Immunonutrition Interactions of

Immunonutrition
Interactions of Diet,
Genetics, and Inflammation
Edited by
Bharat B. Aggarwal • David Heber
Immunonutrition
Interactions
of Diet, Genetics,
and Inflammation
Immunonutrition
Interactions
of Diet, Genetics,
and Inflammation
Edited by
Bharat B. Aggarwal
The University of Texas
Houston, Texas, USA
David Heber
UCLA Center for Human Nutrition
Los Angeles, California, USA
Boca Raton London New York
CRC Press is an imprint of the

Taylor & Francis Group, an informa business
CRC Press
Taylor & Francis Group
6000 Broken Sound Parkway NW, Suite 300
Boca Raton, FL 33487-2742
© 2014 by Taylor & Francis Group, LLC
CRC Press is an imprint of Taylor & Francis Group, an Informa business
No claim to original U.S. Government works

Version Date: 20140127
International Standard Book Number-13: 978-1-4665-0386-1 (eBook - PDF)
This book contains information obtained from authentic and highly regarded sources. Reasonable
efforts have been made to publish reliable data and information, but the author and publisher cannot
assume responsibility for the validity of all materials or the consequences of their use. The authors and
publishers have attempted to trace the copyright holders of all material reproduced in this publication
and apologize to copyright holders if permission to publish in this form has not been obtained. If any
copyright material has not been acknowledged please write and let us know so we may rectify in any
future reprint.
Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced,
transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or
hereafter invented, including photocopying, microfilming, and recording, or in any information stor-
age or retrieval system, without written permission from the publishers.
For permission to photocopy or use material electronically from this work, please access www.copy-
right.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222
Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that pro-
vides licenses and registration for a variety of users. For organizations that have been granted a pho-
tocopy license by the CCC, a separate system of payment has been arranged.
Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are
used only for identification and explanation without intent to infringe.
Visit the Taylor & Francis Web site at
http://www.taylorandfrancis.com
and the CRC Press Web site at
http://www.crcpress.com
Contents
Preface.......................................................................................................................vii
Editors........................................................................................................................ix
Contributors...............................................................................................................xi
Chapter 1 Evolution of Innate and Adaptive Immunity.........................................1

David Heber and Bharat B. Aggarwal
Chapter 2 Cellular Mechanisms of Cytokine Activation..................................... 19

Chapter 3 Cellular Lipids and Inflammation....................................................... 39

David Heber and Susanne Henning
Chapter 4 Biomarkers of Inflammation and the Western Diet............................ 53

Chapter 5 Phytochemicals and Immune Function............................................... 67

David Heber
Chapter 6 Genetic and Environmental Modifiers of Immune Function.............. 85

David Heber
Chapter 7 Cancer and Inflammation.................................................................. 101

David Heber
Chapter 8 Abdominal Obesity: Pathophysiology and Related Metabolic

Complications.................................................................................... 115
Ana F.T.A. Junqueria and Caroline M. Apovian
Chapter 9 Type 2 Diabetes and Inflammation................................................... 141

Zhaoping Li and David Heber
v
vi Contents
Chapter 10 Heart Disease and Inflammation....................................................... 149

Kaveh Daniel Navab
Chapter 11 Chronic Kidney Disease and Inflammation...................................... 167

Karl J. Neff and Carel Le Roux
Chapter 12 Alzheimer’s Disease and Inflammation............................................ 181

Stephen T. Chen and Gary W. Small
Chapter 13 Nutrition in Autoimmunity: A Focus on Systemic Lupus

Erythematosus and Rheumatoid Arthritis........................................ 211
Maureen McMahon
Chapter 14 Asthma and Inflammation................................................................. 229

Andre Nel and David Heber
Chapter 15 Muscle and Immune Function........................................................... 245

Anthony Thomas and David Heber
Chapter 16 Approaches to Reducing Abdominal Obesity................................... 259

Chapter 17 Barriers to Fruit and Vegetable Consumption and Practical

Strategies for Increasing Fruit and Vegetable Intake........................ 279
Susan Bowerman
Chapter 18 Healthy Fats and Oils: Balancing Omega-3 and

Omega-6 Acids in Tissues................................................................. 291
Bill Lands
Chapter 19 Spices and Dietary Supplements with

