have consistently shown an effect of
host genetics on intestinal microbial community structure (Toivanen, Vaahtovuo, and Eerola 2001,
Benson et al. 2010, Kovacs et al. 2011, Spor, Koren, and Ley 2011, McKnite et al. 2012, Parks
et al. 2013) and provide increased power to detect genotype-driven microbial differences. This is
especially relevant since murine studies allow for tight control over environmental factors includ-
ing diet (Benson et al. 2010) For example, numerous genetic studies in mice, including those using
genetic reference panels (Benson et al. 2010, Campbell et al. 2012, McKnite et al. 2012, Hildebrand
et al. 2013) have demonstrated an effect of genetic background on microbial diversity. Inbred strain
surveys in mice also demonstrate a significant effect of host genetic makeup on microbial diversity
(Campbell et al. 2012), and some of these differences have been linked to cardiometabolic phe-
notypes (O’Connor et al. 2014). Interestingly, using an advanced intercross population of mice,
Benson and coworkers demonstrated that a single genetic locus may regulate the abundance of
several taxa (Benson et al. 2010).
However, the relative strength of environmental versus genetic signals on microbial regulation
remains unclear. Much of our knowledge about the environmental effects on the microbiome has
been derived from studies in mice (Spor, Koren, and Ley 2011). Several studies have shown how
the maternal environment, litter effects, cage mates, the location that the mice are housed, and the
commercial vendor can influence microbial populations (Benson et al. 2010, Friswell et al. 2010,
Campbell et al. 2012, Hildebrand et al. 2013, McCafferty et al. 2013). Uterine implantation stud-
ies have also shown that mice of different genetic backgrounds have similar microbial composition
when reared by the same foster mother, indicating that in certain circumstances, environmental driv-
ers can overpower genetic influences at least for nonadherent bacterial populations (Friswell
et al. 2010). These findings are further supported by studies that have demonstrated that bacteria
from diverse sources can colonize the gut of gnotobiotic mice and compete with “normal” micro-
biota (Seedorf et al. 2014). Clearly, more work is needed to understand the interactions between host
genetics and microbial diversity.
Although gene by diet interactions influencing the microbiome is a relatively new field of inves-
tigation (Joseph and Loscalzo 2015), studies using mouse genetic reference populations (Parks et al.
2013) or single gene knockout models (Kashyap et al. 2013b) have demonstrated an interaction
between microbiota and diet that is influenced by host genotype. These investigations have extended
beyond studies utilizing mice to use of novel model organisms such as stickleback fish, in which
there was a modest association between major histocompatibility complex polymorphisms and gut
microbial composition and diversity (Bolnick et al. 2014a). The acceptance of interactions is not
universal as a study of inbred mice showed that diet intervention has reproducible changes on micro-
biome, and overwhelms genetic differences between strains (Carmody et al. 2015). Moreover, gene–
diet interactions on the microbiome have not been clearly documented in humans. A clinical study
reported that despite retained variation in taxonomy following dietary intervention, microbial gene
expression, as assessed by RNA-seq, clustered by diet group and exhibited less between-subject
variation than at baseline (David et al. 2014).
In addition to specific genetic effects, there is also emerging evidence of gender differences in
the composition of the microbiome. In a study of fish, mice, and humans, there were significant
interactions between diet and sex on gut microbiome (Bolnick et al. 2014b). These studies indicate
that attention to gender may be critical to delineating dietary effects on the microbiome. How gender
differences in the microbiome relate to the established gender differences known to occur in cardio-
vascular disease remains to be determined.
ALTERED MICROBIOTA ACROSS LIFE SPAN

Age is an independent risk factor for cardiovascular disease and many of its risk factors. We are
beginning to appreciate the changes to the microbiota that occur as we age, and how perturbations
at specific time points may affect microbial composition of the microbiota.
Newborns and Early Childhood

Among other factors, infants experience tremendous alterations in their microbiome across the first
few years of life. It has been noted that the microbiota are influenced by the type of birth (vagi-
nal or Cesarean). For example, shortly after birth, the microbiota of vaginally delivered infants
resembles the microbiota of their mother’s vagina, while infants delivered via Cesarean section
harbor microbial communities typically found on human skin (Penders et al. 2006). As an infant
progresses through various milestones over the first year of life, such as the initial introduction of
solid foods and transition to table foods, there are significant changes in the microbiota composition
(Yatsunenko et al. 2012). One perturbation that clearly affects the microbiota is antibiotics, and there
is evidence that treatment of newborn mice with antibiotics alters the microbiome and increases
adiposity (Cho et al. 2012). However, whether alterations of the microbiome in childhood affect
cardiometabolic health later in life is unclear.
In addition to changes in microbiota diversity, there is also an effect of age on microbiota gene
ontology. For example, bacterial genes involved in B vitamin metabolism develop over the time
course of early childhood, perhaps in response to the broad changes in diet (Yatsunenko et al. 2012).
This notion of early diet changes driving microbiota diversity is supported by ecological studies
comparing African and European children (De Filippo et al. 2010). In these studies, unique abun-
dance of bacteria from the genus Prevotella and Xylanibacter, known to contain a set of bacterial
genes for cellulose and xylan hydrolysis, were overrepresented in the African population. One can
hypothesize that this particular bacteria coevolved with the microbiota of the African subjects in
response to their diet that is enriched in polysaccharides. Similar effects of coevolution or perhaps
horizontal gene transfer, where bacteria from food invade the microbiota of individuals consuming
that food, are novel and need rigorous testing. It should be noted that at least one group has identi-
fied specific bacteria-associated genes that may be transferred to humans when they consume diets
enriched in seafood (Hehemann et al. 2010).
Gut Microbiome 585
Maternal Diet
Perhaps one of the most interesting concepts regarding horizontal gene transfer and other influences
on the microbiome of children are the effects of the maternal microbiome and perhaps the effects
of maternal diet and exposure to environmental factors. For example, treatment of pregnant mice
with low-dose antibiotics results in a disruption of the microbiota. These altered microbiota in the
offspring affect clinical traits such as body composition and adiposity (Cox et al. 2014). However,
to date these maternal effects have not been thoroughly segregated from potential epigenetic effects
such as DNA methylation.
Microbiota and Elderly Populations

As compared to other age groups, there are relatively few studies of gut microbiomes among
the elderly. Alterations in gut microbial community homeostasis are anticipated based on age-
related physiologic and behavioral changes, including diet, comorbidities, medication use, and
gastrointestinal functioning. Studies have documented significant microbial compositional dif-
ferences between young and older adults, though reported patterns of difference are not consis-
tent. For example, diversity appears to decrease with age (Biagi et al. 2010), and several studies
have shown an increased abundance of Proteobacteria with aging (Biagi et al. 2010, Claesson
et al. 2011). In contrast, there are discordant results with respect to age-related shifts in the
Firmicutes:Bacteriodetes ratio (Biagi et al. 2010, Claesson et al. 2011). The gut microbiome
among healthy, community-dwelling older adults is similar to that of younger adults, with large
population-level variability and high within-person temporal stability (Claesson et al. 2011). The
gut microbiome among samples from older individuals is significantly associated with diet and
health status, including measures of adiposity and inflammation, as well as frailty and cognitive
function (Claesson et al. 2012), and is responsive to probiotic intervention (Eloe-Fadrosh et al.
2015). In contrast to healthy community-dwelling older adults, significant changes in the micro-
biome are apparent among older adults who reside in rehabilitative or residential care facilities
(Claesson et al. 2012, Jeffery, Lynch, and O’Toole 2016), with facility-dwelling individuals
having significantly lower microbial diversity and significant differences in functional measures,
such as decreased SCFA production (Claesson et al. 2012).
SUMMARY
The microbiota represents a critical mediator of the effects of diet on human health and disease
susceptibility. Advances in –omics scale technologies and analysis have provided the fundamental
understanding of the composition of the microbiome and how specific bacteria and overall com-
munity composition change when cardiometabolic disease risk is high, such as in obese individu-
als or diabetic patients. Integrating culture-free microbial diversity analysis with metabolomics
has identified interesting pathways and hypotheses to link dietary changes with specific disease
risk factors. Over time, the goal is that these analyses can be used to refine our dietary recom-
mendations to improve health. These are particularly important as we move toward a personalized
medicine model.
GLOSSARY
HMP Human microbiome project
MetaHIT Metagenomics of the human intestinal tract
OTU Operational taxonomic unit
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31 Associations with Blood Lipids,
Alcohol
Insulin Sensitivity, Diabetes,

Clotting, CVD, and Total Mortality
Charlotte Holst and Janne Schurmann Tolstrup
CONTENTS
Introduction.................................................................................................................................... 593
Blood Lipids.................................................................................................................................. 594
Insulin Sensitivity.......................................................................................................................... 595
Research on Alcohol and Morbidity and Mortality....................................................................... 595
Diabetes.......................................................................................................................................... 596
Clotting.......................................................................................................................................... 596
Cardiovascular Disease.................................................................................................................. 597
Coronary Heart Disease................................................................................................................. 597
All-Cause Mortality....................................................................................................................... 599
Conclusion..................................................................................................................................... 599
References...................................................................................................................................... 600
ABSTRACT
Alcohol is linked to more than 200 diseases, conditions, and injuries. However, extensive scientific
literature indicates that alcohol has beneficial effects on health as well. For example, the association
between alcohol intake and mortality is found to be J-shaped, indicating that moderate consump-
tion of alcohol is associated with a lower risk, when compared to heavy consumption or abstention.
Also, alcohol has been linked to a lower risk of type 2 diabetes mellitus and cardiovascular disease.
Several epidemiological studies suggest that alcohol’s protective effects are due to favorable changes
in anti-inflammatory effects, blood lipids, such as high-density lipoprotein (HDL) cholesterol and
apolipoprotein A1, as well as in insulin sensitivity. Alcohol has also been associated with the level
of fibrinogen, a plasma protein important for the coagulation of blood, which contributes to part
of the protective effects of alcohol on cardiovascular disease by decreasing the risk of thrombosis.
INTRODUCTION
Alcohol (ethanol) drinking has been a part of the human civilization for millennia. It is used
worldwide, and in most Western societies, abstainers constitute the minority. Globally, individuals
above 15 years of age drink on average 6.2 L of pure alcohol per year, corresponding to 13.5 g
of pure alcohol per day. However, there is wide variation in the total alcohol consumption across
the world with the highest consumption levels to be found in the developed world (World Health
Organization 2014).
The use of alcohol is often linked with pleasure and sociability, but it also has potentially harm-
ful consequences. Hence, alcohol contributes substantially to the global burden of disease
593
(Rehm et al. 2010) and in 2012, 5.9% of all deaths were attributable to alcohol (7.6% for men and
4.0% for women) (World Health Organization 2014). Also, alcohol is identified as a causal factor
for more than 200 diseases, conditions, and injuries (Rehm et al. 2010). However, in the context of
public health, the effects of alcohol are not only negative. The association between alcohol intake
and all-cause mortality is J-shaped, indicating that light to moderate drinkers, corresponding to con-
sumers of approximately 1 drink a day in women and 2 drinks a day in men, have a lower mortality
risk, when compared to heavy drinkers (corresponding to consumers of more than 1 drink a day for
women and more than 2 drinks a day for men) and abstainers (Di Castelnuovo et al. 2006, Shekelle
2007, Wang et al. 2014). This reduced mortality risk reflects a relatively lower risk of diabetes and
cardiovascular disease, in particular coronary heart disease, among light to moderate drinkers
compared to abstainers (Roerecke and Rehm 2012, Ronksley et al. 2011) (the definition of a drink
varies from 8 to 14 g, but often a standard drink is defined as containing 12 g of pure alcohol).
However, the authors of a recent study based on more than 150,000 American men and women from
a large nationally representative survey reported that among White men and women, consumption
of moderate levels of alcohol on most days of the week was associated with lowest mortality risk,
but the same risk reduction was not observed among Black men and women with similar drinking
patterns (Jackson et al. 2015).
Alcohol is generally consumed as beer (about 2.5%–6% alc. vol.), wine (about 12% alc. vol.), or
spirits (about 40% alc. vol.). It is quickly absorbed through passive diffusion, primarily in the small
intestine from where it is distributed throughout the total water compartment in the body. The main
part of the absorbed alcohol is oxidized in the body, and the energy liberated from this oxidation corre-
sponds to 29 kJ/g. Additionally, small amounts (5%–10%) are lost through expired air and in the urine.
BLOOD LIPIDS
The protective effects of a low to moderate alcohol intake on cardiovascular disease are partly
thought to be attributable to an increase in high-density lipoprotein (HDL) cholesterol levels. HDL
acquires cholesterol from peripheral tissue and facilitates its transport to the liver for removal in the
bile. HDL cholesterol is secreted as small particles by the liver and the gut and changes composi-
tion in the circulation by exchanging lipids with other lipoproteins or by absorption of cholesterol
from peripheral cells. HDL particles include multiple apolipoproteins, primarily apolipoprotein A1
(Hannuksela et al. 2002).
Substantial evidence from epidemiological studies suggests that HDL cholesterol levels increase
monotonically with the level of consumed alcohol (Foerster et al. 2009, Marques-Vidal et al. 2010,
Tolstrup et al. 2009a). In a comprehensive high-quality meta-analysis of 44 intervention studies by
Brien et al. (2011), the effect of alcohol intake on several biological markers associated with the
risk of coronary heart disease was investigated. In accordance with the findings reported earlier, the
pooled analysis of the effect of alcohol intake on mean HDL cholesterol levels showed a signifi-
cant dose–response relationship (33 studies), and the authors reported that 30 g alcohol consumed
a day was associated with an increase in HDL cholesterol levels of 3.66 mg/dL (95% confidence
interval: 2.22–5.13). Similar to the effect on HDL cholesterol levels, apolipoprotein A1 also signifi-
cantly increased (16 studies). These favorable changes in cardiovascular biomarkers, presumed to be
induced by a low to moderate alcohol intake, provide indirect pathophysiological support for a pro-
tective effect on certain aspects of cardiovascular disease, and in particular coronary heart disease.
However, a high intake of alcohol has been associated with increased levels of nonfasting triglycer-
ides, which, on the contrary, is associated with increased risk of cardiovascular disease. Among 9584
men and women from the Danish general population, Tolstrup et al. (2009a) found a significantly
U-shaped association between alcohol intake and nonfasting triglycerides, with consumption of more
than 14 drinks a week for women being associated with the highest risk. In their meta-analysis, Brien
et al. (2011) also reported a significant increase in the levels of triglycerides among consumers of
more than 60 g alcohol a day, but not at amounts of alcohol intake below 60 g alcohol a day.
Alcohol 595
INSULIN SENSITIVITY
A light to moderate intake of alcohol is associated with a lower risk of type 2 diabetes (Baliunas
et al. 2009, Koppes et al. 2005), an effect possibly mediated by an increase in insulin sensitivity,
which might work through several possible mechanisms, including modulation of the inflammatory
status of several organs, modulation in intermediary metabolism, or modulation of changes in the
endocrine functioning of fat tissue (Hendriks 2007). On the other hand, individuals with reduced
insulin sensitivity (insulin resistance) require larger amounts of insulin, either from the pancreas or
from injections of exogenous insulin, in order to maintain euglycemia.
Epidemiological studies suggest that the relationship between alcohol intake and insulin
sensitivity is either an inverted U-shape, indicating that a moderate alcohol intake is associated
with an increased insulin sensitivity compared to abstention or heavy drinking, or a positive linear
relationship, indicating increased insulin sensitivity by increased alcohol consumption (Ting and
Lautt 2006). However, results of intervention studies examining the relation between alcohol intake
and insulin sensitivity are inconsistent. The inconsistency might be due to methodological issues
such as differences in amount of alcohol intake, duration of alcohol consumption, and duration of
the abstention period (Ting and Lautt 2006). The authors of a systematic review and meta-analysis
including 14 intervention studies, which were all based on nondiabetic study populations, found
that alcohol intake tends to improve insulin sensitivity among women, but not among men (Schrieks
et al. 2015). Furthermore, the authors reported that alcohol intake reduces glycated hemoglobin
(HbA1C, a measure of long-term glycemic control), and fasting insulin concentrations compared
with abstention, but that alcohol intake does not influence fasting glucose. Increasing HbA1c levels
has also been associated with a greater risk of diabetes-related complications and cardiovascular dis-
ease among individuals with type 1 diabetes in the Diabetes Control and Complications trial (DCCT)
(Diabetes Control and Complications Trial (DCCT)/Epidemiology of Diabetes Interventions and
Complications (EDIC) Study Research Group 2016, Stratton et al. 2006) and with mortality among
individuals with type 2 diabetes in the United Kingdom Prospective Diabetes Study (UKPDS)
(Orchard et al. 2015).
RESEARCH ON ALCOHOL AND MORBIDITY AND MORTALITY

As mentioned initially, the scientific literature on alcohol and diseases is extensive. However, for
ethical and logistic reasons, long-term randomized trials have not been performed. The best evi-
dence thus comes from high-quality studies consisting of prospective cohort studies. In such studies,
individuals, often from samples of the general population, have completed a questionnaire about
alcohol intake as well as other lifestyle habits. By correlating this information with incidence of
coronary heart disease in the years following collecting of the baseline data, risk for coronary heart
disease according to alcohol intake can be estimated. Since alcohol is associated with other lifestyle
habits, taking other information into account when calculating such risk estimates is extremely
important. For example, heavy drinkers are more often smoking daily compared with light drinkers.
Concern has also been raised about the validity of self-reported alcohol intake. Are people really
able to report their alcohol intake in a questionnaire? Findings suggest that this is really so. For
instance, correlating self-reported alcohol intake to biomarkers that are known to associate with
alcohol such as alanine aminotransferase, gamma glutamyl transpeptidase, erythrocyte volume, and
alkaline phosphatase shows that stepwise increments of the self-reported alcohol consumption con-
sistently corresponded to stepwise increments in factors expected to correlate with amount of alco-
hol consumption (Tolstrup et al. 2009b). This finding speaks in favor of the validity of self-reported
alcohol consumption—using a questionnaire to assess alcohol intake seems to be effective in sepa-
rating individuals with different levels of alcohol intake. Further, the use of self-reported methods
(questionnaire and personal interviews) has been reviewed extensively and seems to offer a reliable
and valid approach to measuring alcohol consumption (Del Boca and Darkes 2003).
DIABETES
The worldwide prevalence of type 2 diabetes mellitus is rapidly rising with an increase from
382 million individuals in 2013 to an estimated 592 million by 2035 (Guariguata et al. 2014). A light
to moderate alcohol intake (approximately 1 drink a day for women and 2 drinks a day for men)
has been suggested to be a lifestyle factor that might reduce the risk of developing type 2 diabetes
mellitus (Baliunas et al. 2009, Koppes et al. 2005), an effect that has been proposed to be explained
by factors such as increased insulin sensitivity (Pietraszek et al. 2010), anti-inflammatory effects
or effects on adiponectin (Hendriks 2007). A meta-analysis of 44 intervention studies has shown
that alcohol intake significantly increases adiponectin levels, but does not affect inflammatory fac-
tors (Brien et al. 2011), while Schrieks et al. (2015) reported that moderate alcohol consumption
decreases fasting insulin concentrations and HbA1c, but does not affect insulin sensitivity or fasting
glucose levels.
Most studies report a J- or U-shaped association between alcohol consumption and the risk of
developing type 2 diabetes, indicating that moderate drinkers have a lower risk of diabetes compared
to abstainers and heavy drinkers (Pietraszek et al. 2010). Pooling data from 38 observational studies,
Knott et al. (2015) identified a peak risk reduction at 10–14 g alcohol per day with an 18% decrease
in hazards, relative to lifetime and current abstainers. However, the authors reported that reduc-
tions in risk of developing diabetes at moderate levels of alcohol consumption may be restricted to
women and non-Asian populations. A U-shaped relationship for both men and women was found in
a systematic review and meta-analysis undertaken by Baliunas et al. (2009) who examined alcohol
intake as a risk factor for type 2 diabetes based on 20 prospective cohort studies. In comparison
with lifetime abstainers, men consuming 22 g alcohol per day obtained the most protective effect of
alcohol intake with 13% reduced risk of type 2 diabetes. Among women, 24 g alcohol per day was
associated with the lowest risk, corresponding to a 40% reduced risk of type 2 diabetes. For men
consuming above 60 g alcohol per day, the intake became harmful in regard to the risk of type 2
diabetes, whereas this limit was about 50 g alcohol per day for women (Baliunas et al. 2009). The
results from this meta-analysis confirm previous research findings that moderate alcohol intake is
associated with a lower risk of type 2 diabetes in men and women (Carlsson et al. 2005, Howard
et al. 2004, Koppes et al. 2005). Recently, however, it has been suggested that reductions in risk of
developing diabetes may be overestimated in epidemiologic studies by comparing drinkers to a less
healthy current nondrinking reference population (Knott et al. 2015).
CLOTTING
In the blood clotting process (coagulation), the blood forms a clot by changing state from liquid to
gel. This might result in hemostasis, where the blood loss from a damaged vessel stops, followed by
a repair period. Current evidence suggests that alcohol intake is associated with a decrease in levels
of fibrinogen (Brien et al. 2011), a plasma protein that plays an important role in coagulation by
being transformed into an insoluble network of filamentous molecules called fibrin. Alcohol’s effect
on fibrinogen is not well understood; however, a decrease in fibrinogen levels is assumed to impair
the coagulation process and thereby contribute to part of the protective effects of a low to moderate
alcohol intake on cardiovascular disease by decreasing the risk of thrombosis.
Studies investigating the effects of increased alcohol intake on coagulation and fibrinolysis have
mostly been performed on components of blood, rather than on whole blood, with inconsistent
results across studies (Engstrom et al. 2006). Hence, some studies have found that alcohol impairs
primary hemostasis (i.e., the part of hemostasis in which the platelets immediately form a plug
at the site of injury), but that the humoral coagulation factors and the fibrinolytic system are not
affected (this falls under the heading of secondary hemostasis in which additional coagulation fac-
tors respond in a complex cascade to form fibrin strands, which strengthen the platelet plug) (Elmer
et al. 1984, El-Sayed et al. 1999, Serebruany et al. 2000, Zoucas et al. 1982). However, an acute
Alcohol 597
impairment of the fibrinolytic system by increased ethanol intake has been suggested (Olsen and
Osterud 1987, van de Wiel et al. 2001). A single study has also described an increase in fibrinolytic
activity in chronic alcoholics after they have stopped drinking alcohol (Delahousse et al. 2001),
and other authors report that binge drinking increases platelet aggregation (de Lange et al. 2004).
Finally, in whole blood, ethanol intake has been proposed to increasingly impair both coagulation
and fibrinolysis (Engstrom et al. 2006).
CARDIOVASCULAR DISEASE
Cardiovascular disease involves the heart, arteries, and veins. Coronary heart disease accounts for a
major fraction of cardiovascular diseases.
CORONARY HEART DISEASE