Anti-Inflammatory Activity........................................................... 317
Bharat B. Aggarwal and David Heber
Preface
Immune function and nutrition are closely intertwined in human health. The immune
system is composed of an innate immune system and an adaptive immune system.
The latter is only found in vertebrates while the former is an ancient system that goes
back in evolution to insects and plants.
It is the innate immune system that is overactivated in response to the Western
diet and obesity-associated diseases due to chronic low-grade inflammation. These
diseases range from type 2 diabetes to heart disease, which are closely aligned with
the accumulation of visceral and liver fat resulting in insulin resistance. Individuals
who are about 30 lb overweight or have a body mass index (BMI) of 30 or more
have a 30-fold increased risk of type 2 diabetes mellitus. This 3000% increased risk
is not simply another risk factor but an intrinsic part of the pathogenesis of diabetes
bringing us to call this condition diabesity. However, the etiology of diabetes is not
simply linked to weight but to visceral fat. Individuals in India and China can accu-
mulate visceral fat at normal or even low BMI. Some 70 million Americans have
high blood sugar or prediabetes, and the syndrome, called metabolic syndrome,
affects 50% of individuals between the ages of 50 and 65 in the United States and
many other countries.
The interaction of immune function and nutrition underlies the low-grade chronic
inflammation involved in the etiology of many of the common age-related chronic
disease conditions covered in this textbook. The largest portion of the immune sys-
tem is located adjacent to the gastrointestinal tract. Plants, which also have an innate
immune system, live in soil that is made up of both friendly and potentially toxic
bacteria. Plant roots attract helpful bacteria and repel those bacteria that could attack
them. Humans carry their soil with them in the form of trillions of gut bacteria,
which interact with the immune system. Both dietary intake and obesity influence
the gut microflora, called the microbiome. Plants affect the local bacteria in the soil;
it is thus not surprising that dietary phytochemicals and prebiotics in the human diet
also affect gut microflora.
Diet and exercise are necessary strategies in efforts to reduce visceral fat and
modulate systemic immune function through increased intakes of fruits, vegeta-
bles, plant protein, fish oils, prebiotic fibers, and spices. Nutrition in the broadest
sense determines the health of the immune system. When malnutrition results in
death, it is most commonly caused by infections due to loss of immune function.
Therefore, both in obesity and malnutrition, nutritional factors influence immune
function. This close interaction is the genesis of the term immunonutrition, which
represents a new interdisciplinary field of nutritional and medical research.
It is our hope that this textbook will stimulate increased interest in this new inter-
disciplinary field among students and junior investigators who will carry this field
into the future. There is a need for more human studies to complement the exciting
vii
viii Preface
basic research already developed in cell culture and animal models demonstrating
the mechanisms underlying the interaction of nutrition and immune function. We
hope that this book will achieve these objectives.
David Heber MD, PhD, FACP, FACN

Los Angeles, California
Bharat B. Aggarwal, PhD

Houston, Texas
Editors
David Heber, MD, PhD, FACP, FACN, is the direc-
tor of the UCLA Center for Human Nutrition at the
University of California, Los Angeles. He has been
on the faculty of the UCLA School of Medicine
since 1978 and is currently professor of medicine
and public health. Dr. Heber is board certified in
internal medicine and endocrinology and metabo-
lism by the American Board of Internal Medicine
and is certified as a physician nutrition specialist. He
is a former chair of the Medical Nutrition Council
of the American Society of Nutrition. He directed
both the NCI-funded Clinical Nutrition Research Unit and the NIH Nutrition and
Obesity Training Grants at UCLA. He has written over 230 peer-reviewed scientific
articles and 60 book chapters, as well as three professional texts. He has written four
books for the public, including What Color Is Your Diet? (Harper Collins/Regan
Books, 2001) and the L.A. Shape Diet (Harper Collins/Regan Books, 2004). His
main research interests are obesity prevention and treatment and phytonutrients in
cancer prevention and treatment.
Dr. Bharat B. Aggarwal is a Ransom Horne,