The literature on the association between alcohol and coronary heart disease is massive and the
subject has been and still is much debated both in the medical literature and the popular media. The
reason for the controversy is that light to moderate alcohol drinkers have consistently been found to
have a lower risk of coronary heart disease compared to individuals who abstain from alcohol. This
has given rise to the hypothesis that alcohol has a cardioprotective effect.
Evidence linking alcohol with a lower risk of coronary heart disease comes from a high number
of prospective cohort studies, many of which are of high quality. A meta-analysis conducted in 2011
comprised data from 84 prospective cohort studies, totaling 3,159,720 study participants (Ronksley
et al. 2011). This study was conducted following the Meta-analysis of Observational Studies in
Epidemiology (MOOSE) guidelines and is of high quality. The overall adjusted relative risk for
alcohol drinkers relative to nondrinkers was 0.71 (95% CI: 0.66–0.77) for incident coronary heart
disease, that is, the risk for coronary heart disease among drinkers was 29% lower among drinkers
compared with nondrinkers. In analyses exploring dose–response, categories of 2.5–14.9, 15–29.9,
and 30–60 g/day were associated with similar and statistically significant reduction in the relative
risk of coronary heart disease relative to nondrinkers. The highest category (>60 g/day) was associ-
ated with a relative risk of 0.76 (0.52–1.09). Hence, there is evidence of a maximal upper range of
intake for the cardioprotective effect, but no indication of a higher risk among individuals who drink
the most heavily. In contrast, an earlier meta-analysis of 51 studies and a total of 49,640 cases found
that the association between alcohol and coronary heart disease risk is J-shaped, implying a mini-
mum relative risk of 0.80 at 20 g/day, a significant protective effect up to 72 g/day, and a significant
increased risk at intakes above 89 g/day (Corrao et al. 2000). At this level, the statistical power was
limited, which may explain why the larger meta-analysis by Ronksley and colleagues reported a
different association at high levels of alcohol intake (Ronksley et al. 2011). In the Ronksley meta-
analysis, the lower risk for coronary heart disease among drinkers was unchanged after exclusion
of former drinkers from the reference category of nondrinkers, or in practical terms, when the refer-
ence category was changed from nondrinkers to lifelong abstainers (Ronksley et al. 2011). This is
an important result because it is often speculated that individuals who go from drinking to abstain-
ing do so in response to rising health problems. For instance, an individual who has drank heavily
for years may start feeling malaise due to first symptoms of an underlying disease, caused by the
high alcohol intake, which in turn may lead to reduced alcohol intake. Such a mechanism would
lead to an apparent high risk among the nondrinkers, resulting in a relatively lower risk among the
light to moderate drinkers that would not have anything to do with a cardioprotective effect but
simply reflect a systematic bias. This so-called sick quitting hypothesis has been intensely discussed
and tested in the scientific literature, but the overall results do not support the idea that the lower
risk among drinkers is caused by a movement of former drinkers in poor health to the category of
nondrinkers.
Gender generally impacts the way alcohol is metabolized and, hence, the risks associated with
levels of intake. For a given alcohol intake, blood alcohol levels will be higher for women than for
men because women have smaller body sizes and relatively more body fat. This means that any dose
of alcohol will imply a higher effective dose for women than for men, and women are thus gener-
ally more sensitive to any alcohol-related effect. In line with this, a meta-analysis by Corrao and
coworkers indicates differences in the cardioprotective effect of alcohol between men and women
(Corrao et al. 2000). In women, the risk of coronary heart disease decreased for up to 6 drinks/week,
showed evidence of protective effects (i.e., the risk was significantly lower compared with nondrink-
ers) for up to 18 drinks/week, and reached statistical significance of harmful effects (i.e., the risk
was significantly higher compared with nondrinkers), at 30 drinks/week. In comparison, the risk in
men decreased for up to 15 drinks/week, showed evidence of protective effects for up to 51 drinks/
week, and reached statistical significance of harmful effects at 67 drinks/week. Hence, both the pro-
tective and the detrimental effects of alcohol on coronary heart disease are achieved at lower levels
in women than in men.
Drinking pattern has been defined in various ways. These include drinking with meals, on week-
ends only, to intoxication, to a certain blood alcohol level, more than a certain amount per session
(6 drinks, 13 drinks, half a bottle of spirits, etc.), and amount and frequency combined. Evidence
has emerged over the last decade that drinking pattern plays a large role in the association between
alcohol and coronary heart disease. In studies of drinking pattern, the focus is not to compare non-
drinkers and drinkers, but rather to compare drinkers characterized by different drinking patterns.
In a meta-analysis of six epidemiological studies (n = 63,848 participants), the dose–response rela-
tion between alcohol intake and coronary heart disease risk was significantly different in irregular
and regular drinkers. In irregular drinkers who consumed alcohol for two or less days per week,
a J-shaped curve was obtained, with an increasing risk at intakes over 11 drinks/week—a pattern
consistent with a large amount of alcohol consumed per session. In contrast, in regular drinkers who
consumed alcohol on three or more days of the week, a protective effect was observed, even at high
amounts of alcohol intake (Bagnardi et al. 2008). Another meta-analysis concludes that episodic and
chronic heavy drinking does not provide any beneficial effect against coronary heart disease at all
(Roerecke and Rehm 2014).
It has been suggested that wine drinking—especially red wine—is especially beneficial for the
cardiovascular system. This idea originated from the observation that the incidence of coronary heart
disease in wine-loving France was low despite a high prevalence of smoking and high fat intake,
the so-called French paradox (Criqui and Ringel 1994, Renaud and de Lorgeril 1992). Biological
explanations for a more cardioprotective effect of wine compared with beer and spirits include the
fact that substances in wine—besides the ethanol such as resveratrol (Bonnefont-Rousselot 2016)—
have been shown to inhibit platelet aggregation and reduce oxidation of low-density lipoproteins
(Bonnefont-Rousselot 2016, Frankel et al. 1993, Pace-Asciak et al. 1995, 1996, Ramprasath
and Jones 2010, Renaud and Ruf 1996). In support of this appealing theory, it was found in the
Copenhagen City Heart Study that wine drinkers had a much lower mortality than beer and spirit
drinkers (Grønbæk et al. 1995). However, there is evidence that the apparent favorable effect of wine
is an artifact resulting from characteristics of the drinker rather than of the drink itself. Differences
in results from country to country according to wine, beer, and spirit intake may be attributable to
socioeconomic or behavioral characteristics of those individuals who drink wine, beer, and spirits. In
line with this, studies have shown that wine drinkers are more likely to report optimal health, score
higher in intelligence tests, and to eat a more healthy diet than beer and spirits drinkers (Barefoot
et al. 2002, Johansen et al. 2006, Mortensen et al. 2001, Tjønneland et al. 1999). Further speaking
against that wine should have specific effects compared to other beverage types is that randomized
studies found no difference in alcohol’s effect on biomarkers according to type of alcohol (Brien
et al. 2011). In contrast to more popular media where wine is often praised for its health-promoting
effects, the theory that wine has special cardioprotective effects only has a few advocates in the
scientific literature.
Alcohol 599
ALL-CAUSE MORTALITY
The association between alcohol and all-cause mortality is J-shaped (Di Castelnuovo et al. 2006);
the nadir of the J reflects a relatively lower risk of coronary heart disease among light to moderate
drinkers compared with abstainers, and the ascending leg of the J is reflecting an increased risk
of alcohol-related diseases such as cardiomyopathy (Guzzo-Merello et al. 2014), pancreatitis
(Irving et al. 2009), liver cirrhosis, upper gastrointestinal cancers, and deaths from accidents and
violence among excessive alcohol users (Rehm and Shield 2013). Since the association between
alcohol and all-cause mortality thus represents the sum of numerous diseases and outcomes that are
related to alcohol, the shape and nadir of the risk curve depends upon the distribution of other vari-
ables such as age, relative incidences of diseases, the prevalence of drunk-driving, and so on. Hence,
the association between alcohol and all-cause mortality does not have the same causal interpretation
as associations between alcohol and singular endpoints.
A J-shaped relationship between alcohol and total mortality was found in adjusted studies, in
both men and women. The finding that the relative risk of mortality is ≤1 for light to moderate
alcohol intake is consistently observed in population-based cohort studies (Di Castelnuovo et al.
2006). Consumption of alcohol, up to 4 drinks/day in men and 2 drinks/day in women, was inversely
associated with total mortality, maximum protection being 18% in women (99% confidence inter-
val, 13%–22%) and 17% in men (99% confidence interval, 15%–19%). Higher doses of alcohol
were associated with increased mortality. The inverse association of alcohol and total mortality
disappeared at doses lower in women than in men. The same was observed in subanalyses where
ex-drinkers and light drinkers were excluded from the reference category.
Age plays an important role for the association between alcohol and mortality. Since the relative
incidence of alcohol-related diseases and outcomes differs by age, the J-shaped association between
alcohol and all-cause mortality also differs by age. The nadir (representing the alcohol intake at the
lowest risk of mortality) is achieved at a lower alcohol intake in younger individuals; in a British
study, the lowest mortality risk among women 16–34 years old and men 16–24 years old was observed
among the nondrinkers (White et al. 2002). Hence, a beneficial effect of alcohol is not observed
among the young, where alcohol is directly associated with mortality, mainly due to injuries.
Alcohol drinking pattern is also an important consideration and some studies have investigated its
relationship to the risk of all-cause mortality. Results consistently imply an increased mortality risk
associated with drinking large amounts of alcohol per session. Further, there is good evidence that
the protective effect of alcohol on cardiovascular disease only occurs if the pattern of drinking is not
binging (Rehm et al. 2010). Hence, the J-shaped association between alcohol intake and all-cause
mortality depends upon the drinking pattern.
To conclude, light to moderate drinking is not associated with increased mortality risk, or at best
is associated with a lower risk, except among the young. A binge-like drinking pattern may not be
associated with a decrease in risk of mortality among moderate drinkers.
CONCLUSION
Alcohol is linked to a wide range of diseases, conditions, and injuries. In 2012, 5.9% of all deaths
globally were attributable to alcohol. However, the association between alcohol intake and all-cause
mortality is J-shaped, indicating that alcohol consumption at moderate levels is associated with a
lower risk of diseases such as diabetes and cardiovascular disease among light to moderate drinkers
when compared to heavy consumers or abstainers. Beneficial effects on biological markers such as
HDL cholesterol, nonfasting triglycerides, and apolipoprotein A1 are proposed to provide indirect
pathophysiological support for protective effects on certain aspects of cardiovascular disease, and
in particular coronary heart disease among moderate alcohol drinkers. Further, a moderate intake of
alcohol is associated with a decrease in levels of fibrinogen, which is assumed to impair the coagula-
tion process, and thereby decrease the risk of thrombosis. Red wine has previously been suggested
to have especially beneficial effects for the cardiovascular system; however, the scientific literature
is inconclusive. Drinking patterns, on the other hand, have been found to be of importance, in that
there is good evidence that no protective effects of alcohol consumption in relation to cardiovascular
disease are observed among binge drinkers.
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32 Endocrine Disrupting
Chemicals, Obesogens,
and the Obesity Epidemic
Raquel Chamorro-Garcia and Bruce Blumberg
CONTENTS
Abbreviations................................................................................................................................. 604
Endocrine Disrupting Chemicals (EDCs) and Human Health....................................................... 604
The Obesity Epidemic.................................................................................................................... 605
Obesogens, Adipogenesis, and Obesity.................................................................................... 605
In Vitro Exposure to Obesogens and Adipogenesis................................................................... 605
In Vivo Exposure to Obesogens during Adulthood and Obesity............................................... 606
Developmental Origins of Obesity................................................................................................ 606
Energy Balance during Development........................................................................................ 606
Obesogens and the Developmental Origins of Obesity............................................................ 607
Obesogens and the Transgenerational Transmission of Obesity............................................... 608
Obesogens and Cardiovascular Disease......................................................................................... 609
Conclusion and Future Directions................................................................................................. 609
References...................................................................................................................................... 610
ABSTRACT
Obesity and related disorders have become a significant burden worldwide. In 2014, the World
Health Organization estimated that ~2 billion people around the world were overweight and of
those, 600 million people were obese. Obesity is also associated with metabolic conditions such as
hypertension, cardiovascular disease, and type 2 diabetes. The additional health care costs for the
treatment of these conditions are estimated to be $2 trillion annually worldwide. Thus, understand-
ing the factors that are contributing to this epidemic becomes timely and important. The causal fac-
tors associated with obesity were traditionally ascribed to positive energy balance. More recently,
evidence linking obesity with certain genetic mutations and the balance of bacterial flora in the gut
microbiome is mounting. However, the prevalence of obesity and overweight in infants and in other
animal species that live in urban areas indicates that there may be other environmental factors that
should not be overlooked. In this chapter, we review the possible contributions of chemical obeso-
gens to the obesity epidemic and other obesity comorbidities such as cardiovascular diseases. We
discuss in vitro approaches currently used to determine the molecular mechanisms through which
chemicals might act as obesogens as well as in vivo approaches using animal models to test the
capability of obesogens to promote obesity. Since obesity is a condition with a developmental ori-
gin, we particularly focus on the effect of obesogens during in utero development and the potential
transgenerational consequences of this exposure. We review the current knowledge about two differ-
ent obesogens: the organotin tributyltin (TBT), whose mode of action is at least partly understood,
and the estrogenic chemical bisphenol A (BPA), whose widespread use makes exposure ubiquitous.
We provide an overview of the direction this field is following to better understand the relationship
between obesogen exposure and the obesity epidemic.
603
ABBREVIATIONS
BADGE Bisphenol-A diglycidyl ether
BPA Bisphenol A
DDT Dichloro-diphenyl-trichloroethane
DES Diethylstilbestrol
EDCs Endocrine disrupting chemicals
F1–F3 First–third filial generation
FABP4 Fatty acid binding protein-4
MSCs Mesenchymal stem cells
NAFLD Nonalcoholic fatty liver disease
NHANES National Health and Nutrition Examination Survey
NOAEL No observable adverse effect levels
PCBs Polychlorinated biphenyls
PPARγ Peroxisome proliferator activated receptor gamma
RXR Retinoid X receptor
TBT Tributyltin
TPT Triphenyltin
ZFP423 Zinc finger protein 423
ENDOCRINE DISRUPTING CHEMICALS (EDCs) AND HUMAN HEALTH

In the last 60 years, the number of artificial chemicals introduced in our environment has dramati-
cally increased. There is a growing body of research in laboratory animals and in wildlife show-
ing that some of these chemicals used in industry and agriculture may negatively modulate the
endocrine system and, therefore, may be involved in the increasing rates of various health condi-
tions worldwide (Diamanti-Kandarakis et al. 2009). As defined by the Endocrine Society, the term
endocrine disrupting chemicals (EDCs) refers to “an exogenous chemical, or mixture of chemicals,
that interferes with any aspect of hormone action” (Zoeller et al. 2012). This differs from the defi-
nitions promulgated by the United States Environmental Protection Agency and the World Health
Organization that add the qualifier, “and causes adverse health effects in an intact organism, its prog-
eny or subpopulations” (WHO/UNEP 2013). The difference is one of perspective and training. To
an endocrinologist, disrupting the normal function of the endocrine system is de facto, adverse, even
if the results might be subtle and take years to be manifested. To a regulatory toxicologist, adverse
consequences must be demonstrated in a short time frame and subtle changes are typically ignored
or deemphasized. These different perspectives have led to much debate in the scientific literature
between regulators and industrial toxicologists on one side, and the endocrine/environmental health
community on the other side. We adopt the view of the Endocrine Society in the review that follows.
In mammals, the endocrine system consists of a complex network of glands and organs that
regulate physiological functions such as appetite, circadian rhythms, or reproduction. The classical
glands include but are not limited to the pituitary gland, thyroid gland, pancreas, gonads, and adre-
nal glands (Melmed et al. 2011). However, other organs such as fat tissue, liver, and intestines also
have endocrine functions that are critical for the maintenance of body functions, including metabolic
homeostasis (Kershaw and Flier 2004). The link between the classical endocrine glands and other
organs with endocrine functions lies in the fact that all of them secrete hormones into the blood-
stream in response to certain environmental stimuli and will generate a physiological response in a
(usually distant) target tissue. Although the endocrine system has the ability to adapt in response to
hormonal fluctuations, if the changes occur at critical windows in life (e.g., embryonic development)
or last for long periods (e.g., insulin resistance), they may have permanent detrimental effects on the
physiological response of the individual (reviewed by Gore et al. 2015).
Endocrine Disrupting Chemicals, Obesogens, and the Obesity Epidemic 605
The mechanisms through which EDCs act are diverse and include their interactions with nuclear
and nonnuclear hormone receptors (e.g., nuclear and membrane estrogen receptors, respectively),
nonsteroid receptors (e.g., neurotransmitter receptors), or enzymatic pathways involved in the syn-
thesis and metabolism of hormones (e.g., cytochrome P450s). These interactions may activate or
inhibit the pathway they regulate by mimicking or blocking the action of the endogenous molecules
(Gore et al. 2015).
THE OBESITY EPIDEMIC

The endocrine system may participate in the regulation of energy homeostasis by modulating neu-
roendocrine circuits involved in the control of appetite and satiety (Mackay et al. 2013); therefore,
agents altering hormone levels and activity may contribute to the development of obesity. The most
commonly accepted factors involved in obesity are generally ascribed to positive energy balance
(Hall et al. 2012). Other factors such as genetics or gut microbiome may not be directly associated
with lifestyle but their effects can be exacerbated by it (McAllister et al. 2009, Turnbaugh et al.
2006). However, the continuous worldwide increase in global obesity rates is difficult to explain by
only considering the traditional factors associated with this health condition (Ng et al. 2014, Ogden
et al. 2014). These trends open the debate regarding what are all of the major causes contributing to
the obesity epidemic. More alarming is the fact that this increased tendency toward obesity has also
been shown for children below 5 years of age, who, in 2013, numbered at least 40 million world-
wide (Ng et al. 2014). In parallel to the trends observed in humans, it has been shown that wild and
domestic animal populations living in proximity to humans have also undergone significant average
increases in body weight in recent years (Klimentidis et al. 2011). An easy explanation for this trend
would be to assume that animals living around industrialized areas have easy access to unhealthy
food wasted by humans. Interestingly, laboratory animals that are maintained on what are thought
to be tightly controlled, optimal diets are also following the same trend (Klimentidis et al. 2011),
suggesting the existence of additional, unidentified factors that are contributing to the increasing
rates in overweight in different species.
In 2006, our laboratory introduced the “obesogen hypothesis” that proposes the existence of a
subset of EDCs that alter lipid metabolism to inappropriately stimulate an increase in the number
of adipocytes or the amount of fat stored within these cells, which may contribute to disturbances
in metabolic homeostasis (Janesick and Blumberg 2011). This alteration in energy balance may
ultimately lead to the development of obesity.
Obesogens, Adipogenesis, and Obesity

Since the introduction of the obesogen hypothesis, the effects of a growing list of obesogens have
been characterized using both in vitro and in vivo approaches.
Although not all obesogens act through the same pathways, a subset of obesogens act through
the peroxisome proliferator activated receptor gamma (PPARγ), which is considered to be the “mas-
ter regulator of adipogenesis” (Tontonoz and Spiegelman 2008). PPARγ is highly expressed in fat
tissue and functions as an obligate heterodimer with the retinoid X receptor (RXR) (Kliewer et al.
1992). Upon ligand binding, this heterodimer transcriptionally activates downstream genes involved
in lipid synthesis and metabolism such as fatty acid binding protein-4 (FABP4), lipoprotein lipase
(LPL), and leptin.
In Vitro Exposure to Obesogens and Adipogenesis

The organotins tributyltin (TBT) and triphenyltin (TPT) were the first obesogens described and
they both activate PPARγ (Grun et al. 2006, Janesick et al. 2016, Kanayama et al. 2005). More
recently, the pesticides triflumizole, quinoxyfen, spirodiclofen, and zoxamide have been shown
to activate PPARγ and to increase lipid accumulation using in vitro models such as the murine
pre-adipocyte 3T3-L1 cell line and mouse and human mesenchymal stem cells (MSCs) (Janesick
et al. 2016, Li et al. 2012). In the same study, we found that the fungicide fludioxonil is not
a PPARγ activator, but it activates RXR and increases lipid accumulation in 3T3-L1 cells and
MSCs (Janesick et al. 2016). MSCs are able to differentiate into a variety of cell types, includ-
ing adipocytes, osteoblasts, chondrocytes, and myocytes, depending upon the stimuli they receive
(Cristancho and Lazar 2011). By exposing 3T3-L1 cells or MSCs to obesogen candidates in the
presence of an adipogenic cocktail, it is possible to assess the adipogenic capabilities of individual
chemicals by analyzing lipid accumulation and mRNA expression levels of adipogenic marker
genes such as those described earlier (Chamorro-Garcia et al. 2012, Grun et al. 2006, Janesick
et al. 2016, Kirchner et al. 2010). There is a subset of candidate obesogens whose mechanisms of
action remain unknown. One example is bisphenol-A diglycidyl ether (BADGE), which is used
in the manufacture of epoxy resins, paints, and as a coating in food cans. BADGE induces lipid
accumulation in 3T3-L1 pre-adipocytes and MSCs, but the inhibition of PPARγ with the specific
antagonists T0070907 or GW9663 does not interfere with BADGE-induced accumulation of lipids
(Chamorro-Garcia et al. 2012). Other potential obesogens whose mechanisms of action remain
unknown are imazalil, tebupirimfos, florchlorfenuron, flusilazole, acetamiprid, and pymetrozine,
which are not PPARγ or RXR activators but induce adipogenesis in 3T3-L1 cells (Janesick et al.
2016). These studies indicate that further analyses are needed to more fully understand the mecha-
nisms through which obesogens act.
In Vivo Exposure to Obesogens during Adulthood and Obesity

Epidemiological studies showed a positive association between the presence of some EDCs in blood
and an increase in fat storage. Multiple independent epidemiological studies have linked high plasma
levels of polychlorinated biphenyls (PCBs) with obesity (Dhooge et al. 2010, Donat-Vargas et al.
2014, Lee et al. 2011). Cross-sectional analyses showed that increased urine levels of phenols (e.g.,
bisphenol A; BPA), whose use is widespread in industry, and phthalates, used as plasticizers and in
personal care products, are associated with an increase in fat content in the adult human (Carwile
and Michels 2011, Song et al. 2014). In many cases, the increase in fat storage is accompanied by
other metabolic conditions such as insulin resistance, type 2 diabetes, and cardiovascular disease
(James-Todd et al. 2012, Lind, Zethelius, and Lind 2012, Shankar and Teppala 2011).
Likewise, studies performed using laboratory animals have also demonstrated a positive associa-
tion between obesogen exposure during adolescence and adulthood, and obesity and related dis-
eases. Adult exposure to PCB-153 in mice led to increased fat storage, liver steatosis, and abnormal
levels of adipokines in plasma when animals were fed with a high-fat diet (Wahlang et al. 2013).
Mice chronically exposed to a mixture of different PCB congeners and dichloro-diphenyl-trichloro-
ethane (DDT) developed insulin resistance, glucose intolerance, and visceral adiposity (Ibrahim
et al. 2011). Adult mice exposed to BPA had a significant increase in body weight, hyperglycemia,
and insulin resistance (Alonso-Magdalena et al. 2006, Marmugi et al. 2014). Female mice exposed
to phthalates showed increased body weight, visceral adiposity, and food intake (Schmidt et al.
2012). Juvenile exposure to organotins such as TBT induces fat storage, fatty liver, and insulin resis-
tance in rodents (Penza et al. 2011, Zuo et al. 2011). Taken together, both human epidemiological
studies and studies performed in animal models show the positive association between the presence
of obesogens in the body in adult individuals and an increase in fat storage and obesity.
DEVELOPMENTAL ORIGINS OF OBESITY

Energy Balance during Development
There is a growing body of evidence showing that obesity during adulthood may have a develop-
mental origin (Janesick and Blumberg 2011).The developing organism possesses plasticity to adapt
to environmental stimuli and this plasticity may be detrimental for the organism later in life, since
it may promote the development of a variety of health conditions. Thus, environmental insults such
as under- or overnutrition or the presence of artificial chemicals during embryogenesis may lead to
detrimental effects later in life.
Epidemiological studies found a correlation between maternal overweight during pregnancy and
increased body weight at birth (Surkan et al. 2004). Experiments performed in rodents showed
that high-fat diets during pregnancy and lactation lead to obesity, hypertension, and insulin resis-
tance in the offspring even if they were maintained on a normal diet after weaning (Armitage et al.
2005, Khan et al. 2003, Taylor et al. 2005). Experiments performed with cross-fostering approaches
between control dams and dams exposed to a high-fat diet showed that both in utero and lactational
exposure to a high-fat diet are critical factors in the development of metabolic syndrome later in life
and that these effects can be transmitted to subsequent generations (Hoile et al. 2015, Khan
et al. 2005). More strikingly, rats exposed to cafeteria-style “junk-food” during in utero develop-
ment showed a biased preference toward palatable high-fat diets compared to animals exposed to
control rat diet, which indicates that high-fat and high-sugar exposure during critical windows of
development cause an alteration on the central reward pathways that will condition diet choices and
food intake later in life (Ong and Muhlhausler 2011).
Paradoxically, as reviewed in Chapter 28 of this book, undernutrition during critical windows
of development also leads to obesity later in life. Epidemiological observations performed in men
and women born during the “Dutch famine winter” at the end of World War II in the Netherlands
showed that malnutrition during early stages of development led to increased body mass index and
waist circumference in women but not men later in life (Ravelli et al. 1999). Analyses of the same
cohort showed that, when the famine period occurred at later stages of development, both men and
women had lower glucose tolerance, suggesting that undernutrition during development led to per-
manent changes in metabolic homeostasis (Ravelli et al. 1998). In line with these findings, studies
of a British cohort in Hertfordshire showed that poor nutrition during perinatal development was
positively associated with cardiovascular disease and type 2 diabetes during adulthood (Hales and
Barker 1992). These observations led David Barker to propose the “Barker hypothesis,” also known
as the “thrifty phenotype hypothesis.” The fundamental tenet of this hypothesis is that nutritional
deprivation during a very energy-demanding period of life, such as in utero development, will lead
to metabolic adaptations in the fetus that favor energy storage; thus, the metabolism “learns” to
be thrifty with calories. This enables some degree of adaptation to poor nutrition throughout life.
However, if instead this “thrifty phenotype” individual encounters adequate or excess nutrition, the
metabolic adaptations made during fetal life will lead to inappropriate storage of ingested calories,
altering glucose homeostasis and ultimately leading to obesity and related disorders (Hales and
Barker 2001). Thus, the “thrifty adaptations” become detrimental only when the postnatal environ-
ment differs from the prenatal setting.
Obesogens and the Developmental Origins of Obesity