Jr. Distinguished Professor of Cancer Research,
Professor of Cancer Medicine, Professor of
Immunology, Professor of Biochemistry, and
Professor of Experimental Therapeutics, as well
as Chief, Cytokine Research Section, in the
Department of Experimental Therapeutics at the
University of Texas MD Anderson Cancer Center
(MDACC), Houston, Texas. He also serves as a
member of the University of Texas Graduate School
of Biomedical Sciences, Houston; as an adjunct
professor at Albert B. Alkek Institute of Biosciences and Technology, Texas A&M
University, Houston, Texas; and as a member in various institutional committees
of MDACC.
Dr. Aggarwal earned his PhD in biochemistry from the University of California,
Berkeley, and received his postdoctoral training from the Hormone Research
Laboratory at the University of California Medical Center, San Francisco. He
then started his career with Genentech Inc., where he worked for almost 10 years.
His work led to the discovery of TNF-α and TNF-β, essential components of the
immune system, and to the identification of their receptors.
In 1989, Dr. Aggarwal accepted the position of professor and chief of the
Cytokine Research Section at M. D. Anderson Cancer Center, where he currently
ix
x Editors
holds the Ransom Horne, Jr., Endowed Professorship in Cancer Research. Since
then, he has been investigating the role of inflammatory pathways mediated
through TNF, NF-kappaB, and STAT3 for the prevention and therapy of cancer
and other chronic diseases. While searching for novel and safe anti-inflammatory
agents, his group has identified more than 50 novel compounds from dietary
sources and from traditional medicine that interrupt these cell-signaling pathways.
These agents have been tested in various animal models, and some of them are
now in clinical trials. Dr. Aggarwal has published more than 600 papers in peer-
reviewed international journals (including Science, Nature, Cancer Cell, PNAS,
Journal of Experimental Medicine, Blood, JBC, Cancer Research, and Journal of
Immunology), invited reviews, and book chapters.
Dr. Aggarwal is an inventor/coinventor of over 33 patents. He has been included
in ISI Highly Cited among the most popular authors in the immunology category
since 2001. He has also been listed as one of the top 25 researchers worldwide in the
area of apoptosis. His papers exhibit very high citation index (some exceed 1000).
His overall citation is now at 75,900 with an H-index of 106.
Dr. Aggarwal currently serves as a member of the editorial boards of 24 inter-
national journals. He has previously served as a reviewer for more than 160 jour-
nals, various grant proposals, and of several PhD theses. Dr. Aggarwal has edited
12 books and has served as guest editor for special issues of Biotherapy, Cancer
Letters, and Current Opinion in Pharmacology. He has trained over 80 postdoctoral
fellows and visiting professors from around the world. He has co-organized and
served as a member in many national and international conferences and symposia,
started the International Society of Translational Cancer Research, and has delivered
over 350 lectures/seminars/keynote talks in more than 50 countries.
He has recently authored a book entitled Healing Spices (released in January 2011
by Sterling), which is already a bestseller.
Dr. Aggarwal has received numerous awards, including the following:
• ARTOI Award, Association for Research Integrated Oncology Therapies,

Rome, Italy, 2012
• 2011 James A. Duke Award Excellence in Botanical Literature Award,
American Botanical Council, Anaheim, California, 2012
• World Congress Science Prize from Oxygen Club of California, 2010
• Excellence in Research Award of McCormick Research Institute from the
American Association of Nutrition, 2008
• Outstanding Scientist Award from the American Association of Indian
Scientists in Cancer Research, 2006
• Ranbaxy Award for Outstanding Scientist of the Year, 2004
Contributors
Bharat B. Aggarwal Susanne Henning
Department of Experimental Department of Medicine
Therapeutics Center for Human Nutrition
MD Anderson Cancer Center David Geffen School of Medicine
The University of Texas University of California, Los Angeles
Houston, Texas Los Angeles, California
Caroline M. Apovian Ana F.T.A. Junqueria

Section of Endocrinology, Diabetes and Section of Endocrinology, Diabetes and
Nutrition Nutrition
Department of Medicine Department of Medicine
Boston Medical Center Boston Medical Center
School of Medicine Boston, Massachusetts
Boston University
Boston, Massachusetts Bill Lands
American Association for the
Susan Bowerman Advancement of Science
Department of Medicine Washington, DC
Center for Human Nutrition and
David Geffen School of Medicine
American Society for Nutrition
University of California, Los Angeles
Bethesda
and
Stephen T. Chen Society for Free Radical Biology and
Department of Psychiatry and Medicine
Biobehavioral Sciences Indianapolis, Indiana
Division of Geriatric Psychiatry
David Geffen School of Medicine Carel Le Roux
and Diabetes Complications Research Centre
Semel Institute for Neuroscience and Conway Institute of Biomolecular and
Human Behavior Biomedical Research
University of California, Los Angeles University College Dublin
Los Angeles, California Dublin, Ireland
David Heber Zhaoping Li

Department of Medicine Department of Medicine
Center for Human Nutrition Center for Human Nutrition
David Geffen School of Medicine David Geffen School of Medicine
University of California, Los Angeles University of California, Los Angeles
Los Angeles, California Los Angeles, California
xi
xii Contributors
Maureen McMahon Gary W. Small

Division of Rheumatology Department of Psychiatry and
Department of Rheumatology Biobehavioral Sciences
David Geffen School of Medicine Division of Geriatric Psychiatry
University of California, Los Angeles David Geffen School of Medicine
Los Angeles, California and
Semel Institute for Neuroscience and
Kaveh Daniel Navab Human Behavior
Department of Anesthesiology University of California, Los Angeles
David Geffen School of Medicine Los Angeles, California
Los Angeles, California Anthony Thomas
Karl J. Neff Larry L. Hillblom Islet Research
Diabetes Complications Research Centre Center
Conway Institute of Biomolecular and David Geffen School of Medicine
Biomedical Research University of California, Los Angeles
University College Dublin Los Angeles, California
Dublin, Ireland
Andre Nel
Department of Medicine, Pediatrics and
Public Health
Division of NanoMedicine
David Geffen School of Medicine
1 Evolution of Innate and
Adaptive Immunity
CONTENTS
Introduction................................................................................................................. 1
Evolution of Innate Immunity..................................................................................... 5
Innate Immune System in Plants............................................................................ 5
Innate Immune System in Humans........................................................................ 6
Evolution of Cellular Immunity.................................................................................. 6
Immunity and Inflammation................................................................................... 7
Cellular Immunity.................................................................................................. 7
Adaptive Immune System........................................................................................... 9
Malnutrition and Immune Function.......................................................................... 10
Immune Function in Obesity.................................................................................... 11
Macrophage Receptors for Omega-3 Fatty Acids..................................................... 11
Immune Function and Vitamin and Mineral Balance............................................... 12
Practical Considerations for Modulating Immune Function..................................... 14
References................................................................................................................. 15
INTRODUCTION
The human immune system can be divided into two functional entities: the innate
and the adaptive immune systems. The innate immune system appeared early in
evolution prior to the time that plants and animals took separate paths, but the
basic mechanisms of pathogen recognition and activation of the innate immune
response are conserved throughout the evolution of plants and animals includ-
ing humans [1]. Innate immunity is the first line of defense against infectious
microorganisms in humans and relies on germ line–encoded pattern recognition
receptors (PRRs) to recognize pathogen-derived substances [1]. Activation of the
innate immune system through these receptors leads to the expression of a vast
array of antimicrobial effector molecules that attack microorganisms at many
different levels.
The innate immune system has been studied extensively in fruit flies (Drosophila
melanogaster) [2] and even in worms such as Caenorhabditis elegans. These ani-
mals have the same genes as vertebrates, including mice and humans, that encode
intracellular signaling pathways leading to the activation of the transcription fac-
tor nuclear factor-kappa B (NFκB). These gene cassettes encode various proteins
1
2 Immunonutrition: Interactions of Diet, Genetics, and Inflammation
Plants Animals
Adaptive
Angiosperms *Vertebrates* immunity
Gymnosperms Echinoderms
Seed producers Fungi Rotifers
Horsetails Club fungi Anthropods
Club moss Sac fungi Annelids
Ferns Bread mold Mollusks
Bryophytes Worms
Sponges
Protista
Algae
Molds
Amoeba
Innate Flagellates Innate
immunity immunity
Prokaryotes
Cyanobacteria
Eubacteria
Archaeobacteria
Protocells
FIGURE 1.1 Adaptive immune function is a late evolutionary development in vertebrates

while innate immune function can be traced back to the earliest cell types including bacteria.
of signaling pathways modulating NFκB activation and inflammation discussed

elsewhere in this textbook. This evolutionary history combined with other evidence
supports the notion that the activation of NFκB is the central signaling pathway of
activation in innate immunity, leading in turn to the transcription of a set of genes
dependent on NFκB [3]. Moreover, this pathway is a universal pathway that leads to
activation in all host defense systems.
The adaptive immune system evolved much later in higher species (see
Figure 1.1).
In contrast to innate immunity, the adaptive immune system generates
antigen-specific receptors, antibodies, and T-cell receptors by somatic cell DNA
rearrangement [4]. These receptors, found only in higher eukaryotes, recognize
specific pathogen-encoded proteins. Mammals have a complex immune response,
which relies on communication between the innate and adaptive arms of the immune
system.
In the human gut, trillions of bacteria live in symbiosis with the host and affect
both host nutrition and immune function. Studies confirm that gut microbiota carry
on a dynamic interaction with the intestinal innate and adaptive immune systems,
affecting different aspects of its development and function. Communication between
the mucosal immune system and endogenous microflora favors mutual growth, sur-
vival, and inflammatory control of the intestinal microbiome [5].
Since humans evolved in equilibrium with plants, insects, and bacteria, the
innate and adaptive immune systems were clearly influenced by the innate immune
Evolution of Innate and Adaptive Immunity 3
The mammalian The Drosophila