The “obesogen hypothesis” introduces another level of complexity to the developmental origins of
obesity. There is a growing body of research showing that exposure to obesogens during critical
windows of development such as in utero development and during lactation lead to obesity later in
life (Heindel, Newbold, and Schug 2015).
The organotin TBT belongs to the group of obesogens whose in utero effect has been more deeply
characterized (Janesick, Shioda, and Blumberg 2014). In rodents, TBT causes masculinization of
females and infertility in mollusks and fish (Bryan et al. 1986, McAllister and Kime 2003) as well as
toxicity in liver, nervous system, and immune system (Boyer 1989). More recently, it was shown that
exposure to low doses of TBT during tadpole stages in Xenopus laevis and larvae stages in Danio rerio
(zebrafish) increases fat storage (Grun et al. 2006, Tingaud-Sequeira, Ouadah, and Babin 2011). Human
studies have shown that the presence of TBT in placenta is positively associated with weight gain in the
first months of life (Rantakokko et al. 2014). Experiments performed in mice demonstrated that prenatal
exposure to TBT increases adiposity, nonalcoholic fatty liver disease (NAFLD), and the reprogramming
of the MSCs compartment favoring their differentiation into adipocytes at the expense of the bone in the
offspring (Chamorro-Garcia et al. 2013, Grun and Blumberg 2006, Kirchner et al. 2010).
There are other EDCs with shorter half-lives that are in widespread industrial use, such as BPA.
BPA is used in the manufacture of polycarbonate plastics, epoxy resins, and thermal papers; there-
fore, it can be found in a variety of products the public encounters on a daily basis, including plastic
containers, food packaging, thermal papers, medical devices, dental sealants, and so on (Vandenberg
et al. 2010). Despite its ephemeral existence, the widespread use of BPA makes it ubiquitously pres-
ent. BPA has been detected in human samples including urine, serum, breast milk, and fat and is
associated with increased body weight, breast and prostate cancer, and alterations in the reproduc-
tive system (reviewed by Rubin 2011). Data from the National Health and Nutrition Examination
Survey (NHANES), a cross-sectional study with over 2500 participants ≥ 6 years of age, revealed
that BPA was present in 92.6% of the samples at an average level in urine of 2.6 ng/mL (Calafat
et al. 2008). Interestingly, participants between 6 and 11 years of age showed an average BPA level
of 4.5 ng/mL. These data raise concern about the impact of obesogens during childhood, when
metabolic setpoints are being programmed and are, therefore, more susceptible to environmental
insults. Although BPA is an estrogen, its mechanism of action in promoting obesity is not known
but it is notable that perinatal estrogen exposure can predispose animals to obesity later in life
(Newbold et al. 2007). Experiments performed using animal models showed that in utero exposure
to low doses of BPA leads to increased body weight and abdominal fat and the disruption of lipid
homeostasis later in life (Alonso-Magdalena et al. 2010, Howdeshell et al. 1999, Somm et al. 2009).
Similar results regarding increased body weight during adulthood were found in rodents exposed to
the estrogen diethylstilbestrol (DES) during in utero development (Newbold et al. 2004).
Obesogens and the Transgenerational Transmission of Obesity

Recent studies performed in animal models have revealed that ancestral perinatal exposure to obeso-
gens leads to obesity in subsequent generations (Chamorro-Garcia et al. 2013, Manikkam et al. 2013).
The transgenerational transmission of diseases implies that the germ line genome has been modified
in nucleotide sequence (mutations) in epigenomic marks (epimutations) or both. As discussed more
extensively in Chapter 28, the mechanisms involved in the modification of the epigenomic profile
include covalent modifications of the DNA (e.g., methylation and hydroxymethylation of cytosines)
and histones (e.g., methylation of lysines), and the presence of noncoding RNAs (Xin, Susiarjo,
and Bartolomei 2015). By modifying the epigenomic profile of the cell, it is possible to modulate
the functional output of the information stored in the genome sequence. During early stages of the
development, the primordial germ cells go through a genome-wide demethylation/remethylation
cycle before implantation. At this stage, the genome is extremely sensitive to the exposure of agents
that may permanently change the original methylation pattern (Heard and Martienssen 2014). Thus,
any changes in the epigenomic profile in the germ line at these developmental stages and the biologi-
cal traits associated with them may be transmitted to subsequent generations, although, the precise
mechanisms remain to be elucidated.
Experiments performed in our laboratory showed that in utero exposure to environmentally rel-
evant doses of TBT (5.42, 54.2, and 542 nM) of the first generation (F1) led to the development of
obesity in subsequent generations with a sexually dimorphic penetrance (Chamorro-Garcia et al.
2013). F1 female mice exposed to TBT exhibited increased adiposity in a dose-dependent manner,
whereas in F1 male mice this effect was not as strong as in subsequent generations. Both males
and females developed nonalcoholic fatty liver with the phenotype stronger in females. F2 females
and F2 and F3 males derived from TBT-exposed animals showed a dramatic increase in adipos-
ity, whereas F3 females did not show significant changes. Gene expression analyses in the MSCs
showed a significant increase in the gene expression levels of adipogenic markers such as ZFP423,
FABP4, LPL, and PPARγ in both genders. These effects were stronger in the F3 males than in
previous generations, suggesting the existence of an epigenetic mechanism involved in the regula-
tion of this phenotype (Chamorro-Garcia and Blumberg 2014, Chamorro-Garcia et al. 2013). It is
worth noting that two of the TBT concentrations used in this experiment (5.42 and 54.2 nM) were
lower than the established no observable adverse effect levels (NOAEL) (Vos et al. 1990). Other
obesogens involved in the transgenerational inheritance of obesity include BPA, phthalates, hydro-
carbons, and DDT. In all cases, the exposure to obesogens led to sperm epimutations in regions
associated to obesity (Manikkam et al. 2013, Skinner et al. 2013, Tracey et al. 2013).
A recent study showed that surgery-induced weight loss in obese men led to a significant change
in the DNA methylation profile of genes involved in central control of appetite in the sperm (Donkin
et al. 2016). Studies performed in a different cohort of obese men showed that noncoding RNA
expression levels and DNA methylation profiles in sperm were significantly different when com-
pared to the profiles in lean men (Donkin et al. 2016). These data indicate that the epigenome of the
male germ line is susceptible to environmental changes.
OBESOGENS AND CARDIOVASCULAR DISEASE

Obesity, NAFLD, and type 2 diabetes are just a few examples of risk factors for future cardiovas-
cular diseases such as heart failure, coronary heart disease, or atrial fibrillation (Mozaffarian et al.
2016). Therefore, it should come as no surprise that factors that are contributing to obesity, NAFLD,
and type 2 diabetes also contribute to cardiovascular comorbidities.
In the last two decades, there is increasing evidence showing an association between obesogen
exposure and cardiovascular disease. Independent cross-sectional and longitudinal epidemiologi-
cal studies revealed that urine BPA levels in adults is associated with an increasing risk for future
coronary artery disease and high blood pressure (Bae et al. 2012, Lang et al. 2008, Melzer et al.
2010, 2012). Ex vivo analyses of rat cardiomyocytes showed that exposure to low doses of BPA
alters electrical conduction causing arrhythmias in females; moreover, this effect was exacerbated
when other conditions such as stress and previous heart damage were also present (Posnack et al.
2014, Yan et al. 2011, 2013). Two independent experiments showed that chronic BPA exposure in
adult mice caused increased blood pressure and atherosclerosis (Saura et al. 2014, Sui et al. 2014),
whereas perinatal chronic exposure to low doses of BPA modified the epigenetic profile of cardiac
cells, leading to remodeling of cardiac structure and function (Patel et al. 2013). Although the
mechanism underlying this phenotype remains unclear, alterations in the activity of the estrogen
receptor and the pregnane X receptor are two hypotheses under investigation (Sui et al. 2014, Yan
et al. 2013).
As mentioned earlier, our lab showed that in utero exposure to TBT causes increased fat storage
and NAFLD in mice and that this phenotype is transmitted to future generations (Chamorro-Garcia
et al. 2013). It has been reported that TBT alters cardiac function by increasing coronary perfusion
pressure and cardiac hypertrophy in adult Wistar rats exposed to TBT for 15 days via gavage feeding
(dos Santos et al. 2012). There is a notable lack of epidemiological studies associating TBT with
cardiovascular disease, but these experiments performed in rodents suggest the potential contribu-
tion of TBT to the development of these health conditions.
Other EDCs that have been shown to alter cardiac function are dioxins, whose presence in blood
in humans has been associated with artery disease when combined with other risk factors such as
obesity (Min et al. 2011).
CONCLUSION AND FUTURE DIRECTIONS

Evidence is mounting regarding the existence of factors relevant to the obesity epidemic other than
nutrition and exercise, which have been typically considered to be the most important factors in
obesity. However, understanding the potential interactions between nutrition and other factors such
as stress, exercise, genetics, and so on has become an important challenge.
The obesogen hypothesis introduces another level of complexity to the current understanding
of obesity and related disorders and offers a different perspective. One key point is that there is
now abundant evidence showing that obesogens are ubiquitously present in industrialized societies,
occurring in water pipes, personal care products, food packaging, and agriculture. As a result, it
makes sense to devote resources to understanding how obesogens act in biological systems in order
that their effects on human, animal, and wildlife health be better assessed.
Experiments performed using in vitro models such as cell lines and primary cultures are con-
tributing to the detection of new EDCs with obesogenic properties. Although such experiments
have obvious limitations, they can contribute to a deeper, mechanistic understanding of how obe-
sogens act. In vivo experiments performed in animal models will be required to definitively prove
that chemicals are bona fide obesogens. Moreover, transgenerational studies in animal models are
changing the genetic determinism dogma that the inheritance of certain phenotypes results solely
from genomic mutations by providing evidence that epigenomic changes in DNA and histone
methylation also play an important role in phenotypic inheritance. These new approaches demon-
strate that, despite efforts to reduce or ban the use of certain artificial chemicals, the effects of such
chemicals could last for generations not only as residues in nature but even more alarming, in our
epigenome.
Further studies are needed to determine what interactions exist between obesogens and tradi-
tional factors involved in obesity such as diet. We currently know very little about the effects of
exercise in fat mobilization and thermogenesis after obesogen exposure, the interactions of obeso-
gens with diet composition, or the potential effects of obesogens on the balance of bacterial strains
present in the gut microbiome.
Obesity is a multifactorial disease that is difficult to treat once it has developed. Obesity affects a
significant fraction of the world population irrespective of the country, average income, age, gender,
or lifestyle (Ng et al. 2014). The prevalence of other conditions associated with obesity (e.g., type 2
diabetes, hypertension, and cardiovascular disease) continues to increase in parallel with the obesity
epidemic. Since obesity is largely refractory to treatment, new approaches are needed to prevent
obesity from developing. The obesogen hypothesis offers a new perspective for understanding this
global problem and proposes that the reduction of chemical exposure during critical windows of
development may offer an additional approach for prevention.
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Index
A coronary heart disease, 597–598
diabetes, 596
AA, see Arachidonic acid incidence, 594
Adenosine monophosphate kinase (AMPK), 339 insulin sensitivity, 595
Adipose tissue (AT) morbidity and mortality, 595
aerobic exercise, 103–106 Alphalinolenic acid (ALA), 423
resistance exercise, 106–108 Alternate healthy eating index (AHEI), 409–410
waist circumference, 102 Ambulatory blood pressure, 468
waist-to-hip ratio, 102 American Heart Association (AHA), 239
Adiposity rebound, 152 Aminostatic theory, 320
Adjustable gastric banding (AGB), see Laparoscopic AMPK, see Adenosine monophosphate kinase
adjustable gastric banding Anabolic resistance, 337
Adolescent obesity Apolipoprotein A2 (APOA2) gene, 563
definition, 150–151 Apolipoprotein CIII (apoCIII), 257
treatment Arachidonic acid (AA), 195
bariatric surgery, 156–157 Arcuate nucleus (ARC), 6
dietary, 153 Areas under the curve (AUC), 304
fruit and vegetables, 155 Arterial stiffness, 469
macronutrient, 155 Aspartate aminotransferase (AST) levels, 278, 280,
meal replacements, 155 286–287
medications, 156 AT, see Adipose tissue
metabolic surgery, 157 Automating behavior, 37
physical activity, 156
screen time, 156
sedentary activity, 156 B
semi-structured diet regimens, 154
sugar-sweetened beverages, 155 Bariatric surgeries, 78
surgical criteria, 157–158 biliopancreatic diversion, 80, 83–85, 88–91
weight management goals, 153–154 caloric intake reduction, 84
Aging dietary compliance, 85
age-adjusted death rate, 174 eating behavior, 83–84
calorie restriction on gut harmones
alternate-day fasting paradigms, 183–184 hormones from adipose tissue, 16
Biosphere 2, 178 surgical procedures, 15
CALERIE 1, 179–181 initial consultation, 85
CALERIE 2, 181–183 laparoscopic adjustable gastric banding
clinical controlled intervention trials, 176 advantages of, 81
CRON, 178–179 vs. Roux-en-Y gastric bypass, 81–83
epidemiological data, 176 postoperative screening, 87
hierarchical model, 185 postoperative weight loss outcomes, 85–86
impact of, 185–186 pre-and postoperative interventions, 87–88
life span extension, 176–178 preoperative screening, 86–87
mimetics, 184 protein and calcium intake, 84
nonhuman primates, 176 resistance exercise, 84
short-duration interventions, 179 Roux-en-Y gastric bypass, 79–85, 88–91
timed eating, 183 sleeve gastrectomy, 81
health care expenditures, 174 vitamins and minerals
mortality, 174 folate, 90
pathologies and clinical manifestations, 174–175 iron, 89
prevalence of, 174 thiamine, 91
primary and secondary aging, 175 vitamin A, 90
AHEI, see Alternate healthy eating index vitamin B6, 90–91
Alanine aminotransferase (ALT) levels, 278, 280, 286–287 vitamin B12, 90
Alcohol, 378–379 vitamin D, 88–89
all-cause mortality, 599 vitamin K, 89
blood lipids, 594 zinc, 89
cardiovascular disease, 597 Barrier function, 575
clotting, 596–597 BCAA, see Branched-chain amino acids
615
616 Index
BED, see Binge eating disorder Food Guide Pyramid, 340–341