Toll-like receptor Toll signaling
signaling pathway pathway
TLR Toll
MyD88 dMyD88
IRAK TRAF6 Pelle
Cactus
IKK kinase
IκB Cactus
NFκB DIF
FIGURE 1.2 Comparison of the mammalian and fruit fly Toll-like receptor signaling path-
ways. The intracellular domain of the toll-like receptors in flies and mammals interact with
a homologous domain in the adaptor protein MyD88. The terminal parts of the pathway
are also homologous between Drosophila and mammals; phosphorylation of Cactus initiates
its degradation and the release of the Dif/Relish dimer, which is a transcription factor and
homologue of NFκB. (From Janeway, C.A., Jr. et al., Immunobiology: The Immune System in
Health and Disease, 6th edn., pp. 52–53, Garland Science, New York, 2005.)
system of plants. Toll receptors in fruit flies perform the same defensive function
as in mice, and there are analogous signaling pathways in both mice and fruit
flies largely conserved through evolution in the human innate immune system
(see Figure 1.2).
The genes of innate immune function in plants are not arranged in the same
order as in fruit flies, but all the signaling elements can be identified in plants as
separate or fused genes [6]. During pathogen-initiated or environmental stress, plant
hormonal signaling pathways prioritize defense over other cellular functions. This
connection, in turn, provides the necessary background for the immune modula-
tory effects of plant substances under the general rubric of phytochemicals found in
fruits, vegetables, grains, spices, and herbs. Typically, plants raised under stressful
conditions such as low light and water produce increased concentrations of specific
molecules. In other situations, a specific external stress such as ultraviolet light in
dark-adapted mushrooms can lead to the elaboration of defensive toxins.
The cloning of the obese gene (ob) in mice, leading to the discovery of leptin,
began a decade of intensive cellular and molecular investigation on the regulation of
body weight, food intake, and physical activity [7]. The role of leptin has largely been
misunderstood due to the observation that when this protein is administered to obese
mice lacking only this gene in a homozygous condition (the ob/ob mouse), these mice
become thin. A very small number of humans with this genetic condition have also
been shown to become thin following leptin administration. However, despite the ori-
gin of the name leptin (from the Greek root for thinning), its primary role is in the
recovery from starvation and as a cytokine in the immune response. As levels of leptin
in the circulation and central nervous system fall with malnutrition, food intake is
increased and physical activity is decreased. In fact, low leptin levels are a biomarker
of malnutrition in elderly hospitalized patients, and levels rise as malnourished indi-
viduals are renourished [8]. The circulating levels of leptin are proportional to fat mass
but are lowered rapidly by fasting or increased by inflammatory mediators.
The impaired T-cell immunity of mice now known to be defective in leptin (ob/
ob) or its receptor (db/db) is related to the absence of functional leptin signaling due
to the absence of a functional leptin protein (ob/ob) or the leptin receptor protein
(db/db) [9]. Impaired cell-mediated immunity and reduced levels of leptin are both
features of low body weight in humans. Indeed, malnutrition predisposes to death
from infectious diseases, and the impaired immune function resulting from protein-
energy malnutrition and HIV infection is well documented [10]. On an evolutionary
basis, the ability to fight off infection and the ability to store fat in cells were both
critical to survival. While the adaptation to starvation and associated gradual weight
loss do not impair immune function, rapid weight loss does [11]. Moreover, the close
interrelationship of immune function and nutrition has been confirmed with modern
molecular-nutrition tools.
Over the last century, the intricate interaction between human immunity and
metabolism has been recognized and investigated extensively [12]. Indeed, it has
been demonstrated that adipose tissue is not merely the site of energy storage, but
can be considered as an immune-related organ producing a series of molecules
named adipocytokines. Nutritional depletion, specific deficiencies, and kwashiorkor-
like malnutrition suppress immune function [13].
The immune system in humans is an integral part of the adaptation to starvation.
The observation that depletion of the body cell mass to less than half of its normal
mass is incompatible with life regardless of the etiology of malnutrition hinges on
the decompensation of immune function. On the other hand, staying with the theme
that humans are well adapted to starvation but poorly adapted to overnutrition, there
is chronic low-grade inflammation associated with overweight and obesity, medi-
ated by intra-abdominal fat-resident immune cells that cause a systemic inflamma-
tion [14]. In order to understand these two poles of the interaction of nutrition and
immune function, it is necessary to understand the difference between innate and
adaptive immune mechanisms and their evolution.
EVOLUTION OF INNATE IMMUNITY

Genetic studies of plants and animals have demonstrated that the innate immune
system existed at the time the ancestors of plants and animals separated in evolu-
tion. The Toll pathway of NFκB activation has been demonstrated conclusively in
fruit flies, mice, and humans and is also believed to occur in plants [15]. The DNA
sequences of this pathway are found in invertebrates, vertebrates, and plants.
Insects have a very potent innate immune response that effectively combats a
broad spectrum of pathogens. For example, Drosophila can withstand, and clear,
bacterial burdens that, relative to their size, would be lethal to mammals [16].
Induction of innate immunity in both mammals and insects leads to the activation of
similar effector mechanisms, such as stimulation of cell-based phagocytic activity
and expression of antimicrobial peptides [17]. For example, Drosophila produces
a wide range of potent antimicrobial peptides in response to infection by fungi or
bacteria [18]. Induction of the antimicrobial peptides is regulated at the level of tran-
scription, and they are expressed primarily in the fat body, the insect liver analog.
In fruit flies, immunity to infection by various microorganisms can be affected
by making different Toll-pathway genetic mutations. An immune specificity sys-
tem based on variations in the Toll gene and other PRRs appears to exist in the
fruit fly. Whether this same genetic variation exists in mice and humans is as yet
unknown. However, there are significant similarities in the signaling pathways used
by humans and flies to activate the innate immune response. In both cases, infection
leads to the activation of toll-like receptors (TLRs), which in turn initiate intracel-
lular signaling cascades that culminate in the activation of NF-κB-related transcrip-
tion factors.
Innate Immune System in Plants

Plants must survive complex environments in which they interact with a broad range
of microbial pathogens such as fungi, bacteria, and viruses with different infec-
tion strategies as well as herbivore insects and animals. The evolutionary arms
race between plants and their attackers provided plants with a highly sophisticated
defense system that, like the animal innate immune system, recognizes pathogen
molecules and responds by activating specific defenses that are directed against the
invader [19]. Recent advances in plant immunity research have provided exciting
new insights into the underlying defense-signaling network. Diverse small-molecule
hormones play pivotal roles in the regulation of this network. Their signaling path-
ways cross-communicate in an antagonistic or synergistic manner, providing the
plant with a powerful capacity to finely regulate its immune response.
Phytohormones are small molecules that are essential for the regulation of plant
growth, development, reproduction, and survival. They act as signal molecules and
occur in low concentrations. Classic phytohormones are abscisic acid (ABA), auxins,
cytokinins, ethylene (ET) and gibberellins, brassinosteroids, jasmonates (JAs), and
salicylic acid (SA). Changes in hormone concentration or sensitivity, which can be
triggered under stress conditions, mediate a whole range of adaptive plant responses.
The importance of SA, JAs, and ET as primary signals in the regulation of the plant’s
immune response is well established [20–24]. More recently, ABA [25,26], auxins
[27,28], gibberellins [29], cytokinins [30,31], and brassinosteroids [32] have also
been demonstrated to play important roles in innate immunity. The involvement of
so many plant-growth regulators in plant immunity suggests that the control of plant
growth, development, and defense is interconnected in a complex network of cross-
communicating hormone-signaling pathways. Therefore, the innate immune system
enables plants to utilize their resources in a cost-efficient manner by regulating the
amounts of substrates committed to cell formation. These defense responses may have
evolved to save energy under enemy-free conditions, as they only involve costs when
defenses are activated upon pathogen or insect attack. Trade-offs between plant-growth
rate and disease resistance are established by numerous studies and are consistent with
the idea that plant growth and defense are interconnected via common signaling path-
ways just as human nutritional status and immune function are interconnected.
Innate Immune System in Humans

The innate immune system consists of the cells and mechanisms that defend the
host from infection by other organisms, in a nonspecific manner. This means that
the cells of the innate system recognize and respond to pathogens in a generic way,
but unlike the adaptive immune system, it does not confer long-lasting or protective
immunity to the host. Innate immune systems provide immediate defense against
infection and are the dominant immune systems found in plants, fungi, insects, and
primitive multicellular organisms. It is thought to be an older evolutionary develop-
ment than the adaptive immune system. The innate immune system is activated by
danger signals and can be likened to firemen playing cards in the fire station until
the alarm goes off, and they all jump down the pole and onto fire trucks to put out
the fire. A fresh perspective is offered by approaching the immune system by the
premises laid down by Polly Matzinger in her danger model [33,34]. Even today,
more than a decade after its emergence, this model offers a fundamentally different
interpretation of classic and newly emerging concepts in immunology.
The innate immune system in humans carries out the following functions:
(a) recruitment of immune cells to sites of infection, through the production of
chemical factors, including specialized chemical mediators called cytokines and
chemokines; (b) activation of the complement cascade to identify bacteria, activate
cells, and promote clearance of dead cells or antibody complexes; (c) identification
and removal of foreign substances present in organs, tissues, blood, and lymph by
specialized white blood cells; and (d) activation of the adaptive immune system
through a process known as antigen presentation.
EVOLUTION OF CELLULAR IMMUNITY

It has been proposed that single-cell protozoa such as the amoeba may be the evo-
lutionary ancestors of the macrophage and other mobile cells of the immune system
[15]. Protozoa such as the amoeba need to discriminate between food and other
amoebas. If amoebas could not make this distinction they would consume them-
selves and the other members of their species, leading to extinction. Specific surface
receptors on amoebas discriminate between food to be engulfed and digested from

another amoeba. The nature of this hypothesized receptor is not yet established, but
it would have to be highly specific in order to discriminate self from nonself, the
most basic functions of immune function.
Macrophages, like protozoa, move at random unless exposed to a chemoattrac-
tant. Then, they all head in the same direction. In this respect also, protozoa behave
like macrophages and may have migrated into the cavities of early multicellular
organisms where they performed some symbiotic role. There are many areas of this
evolutionary hypothesis that remain unknown. For example, did the early macro-
phages lead to the evolution of lymphocytes or dendritic cells? The dendritic cells
are named for their long projections of cytoplasm resembling the dendritic processes
on nerve cells. Their one function is to seek out antigens and present them to the
subpopulation of T-lymphocytes as part of the adaptive immune response.
Immunity and Inflammation