Beverages HC/LP diet, 340–341
alcohol, 378–379 mPS, 339
coffee, 377–378 mTORC1 activation, 339–340
SSBs mTORC1 signal vs. BCKAD pathway, 340
coronary heart disease, 376 muscle full response, 340
fructose, 377 roles of, 339
Hill’s criteria, 377 plasma amino acid profile, 130
NHANES, 375 Branchedchain ketoacid dehydrogenase (BCKAD)
T2DM, 375 complex, 338–340
weight gain, 376
Bifidobacterium longum, 575
Biliopancreatic diversion (BPD) C
with duodenal switch, 80
thiamine deficiency, 91 Calorie restriction (CR)
vitamin A deficiency, 90 alternate-day fasting paradigms, 183–184
vitamin B6 deficiency, 90–91 Biosphere 2, 178
vitamin D deficiency, 88 CALERIE 1, 179–181
vitamin K deficiency, 89 CALERIE 2, 181–183
vitamin supplementation, 85 clinical controlled intervention trials, 176
eating behavior, 83–84 CRON, 178–179
malabsorptive procedure, 80 epidemiological data, 176
restrictive procedure, 80 hierarchical model, 185
side effects and complications, 80–81 impact of, 185–186
vitamin D deficiency, 88–89 life span extension, 176–178
vitamin K deficiency, 89 mimetics, 184
vitamin 12 deficiency, 90 nonhuman primates, 176
zinc concentrations, 89 short-duration interventions, 179
Binge eating disorder (BED), 25, 38 timed eating, 183
Binge eating scale (BES), 31 Calorie Restriction Optimal Nutrition (CRON), 178–179
Blood lipids, 410 Carbohydrate–insulin model, 54–55
Blood pressure, 410 Carbohydrate quantity
BMI, see Body mass index CVD risk, 299–300
Body fat distribution diabetes risk, 301
alcohol, 134–135 glycemic control, 300–301
carbohydrates, 128–129 lipids and lipoproteins, 298–299
dairy products, 135–136 Carbohydrate (CHO) restriction
dietary factors, 139 low-carbohydrate diets
dietary fiber, 136–137 caloric restriction diet, 284
dietary patterns, 131–132 characteristics and results, 281–283
food items, 132–133 macronutrient composition, 281, 284
gut microbiota, 137 soy, 285
infant feeding, 137–139 quality, 285
isoflavones, 136 recommendations, 286
lipids Cardiometabolic health, 335–336
CLA, 127–128 Cardiometabolic risk
fatty acids, 125–126 dairy fat, 488
MCT vs. LCT, 126 dairy product consumption
n-3 FA, 126–127 blood pressure, 486–488
Mediterranean diet, 133–134 glucose–insulin homeostasis, 485–486
probiotics, 137 low-grade systemic inflammation, 485
protein, 129–130 plasma lipid levels, 483–485
soy, 136 vascular function, 486–488
vitamin A and C, 137 Cardiometabolic Think Tank, 253–254
whole grain intake, 136–137 Cardiovascular disease (CVD)
Body fatness, 410 age-adjusted death rate, 174
Body mass index (BMI), 102, 150–151, 410 AHEI, 409–410
BPD, see Biliopancreatic diversion alcohol, 597
Brainstem dorsal vagal complex (DVC), 4 a posteriori patterns
Branched-chain α-keto acid dehydrogenase (BCKD) cohort and case–control studies, 411
enzyme, 130 CVD risk factors, 412–413
Branched-chain amino acids (BCAA) dairy product, 412
leucine healthy/prudent, 411–412
AMPK, 339–340 meta-analysis, 403, 411
Index 617
reduced rank regression, 412 pediatric pharmacotherapy, 165

unhealthy/Western, 411–412 physical activity, 111–113, 164–165
atherosclerosis, 161 positive associations, 211–212
beverages prevalence of, 174
alcohol, 378–379 primary and secondary aging, 175
coffee, 377–378 protein intake, 325–326
SSBs, 374–377 replication, 557
Bogalusa Heart study, 161 risk assessment, 212–213
calorie restriction risk factors, 558, 566
alternate-day fasting paradigms, 183–184 structural and hemodynamic changes, 161
Biosphere 2, 178 surgery, 165
CALERIE 1, 179–181 TFA
CALERIE 2, 181–183 blood pressure, 228
clinical controlled intervention trials, 176 consumption, 229
CRON, 178–179 glucose homeostasis, 228
epidemiological data, 176 hemostatic function, 228–229
hierarchical model, 185 inflammation, 229
impact of, 185–186 intakes, 230–231
life span extension, 176–178 LDL, 227
mimetics, 184 lipoproteins, 225–226
nonhuman primates, 176 Lp(a), 227–228
short-duration interventions, 179 mechanistic studies, 226–227
timed eating, 183 prospective cohort studies, 230
carbohydrate quantity, 299–300 recommendations, 231
cardiometabolic effects serum lipids, 225–226
caffeine, 245–246 sources of errors, 230
epidemiology, 242–243 vascular function, 229
sucrose and fructose, 244–245 T2DM, 163
clinical trial, 212 whole grains, 307
dairy, 381–382 CBT, see Cognitive behavioral therapy
acute feeding studies, 359–360 Central nervous system (CNS) regulation of food intake
longer-term feeding studies, 358–359 brainstem regulation, 4–6
dietary intervention, 164–165 brainstem dorsal vagal complex, 4
dyslipidemia, 161–162 CCK, 5
eggs, 382–383 central regulation of feeding, 5
energy balance DMH, 6
APOA2 gene, 563 links between peripheral signals, 5
dietary macronutrient distribution, 561 NTS, 4–5
FTO, 559–561 hypothalamus
GRS, 559–560 ARC, 6
GWAS, 559 dorsomedial and ventromedial nucleus, 6–7
obesity, 558 lateral hypothalamus, 6
PLIN1, 562 paraventricular nucleus, 6
SFA intake, 561 neurotransmitters and homeostatic control
weight-loss diets, 561 amphetamine-regulated transcript, 7–8
epidemiological trial, 212 cocaine-regulated transcript, 7–8
fish, 361–362, 384–385 neuropeptide Y and agouti-related peptide, 7
fruits and vegetables, 379–380 proopiomelanocortin, 7–8
glucose-related risk factors, 563–565 CETP, see Cholesteryl ester transfer protein
glycemic index, 303–304 CHD, see Coronary heart disease
grains and fiber, 385–386 Childhood obesity
health care expenditures, 174 causes and conditions, 152–153
hypertension, 158–160 CVD
insulin resistance, 163 atherosclerosis, 161
lipid metabolism, 565–566 Bogalusa Heart study, 161
meats, 383–384 dietary intervention, 164–165
meta-analysis, 374 dyslipidemia, 161–162
metabolic trial, 212 hypertension, 158–160
MetSyn, 162–163 insulin resistance, 163
mortality, 174 MetSyn, 162–163
NAFLD, 164 NAFLD, 164
nuts and legumes, 386–387 pediatric pharmacotherapy, 165
obesity, 609 physical activity, 164–165
pathologies and clinical manifestations, 174–175 structural and hemodynamic changes, 161
618 Index
surgery, 165 Dairy product consumption

T2DM, 163 blood pressure, 486–488
definition, 150–151 glucose–insulin homeostasis, 485–486
prevalence, 151 low-grade systemic inflammation, 485
risk factors, 151–152 plasma lipid levels, 483–485
treatment vascular function, 486–488
bariatric surgery, 156–157 DCCT, see Diabetes Control and Complications trial
dietary, 153 De novo lipogenesis (DNL), 276, 278
fruit and vegetables, 155 Developmental origins of health and disease (DOHaD), 532
macronutrient, 155 Diabetes Control and Complications trial (DCCT), 595
meal replacements, 155 Diabetes Prevention Program (DPP), 38–39
medications, 156 Dialectical behavioral therapy (DBT), 39
metabolic surgery, 157 Diastolic blood pressure (DBP), 112–113
physical activity, 156 Dietary Approaches to Stop Hypertension (DASH) diet,
screen time, 156 160, 215, 407–408
sedentary activity, 156 blood pressure changes, 433–434
semi-structured diet regimens, 154 cardiometabolic health
sugar-sweetened beverages, 155 lipids, lipoproteins, and apolipoproteins, 445
surgical criteria, 157–158 meta-regression analyses, 437
weight management goals, 153–154 study objectives, design, study population, and
Cholecystokinin (CCK), 5, 11 outcome, 437–446
Cholesteryl ester transfer protein (CETP), 226–227, 257 DASH-Sodium trial, 434–435
Coffee consumption, 377–378 fat and saturated fat, 433
Cognitive behavioral therapy (CBT) fruit and vegetable diet, 432
BED, 25, 38 high-fat DASH diet, 436
for bulimia nervosa, 38 hypertension, 431
CBT-E, 38 lower blood pressure, 432
Cognitive restructuring, 35–36 Omni-Heart Study, 436–437
Comprehensive Assessment of Long-Term Effects of PREMIER study, 435
Reducing Intake of Energy (CALERIE) protein sources, 436
CALERIE 1, 179–181 standard American diet, 432
CALERIE 2, 181–183 systolic and diastolic blood pressures, 433
Compulsive overeating Dietary fat model, 56
BES, 31 Dietary intervention studies
cardiometabolic health risks, 32 blood pressure, 261–262
definition, 25 fasting glucose concentrations, 262–263
drug addiction, behavioral similarities to, 30–31 HDL concentration, 261
neurobiological mechanisms, 31–32 HFCS consumption, 264
YFAS, 31 insulin resistance, 263–264
Conjugated linoleic acid (CLA), 127–128 liver lipid accumulation, 263
Coronary heart disease (CHD) TG concentrations, 260–261
alcohol, 597–598 Waist circumference, 262
body weight, 470–472 Dietary patterns
CVD events, 457–458 AHEI, 409–410
insulin resistance and glycemic response, 470 a posteriori patterns, 399
lipoprotein function, 466–467 cardiovascular disease, 411–412
lipoprotein particles, 459, 466 CVD risk factors, 412–413
major CVD risk factors, 458–465 a priori, 406
oxidation and inflammation, 467–468 DASH diet, 407–408
vascular health, 468–470 HEI, 408–409
CR, see Calorie restriction implications and directions, 413–414
C-reactive protein (CRP), 354 Mediterranean diet
aMED, 400–401, 404–405
D cochrane meta-analysis, randomized trials, 406
cohort studies, 403
Dairy exceptional longevity, 402
cheese, butter, and yogurt, 381–382 intervention trials, 406
CVD and diabetes lower incidence, CVD, 402–403
acute feeding studies, 358–359 lower risk, metabolic syndrome, 405
longer-term feeding studies, 358–359 meta-analysis, 405
fat, 488 meta-analysis forest plot, 404
paleolithic diets, 500–501 vs. PREDIMED, 403–404
total mortality/CVD mortality/CVD events, 356–357 protective effects, 404
type 2 diabetes, 357–358 randomized controlled trial, 403
Index 619
T2D, 405 positive energy balance, 324