The activation of the innate immune system is accompanied by inflammation (Latin,
inflammatio, a setting on fire), a complex biological response of vascular tissues to
harmful stimuli, such as pathogens, damaged cells, or irritants. The inflammatory
response is characterized by the following symptoms: redness (rubor), heat (calor),
swelling (tumor), and pain (dolor). Inflammation is a protective attempt by the organism
to remove the injuring stimulus as well as initiate the healing process for the tissue.
Inflammation can be classified as either acute or chronic. Acute inflammation is
the initial response of the body to harmful stimuli and is achieved by the increased
movement of plasma and leukocytes from the blood into the injured tissues. A cas-
cade of biochemical events propagates and matures the inflammatory response,
involving the local vascular system, the immune system, and various cells within
the injured tissue [35]. Prolonged inflammation, known as chronic inflammation,
leads to a progressive shift in the type of cells which are present at the site of inflam-
mation and is characterized by simultaneous destruction and healing of the tissue
from the inflammatory process. Chronic inflammation is associated with many
diseases including heart disease, diabetes, and common forms of cancer. So while
acute inflammation and healing is a life-saving adaptation analogous to the ability
to store energy as fat, prolonged inflammation damages critical tissues and organs in
the body. Obesity is associated with increased chronic inflammation while starvation
is associated with impaired immune function.
In the absence of inflammation, wounds and infections would never heal, and
progressive destruction of the tissue would compromise the survival of the organism
[36]. However, chronic inflammation can also lead to a host of diseases, such as hay-
fever, atherosclerosis, and rheumatoid arthritis. It is for that reason that inflammation
is normally closely regulated by the body.
Cellular Immunity
White blood cells (WBCs) are called leukocytes and are able to move freely through
the circulation, lymphatics, and tissues in order to capture cellular breakdown
products, foreign materials, or invading pathogens. Most leukocytes cannot divide or

reproduce on their own but are the products of stem cells found in the bone marrow
which develop into one or another type of white cell. Leukemia is the unregulated
overproduction of cells from one step of the process such as promyelocytic leukemia
where the promyelocyte crowds out the development of other white cells leading
to infection, other red cells leading to anemia, or platelets leading to bleeding. The
different leukocytes of the innate immune system include natural killer cells or NK
cells, mast cells, eosinophils, basophils, and the phagocytic cells including macro-
phages, neutrophils, and dendritic cells and function within the immune system by
identifying and eliminating pathogens that might cause infection.
When activated, mast cells rapidly release characteristic granules, rich in hista-
mine and heparin, along with various hormonal mediators and chemokines or che-
motactic cytokines into the environment. Histamine dilates blood vessels, causing
the characteristic signs of inflammation, and recruits neutrophils and macrophages.
Macrophages, neutrophils, and dendritic cells are called phagocytic cells because
they engulf and internalize foreign matter, cell debris, or pathogens.
Macrophages, from the Greek, meaning large eating cell, are large phagocytic
white blood cells that move outside the vascular system across the cell membranes
of capillary vessels and enter the areas between cells in response to danger signals
from invading pathogens, foreign materials, or dying cells. In tissues, organ-specific
macrophages are differentiated from phagocytic cells present in the blood called
monocytes. Macrophages are the most efficient phagocytes and can engulf and
digest substantial numbers of bacteria, cellular material, or microbes. The binding
of molecules to receptors on the surface of a macrophage triggers it to engulf and
destroy its meal through the generation of a respiratory burst, causing the release
of reactive oxygen species. The stimulated macrophage also produces chemokines,
proteins that summon other cells to the site of infection or injury.
Neutrophils, along with eosinophils and basophils, are known as granulocytes
due to the presence of granules in their cytoplasm. Polymorphonuclear cells (PMNs)
are white cells with distinctive lobed nuclei. Neutrophil granules contain a variety
of toxic substances that kill or inhibit growth of bacteria and fungi. Similar to mac-
rophages, neutrophils attack pathogens by activating a respiratory burst. The main
products of the neutrophil respiratory burst are strong oxidizing agents including
hydrogen peroxide, free oxygen radicals, and hypochlorite. Neutrophils are the most
abundant type of phagocyte, normally representing 50%–60% of the total circulating
leukocytes, and are usually the first cells to arrive at the site of an infection. The
bone marrow of a normal healthy adult produces more than 100 billion neutrophils
per day and more than 10 times that many per day during acute inflammation.
Basophils and eosinophils are similar to the neutrophil in containing granules.
When activated by a pathogen encounter, basophils release histamine, which is
important in the defense against parasites, and plays a role in allergic reactions (such
as asthma). Upon activation, eosinophils secrete a range of highly toxic proteins and
free radicals that are highly effective in killing bacteria and parasites, but are also
responsible for tissue damage occurring during allergic reactions. Activation and
toxin release by eosinophils are therefore tightly regulated to prevent any inappropri-
ate tissue destruction.
Natural killer cells, or NK cells, are a component of the innate immune system,
which does not directly attack invading microbes. Rather, NK cells destroy compro-
mised host cells, such as tumor cells or virus-infected cells, recognizing such cells
by a condition known as missing self. This term describes cells with low levels of
a cell-surface marker called MHC I (major histocompatibility complex)—a situa-
tion that can arise in viral infections of host cells. They were named natural killer
because of the initial notion that they do not require activation in order to kill cells
that are missing self.
Dendritic cells (DCs) are phagocytic cells present in tissues that are in con-
tact with the external environment, mainly the skin (where they are often called
Langerhans cells), and the inner mucosal lining of the nose, lungs, stomach, and
intestines. They are named for their resemblance to neuronal dendrites, but dendritic
cells are not connected to the nervous system. Dendritic cells are very important in
the process of antigen presentation and serve as a link between the innate and adap-
tive immune systems.
Dendritic cells are present in all tissues, where they gather antigens from the
local environment but are not in an immunostimulatory state. In Janeway’s stranger
model, antigen-presenting cells (later appreciated to be DCs) were endowed with
PRRs that recognize the unique features of microbial molecules (pathogen-associ-
ated molecular patterns, PAMPs). When PAMPs were present—for example, from
an infection or adjuvant—then DCs were stimulated to migrate to lymphoid tis-
sues and present both antigen and costimulatory molecules (CD80 and/or CD86)
to T-cells. In Matzinger’s danger model [33,34], the crucial event controlling the
initiation of an immune response was not infection, but the production of danger sig-
nals known as damage-associated molecular patterns (DAMPs) from cells stressed,
damaged, and/or dying in the local tissue. These were postulated to act on DCs in a
manner that also caused them to migrate to lymphoid tissue and present antigens to
T-cells in an immunostimulatory manner. It has been speculated that DAMPs might
be produced in response to PAMPs and therefore that DAMPs might be the final
mediator promoting immune responses in all situations, including infection. This
might occur; however, it is also possible, and in our view probable, that DAMPs and
PAMPs can alert the immune system to a problem independently and possibly even
in a synergistic manner.
ADAPTIVE IMMUNE SYSTEM