Women’s Health Initiative observational study, 406 protein turnover, 323
methodology Fatty acids (FA), 125–126
analysis, statistical methods, 398 Fish consumption, 361–362
common characteristics, empirical pattern, 399 Flow-mediated dilation (FMD), 215–216, 229, 487
components and scoring criteria, major numerical Folate, 90
indices, 399–402 Food consumption
reduced rank regression, 399 beverages
multiple study designs, 402–403 alcohol, 378–379
Dietary protein, see Protein intake coffee, 377–378
Dietary self-monitoring, 36 SSBs, 374–377
Diet-induced thermogenesis (DIT), 320–322 dairy, 381–382
The Diogenes study, 303 egg, 382–383
Distracted eating, 27–28 fish, 384–385
Docosahexaenoic acid (DHA), 195–196 fruits and vegetables, 379–380
DOHaD, see Developmental Origins of Health and Disease grains and fiber, 385–386
Dorsomedial nucleus of hypothalamus (DMH), 6 meats, 383–384
Dual-energy X-ray absorptiometry (DXA), 130 meta-analysis, 374
Dumping syndrome, 83–84 nuts and legumes, 386–387
Food intake, regulation of
CNS
E brainstem regulation, 4–6
hypothalamus, 6–7
Early life nutrition, see Epigenetics neurotransmitters and homeostatic control, 7–8
Eggs, 360, 382–383 energy homeostatic mechanisms, 4
Eicosapentaenoic acid (EPA), 195–196 gastrointestinal signals
Endocrine disrupting chemicals (EDCs), 604–605 EECs, 9
Endothelial dysfunction, 469 endocrine action of gut hormones, 8
Energy balance gut harmones, 10–15
and body weight regulation gut peptides effects on appetite, 9
feedback circuits, 56–57 maintenance of energy equilibrium, 9
macronutrient metabolism, regulation of, 52–53 physiological effects, 9
metabolic adaptation/adaptive thermogenesis, 56 GI system, 4
obesity, macronutrient models, 54–56 gut–brain axis, 4
negative, 318–319 leptin, 4
neutral, 319 mechanical mechanisms
positive, 324 bariatric surgery on gut hormones, effect of,
Energy intake, 321–322 15–16
Enhanced cognitive behavioral therapy (CBT-E), 38 diet and gut–brain axis, 16–17
Enteroendocrine cells (EECs), 9 ileal-brake reflex, 15
Epigenetics mchanoreception in apetite control, role of, 15
animal models non-homeostatic mechanism, 4
cardiometabolic outcomes, 535–536 Fructose, 129
DNA methylation, 534–535 Fructose-containing sugar
IGF2 gene, 535 DNL, 255–256
maternal overweight/obesity, 537–538 dyslipidemia, 257
maternal undernutrition, 536–537 hepatic glucose metabolism, 255
secondary trigger, 534 hepatic lipid, 255
strategies, 542–544 hyperuricemia, 258
DNA methylation patterns, 532 inflammation, 258–259
DOHaD, 532 intramyocellular lipid accumulation, 257–258
epidemiology and clinical cohorts, 533–534 potential mechanisms, 255–256
inflammation and programming, 538–540 visceral adipose accumulation, 258
metabolic stressors, 533 VLDL1 production and secretion, 257
paternal effects, 542
transgenerational effects, 541–542
Essential amino acids (EAA), 337–338 G
Gastric bypass (GB), 156–157
F Gastric inhibitory polypeptide (GIP), 14–15
Gastrointestinal (GI) system, 4
Fat-free mass (FFM) Gene–diet interactions
body composition, 322 CVD risk factors, 558, 566
negative energy balance, 318–319 energy balance
620 Index
APOA2 gene, 563 metagenomics, 572–574

dietary macronutrient distribution, 561 newborns and early childhood, 584
FTO, 559–561 16S rRNA sequences, 573
GRS, 559–560
GWAS, 559 H
obesity, 558
PLIN1, 562 Hazard ratios (HRs), 242–243
SFA intake, 561 Health Professional Follow-Up Study (HPFS), 307, 353
weight-loss diets, 561 Healthy eating index (HEI), 408–409
glucose-related risk factors, 563–565 Heart disease
lipid metabolism, 565–566 case–control study, 362
replication, 557 isoflavonoid excretion, 363
Genome-wide association studies, 279 LDL cholesterol, 363
Glycemic control (GC) nontraditional risk factors, 363–364
carbohydrate quantity, 300–301 population-based study, 362
glycemic index, 304 Hepatocellular carcinoma, 278
whole grains, 307–308 High-carbohydrate, low-protein (HC/LP) diet
Glycemic index (GI), 128–129 blood lipids, 342–343
CVD, 303–304 glycemic regulations, 342
diabetes risk, 305–306 leucine, 340–341
glucose homeostasis, 304 High-density lipoprotein (HDL), 195
glycemic control, 304 High-density lipoprotein-cholesterol (HDL-C)
inflammation, 302–303 aerobic exercise, 104–105, 110–111
lipids and lipoproteins, 301–302 atherogenic lipid profile, 110
Glycemic load (GL), 128–129 blood lipid concentrations, 109
Glycemic regulations, 341–342 resistance exercise, 111–112
Gut–brain axis, 4, 9, 12–14, 16–17 Higher-protein diets, 70–71
Gut harmone on regulation of food intake High fructose corn syrup (HFCS), 239, 242, 246, 255
amylin, 15 High-intensity interval training (HIIT), 114
CCK, 11 High meal frequency (HMF)
ghrelin, 10–11 dietary patterns, 517–518
GIP, 14–15 effects of, 521–523
glucagon-like peptide-1, 12–13 mechanisms, 522, 524
oxyntomodulin, 14 High-protein diets, 318–319
pancreatic polypeptide, 11–12 Homeostasis model assessment of insulin resistance
peptide YY, 13–14 (HOMA-IR), 264, 521
Gut microbiome β-Hydroxybutyrate, 321
acute changes, 575 Hypertension, 431
Bacteroides, 573
cardio-metabolic health
I
causality, 578–579
gnotobiotics, 579 Inflammation, 410
human fecal transplants, 579–580 Insulin receptor substrate 1 (IRS1), 564
“Koch’s postulates,”, 578 Intermittent fasting (IF)
cardiometabolic risk factors, 578 ADMF, 517
“culture-free” methods, 572 dietary patterns, 517–518
CVD therapies, 577–578 effects of, 519–520
diversity, 574 mechanisms, 519
effects of diet, 580–581 methods, 518–519
fiber, 581–582 Intima-media thickness (IMT), 356
food additives, 582 Intrahepatic triglyceride (IHTG) content, 322–323
polyphenols, 582 Intrauterine growth restriction (IUGR), 536
pre-/probiotics, 582 Iron, 89
and elderly populations, 585
functional characterization, 573
healthy adults, 574 L
and host genetics interactions, 582–584 Laparoscopic adjustable gastric banding
hypothesized mechanisms, 572 advantages of, 81
individual variability, 573 calcium and vitamin D, 89
and inflammation, 575–576 vs. Roux-en-Y gastric bypass, 81–83
maternal diet, 585 vitamin supplementation, 85
metabolically active tissue, 576–577 Laparoscopic sleeve gastrectomy (LSG), 156–157
Index 621
Lifestyle, Exercise, Attitudes, Relationships, Nutrition Minimally modified LDL (mmLDL), 227
(LEARN) program, 34–35 Monounsaturated fatty acids (MUFA), 125–126
Limbic hypothalamic-pituitary-adrenocortical (L-HPA) Multi-Ethnic Study of Atherosclerosis, 409
axis, 29 Muscle protein synthesis (mPS), 336, 338–339
Linoleic acid (LA) metabolites, 196–198
Lipoprotein(a) (Lp(a)), 227–228 N
Lipoprotein-associated phospholipase A2 (LpPLA-2), 201
Lipoprotein lipase (LPL) gene, 565 National Cholesterol Education Program (NCEP), 483
Load diets, 71 National Health and Nutrition Examination Survey
Long-chain triglycerides (LCT), 126 (NHANES), 151, 375, 409, 457
Low-carbohydrate diets, 326 Nitrogen balance methods, 337
Low-density lipoprotein (LDL), 195, 227 NNR, see Nordic Nutrition Recommendations
Low-density lipoprotein–cholesterol (LDL-C), 216 Nonalcoholic fatty liver disease (NAFLD), 155, 164
aerobic exercise, 104–105, 110–111 antioxidants, 286–287
atherogenic lipid profile, 110 in children, 280
blood lipid concentrations, 109 CHO restriction
resistance exercise, 111–112 low-carbohydrate diets, 281–285
Low-glycemic-index (GI) diet, 71, 155 quality, 285
recommendations, 286
M epidemiology, 279–280
hepatic triglyceride accumulation, 276–278
Mediterranean diet (MD), 125 histological features, 276–277
body fat distribution, 133–134 pharmacological treatments
dietary fat issue, 422 bile acid, 288
epidemiology, 424 insulin-sensitizing agents, 287–288
history, 423–424 pentoxifylline, 288–289
randomized trials, 425 prebiotics, 287
T2D, 422, 425 probiotics, 287
weight loss, 71–72 risk factors, 278–279
Mediterranean score (MEDS), 133–134 treatments, 280–281
Mediterranean-style diet (MSD), 155 Nonesterified fatty acids (NEFAs), 276
Medium-chain triglycerides (MCT), 126 Non-homeostatic CNS pathways, 8
Metabolic equivalents (METs), 114 Nordic Nutrition Recommendations (NNR), 506
Metabolic syndrome (MetS), 155, 212; see also N-3 fatty acids (FA), 126–127
Nonalcoholic fatty liver disease (NAFLD) Nuclear factor kappa B (NFκB), 201–203
added sugars Nucleus of the tractus solitarius (NTS), 4–5
consumption levels, 254 Nurses’ Health Study (NHS), 451
established paradigm, 259 Nut consumption
fructose proportion, 255–259 coronary heart disease
sources, 254–255 body weight, 470–472
affirmed concepts, 252–253 CVD events, 457–458
CVD, 162–163 insulin resistance and glycemic response, 470
dietary sugar consumption lipoprotein function, 466–467
dietary intervention studies, 260–265 lipoprotein particles, 459, 466
observational studies, 259–260 major CVD risk factors, 458–465
nut consumption, 456–457 oxidation and inflammation, 467–468
risk factors vascular health, 468–470
formally defined diagnostic criteria, 253 Dietary Guidelines for Americans, 450
insulin resistance, 253 early epidemiological associations
lifestyle risk factors, 254 Adventist Health Study, 450
residual risk indicators, 254 hazard ratio, 450
Microbiota, see Gut microbiome Iowa Women’s Health Study, 450–451
MicroRNAs (miRNAs), 565 NHS, 451
Mindless overeating, 25–26 MetS risk, 456–457
ambient factors, 26–27 recent epidemiological studies, 451, 455
categorization cues and health halo effects, 27 risk of type 2 diabetes, 455–456
definition, 25
distracted eating, 27–28 O
food proximity and visibility, 26
palatability, 27 Obesity
portion size, 26 epidemic
social facilitation and matching, 28 cardiovascular disease, 609
622 Index
developmental origins, 607–608 stress-induced overeating, 28–29

energy balance, 606–607 abdominal adiposity, risk for, 25
in vitro exposure, 605–606 and cardiometabolic outcomes, 30
in vivo exposure, 606 definition, 25
obesogens and adipogenesis, 605 neurobiological mechanisms, 29
transgenerational transmission, 608–609 TSST, 30
macronutrient models Oxidized LDL (oxLDL), 227
carbohydrate–insulin model, 54–55
dietary fat model, 56 P
protein leverage model, 55
Obesogens, see Obesity, epidemic Paleolithic diets, 411
Omega-3 fatty acids alcohol, 497
inflammatory marker, 201–202 carbohydrates, 495
lipids, 200 controlled trials
omega status, 203 kidney function, 507
prospective cohort studies, 201–202 metabolically controlled study, 507
RCTs, 202–203 nonrandomized controlled study, 506
structures, 194 randomized controlled trials, 505
Omega-6 fatty acids type 2 diabetes, 506
inflammatory diseases, 195–196 dairy, 500–501
LA metabolite, 196–198 fish and shellfish, 497
lipids, 195 grains
omega status, 203 DART, 500
prospective cohort study, 196, 199 fiber intake, 500
RCT, 199–200 lectins, 498–499
structures, 194 protease inhibitors, 499
OmniCarb study, 302 sporadic consumption, 498
Oral glucose tolerance test (OGTT), 108, 117 WGA, 498–499
Overeating human nutrition, 494
cognitive-behavioral techniques hunter–gatherers, 502–503
and cardiometabolic outcomes, 38–39 insects and larvae, 497
CBT-BN, CBT-BED, and CBT-E protocols, 38 Kitava study, 503–504
cognitive restructuring, 35–36 meat, 496–497
goal setting and striving, 36–37 nutritional characteristics, 501–502
lifestyle interventions, 35 tree nuts, 496
problem-solving, 37–38 urbanization, 504–505
self-monitoring, 36 Pancreatic polypeptide (PP), 11–12
stimulus control, 37 Perilipin 1 (PLIN1), 562
compulsive overeating Peripheral blood monocytes (PBMCs), 539
BES, 31 Peroxisome proliferator activated receptor gamma
cardiometabolic health risks, 32 (PPARγ), 605
definition, 25 Physical activity (PA)
drug addiction, behavioral similarities to, 30–31 abdominal obesity
neurobiological mechanisms, 31–32 aerobic exercise, 103–106
YFAS, 31 resistance exercise, 106–108
environmental interventions waist circumference, 102
behavioral principles, 32–33 waist-to-hip ratio, 102
and cardiometabolic outcomes, 35 CVD, 113
institutional level, 33–34 dyslipidemia
“small-change” interventions, 34 aerobic exercise, 104–105, 110–111
vs. traditional CBT lifestyle interventions, 34–35 atherogenic lipid profile, 110
mindfulness interventions blood lipid concentrations, 109
cardiometabolic outcomes, effects on, 40–41 resistance exercise, 111–112
combination mindfulness approaches, 40 efficacy studies, 113–114
independent mindfulness approaches, 39–40 HIIT, 114
mindless overeating, 25–26 hypertension
ambient factors, 26–27 aerobic exercise, 112–113
categorization cues and health halo effects, 27 resistance exercise, 113
definition, 25 insulin resistance
distracted eating, 27–28 aerobic exercise, 108–109
food proximity and visibility, 26 resistance training, 109–110
palatability, 27 sedentary behavior
portion size, 26 epidemiological evidence, 115
social facilitation and matching, 28 independent risk factor, 115
Index 623
intervention studies, 116–117 telomeres, 354

METs, 114 TMAO, 354
North American adults, 114–115 total mortality, 351
Pistachios, 466 weight, 353–354
Polyphenols, 425 Resting energy expenditure (REE), 324
Polyunsaturated fatty acids (PUFA), 125–126, 138–139 RISCK study, 302
Portion distortion, 26 Roux-en-Y gastric bypass (RYGB)
Poultry, 360 caloric intake, 84
Prebiotics, 287 dietary compliance, 85
Prevención con Dieta Mediterránea dumping syndrome, 83
(PREDIMED) trial, 457 eating behavior, 83
Probiotics, 287 folate deficiencies, 90
Problem solving techniques, 37–38 iron deficiency, 89
Proliferator activator receptor-gamma (PPAR-γ), 196 vs. laparoscopic adjustable gastric banding, 81–83
Protein-centric perspective long-limb/short-limb procedure, 79–80
body composition and sarcopenia, 337–338 protein intake, 84
cardiometabolic health, 335–336 purpose of, 79
cardiometabolic risk factors thiamine deficiency, 91
blood lipids, 342–343 vitamin B6 deficiency, 90–91
glycemic regulations, 341–342 vitamin D, 88–89
leucine vitamin 12 deficiency, 90
AMPK, 339–340 zinc concentrations, 89
Food Guide Pyramid, 340–341 Rumminococcus gnavus, 575
HC/LP diet, 340–341
mPS, 339 S
mTORC1 activation, 339–340
mTORC1 signal vs. BCKAD pathway, 340 Satiety-stimulating effect, 320
muscle full response, 340 Saturated fatty acid (SFA), 125–126
roles of, 339 atherogenic dyslipidemia, 214
metabolic syndrome, 334 dietary guidelines, 216–217
weight loss and muscle mass, 335–336 diets
Protein-induced satiety, 320–321 animal models, 210
Protein intake CVD, 211–213
adverse effects, 326 lipid profiles, 211
appetite, 319–321 food sources, 214–215
body composition, 322–323 LDL-C, 216
cardiovascular diseases, 325–326 primer, 209–210
energy expenditure, 322 replacement nutrients, 213–214
energy intake, 321–322 risk factors, 215–216
negative energy balance, 318–319 Seguimiento Universidad de Navarra (SUN)
neutral energy balance, 319 cohort study, 457
positive energy balance, 324 Self-monitoring, 36
protein turnover, 323–324 Serum/Urine Trimethylamine Oxide (TMAO), 354
reward homeostasis, 325 Sleeping metabolic rate (SMR), 322
Protein leverage model, 55 Sleeve gastrectomy (SG), 81
calcium and vitamin D dosage, 89
R caloric intake, 84
folate deficiencies, 90
Randomized controlled trial (RCT) iron deficiency, 89
aerobic exercise, 103–106 mineral supplement, 85
omega-3 fatty acids, 202–203 multivitamins supplement, 85
omega-6 fatty acids, 199–200 vitamin B6 deficiency, 90
resistance exercise, 106–108 vitamin D deficiency, 88
waist circumference, 102 vitamin 12 deficiency, 90
waist-to-hip ratio, 102 zinc concentrations, 89
Red and processed meat Soy intake
blood pressure, 354 case–control study, 362
characteristics, 353 isoflavonoid excretion, 363
CVD mortality and events, 351–352 LDL cholesterol, 363
diabetes, 352–353 nontraditional risk factors, 363–364
harmful effect, 353 population-based study, 362
inflammatory markers, 354 Spanish Ketogenic Mediterranean Diet (SKMD), 285
metabolomics, 354 Standard mean difference (SMD), 126
saturated fat/cholesterol, 354 Stearoyl coenzyme A desaturase 1 (SCD1), 288
624 Index
Stimulus control, 37 nontraditional risk factors, 363–364

Stress-induced overeating, 28–29 paleolithic diets, 506
abdominal adiposity, risk for, 25 population-based study, 362
and cardiometabolic outcomes, 30 risk factors, 357–358
definition, 25 Type 2 diabetes mellitus (T2DM), 163, 212, 376, 596
neurobiological mechanisms, 29
TSST, 30 U
Sugar-sweetened beverages (SSBs), 129
cardiometabolic effects United States Department of Agriculture (USDA), 154
caffeine, 245–246
epidemiology, 242–243 V
sucrose and fructose, 244–245
coronary heart disease, 376 Very-low-density lipoprotein (VLDL), 195, 276
energy intake Very-low-density lipoprotein 1 (VLDL1), 257
clinical studies, 240 Visceral adipose tissue (VAT), 258, 322–323
meta-analyses, 239–240 Vitamin A, 90
randomized controlled trials, 241 Vitamin B6, 90–91
weight gain, soft drinks, 241 Vitamin B12, 90
fructose, 377 Vitamin D, 88–89
gene–environment interaction, 238–239 Vitamin K, 89
Hill’s criteria, 377
NHANES, 375 W
T2DM, 375
weight gain, 376 Waist circumference (WC), 102–103
Sympathetic-adrenal-medullary (SAM) axis, 29 Waist-to-hip ratio (WHR), 127
Systolic blood pressure (SBP), 112–113 Weighted mean difference (WMD), 126
Weight loss
T balanced-deficit diets/prudent diets, 65, 67
bariatric procedures, 78, 82–91
Thiamine, 91 calorie intake reduction, 62
Total cholesterol (TC), 112, 211 computer models, 62
Trans fatty acids (TFA) diet composition, effect of, 63
CVD genetic influences, 63, 66
blood pressure, 228 higher-protein diets, 70–71
consumption, 229 initial rate of, 63
glucose homeostasis, 228 intermittent/alternate-day fasting regimens, 67
hemostatic function, 228–229 lifestyle/medical therapy, 78
inflammation, 229 low-carbohydrate diet, 61, 64–65, 67–70
intakes, 230–231 low-fat diets, 62, 64–65, 67–70
LDL, 227 low–glycemic index/load diets, 71
lipoproteins, 225–226 meal replacements, 63, 67
Lp(a), 227–228 Mediterranean diet, 71–72
mechanistic studies, 226–227 program attendance, effect of, 63
prospective cohort studies, 230 very-low-calorie diets, 63, 65
recommendations, 231 Wheat germ agglutinin (WGA), 498–499
serum lipids, 225–226 Whole grains
sources of errors, 230 CVD risk, 307
vascular function, 229 diabetes risk, 308
partial and full hydrogenation, 224 glycemic control, 307–308
Trier Social Stress Test (TSST), 30 lipids and lipoproteins, 306–307
Trimethylamine (TMA) formation, 577
Trimethylamine N-oxide (TMAO), 577 Y
Type 2 diabetes (T2D), 342, 403, 405, 410
case–control study, 362 Yale Food Addiction Scale (YFAS), 31–32
dairy, 357–358
gene–diet interactions, 563–565 Z
insulin resistance, 422, 425
isoflavonoid excretion, 363 Zinc, 89
LDL cholesterol, 363
Mediterranean diet, 422

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What are some of the nutritional factors discussed in the book?

What are some of the nutritional factors discussed in the book?

What medical procedures are mentioned for weight loss?

What medical procedures are mentioned for weight loss?

Nutrition and

© 2018 by Taylor & Francis Group, LLC

No claim to original U.S. Government works

Printed on acid-free paper

International Standard Book Number-13: 978-1-4987-0426-7 (Hardback)

Library of Congress Cataloging-in-Publication Data

Names: Bergeron, Nathalie, editor. | Siri-Tarino, Patty W., editor. | Bray,

Visit the Taylor & Francis Web site at

and the CRC Press Web site at

Section I Energy Balance, Adiposity, and

Chapter 1 Regulation of Food Intake: The Gut–Brain Axis..........................................................3

Chapter 2 Overeating Behavior and Cardiometabolic Health: Mechanisms and Treatments......23

Chapter 4 Diets for Weight Loss..................................................................................................61

Chapter 5 Weight Loss by Surgical Intervention: Nutritional Considerations and Influence

Chapter 6 Physical Activity and Cardiometabolic Health......................................................... 101

Chapter 7 Diet as a Potential Modulator of Body Fat Distribution........................................... 123

Chapter 8 Nutritional Considerations for Cardiometabolic Health in Childhood and

Chapter 9 Aging and Cardiovascular Disease: Lessons from Calorie Restriction.....................173

Section II Dietary Fats and Cardiometabolic Health

Chapter 11 Evolving Role of Saturated Fatty Acids....................................................................209

Chapter 12 Effects of Dietary Trans Fatty Acids on Cardiovascular Risk...................................223

Chapter 14 Effects and Mechanisms of Fructose-Containing Sugars in the Pathophysiology

Chapter 15 Dietary Carbohydrate Restriction in the Management of NAFLD and

Chapter 16 Dietary Starches and Grains: Effects on Cardiometabolic Risk................................297

Section IV Dietary Protein and Cardiometabolic Health

Chapter 18 A Protein-Centric Perspective for Skeletal Muscle Metabolism and

Chapter 19 Protein Sources, CVD, Type 2 Diabetes, and Total Mortality..................................349

Chapter 20 Consumption of Foods, Food Groups, and Cardiometabolic Risk............................373

Chapter 21 Dietary Patterns and Cardiometabolic Disease.........................................................397

Chapter 23 The DASH Diet.........................................................................................................431

Chapter 25 Dairy Product Consumption, Dairy Fat, and Cardiometabolic Health......................481

Chapter 26 Paleolithic Diets........................................................................................................493

Chapter 27 Fasting Intermittently or Altering Meal Frequency: Effects on Plasma Lipids.........517

Section VI Other Nutritional Influences

Chapter 28 Early-Life Nutrition, Epigenetics, and Later Cardiometabolic Health.....................531

Chapter 29 Gene–Diet Interactions..............................................................................................555

Chapter 31 Alcohol: Associations with Blood Lipids, Insulin Sensitivity, Diabetes,

Patty W. Siri-Tarino, PhD, is an associate staff scientist in the

George A. Bray, MD, MACP, MACE, is a Boyd professor emeri-

Ronald M. Krauss, MD, is senior scientist and Dorothy Jordan

Andrea M. Brennan Lynda Frassetto

Clint Gray Karim Kheniser

Geneviève B. Marchand Jose M. Ordovas

Jordi Salas-Salvadó Janne Schurmann Tolstrup

Surya Panicker Rajeev, Ian W. Seetho, and John P.H. Wilding

CENTRAL NERVOUS SYSTEM REGULATION OF FOOD INTAKE

ARC (NPY/AgRP and

Adipose tissue Leptin

LHA and perifornical area

Arcuate POMC (α-MSH)

Ghrelin PYY3–36 GLP–1 Leptin

GI tract (gut hormones) Adipose tissue

Neurotransmitters and Homeostatic Control of Food Intake

Proopiomelanocortin and Cocaine- and Amphetamine-Regulated Transcript

Non-Homeostatic CNS Pathways

Section I Energy Balance, Adiposity, and

Section VI Other Nutritional Influences