The adaptive immune response provides the immune system with the ability to recog-
nize and remember specific pathogens (to generate immunity) and to mount stronger
attacks each time the pathogen is encountered [37]. It is adaptive immunity because
the body’s immune system prepares itself for future challenges. It is believed to have
evolved at the time of the first vertebrates with jaws including various types of fish,
reptiles, and amphibians. One can speculate that eating led to potential repeated
exposure to toxic substances requiring a specific memory for antigens and the ability
to mount a more robust response to eliminate threatening substances.
This immune response system is highly adaptable because of a process of acceler-
ated somatic mutations and irreversible genetic recombinations of antigen-receptor
gene segments. This mechanism allows a small number of genes to generate a vast
number of different antigen receptors, which are then uniquely expressed on each
individual lymphocyte. Because the gene rearrangement leads to an irreversible
change in the DNA of each cell, all of the offspring of that cell will then inherit genes
encoding the same receptor specificity, including the memory B-cells and memory
T-cells that are the keys to long-lived specific immunity.
The host’s cells express self antigens. These antigens are different from those on
the surface of bacteria (non-self antigens) or on the surface of virally infected host
cells (missing-self). The adaptive response is triggered by recognizing nonself and
missing-self antigens.
With the exception of nonnucleated cells (including red blood cells), all cells are
capable of presenting antigens and of activating the adaptive response. Some cells
are specially equipped to present antigens and to prime naive T-cells. Dendritic cells
and B-cells (and to a lesser extent macrophages) are equipped with special immu-
nostimulatory receptors that allow for enhanced activation of T-cells and are termed
professional antigen-presenting cells (APCs). Several T-cell subgroups can be acti-
vated by professional APCs, and each type of T-cell is specially equipped to deal
with each unique toxin or bacterial and viral pathogen. The type of T-cell activated
and the type of response generated depend, in part, on the context in which the APC
first encountered the antigen.
MALNUTRITION AND IMMUNE FUNCTION

Nutrient deficiencies can impair immune function, and nutrient supplementation can
restore normal immune capacity [38]. The association of malnutrition and infection
has been recorded in ancient historical accounts. For example, an examination of
church records in England in the twelfth century shows an interesting association
between consecutive years of famine and epidemics of pestilence or communicable
diseases. Recent epidemiological studies in the Americas and Asia have confirmed
that infection, often added on malnutrition, is a major cause of morbidity and is
responsible for about two-thirds of all deaths among children under five years of
age. In 1968, Scrimshaw summarized the human and animal data on interactions,
often synergistic but occasionally antagonistic, between nutritional deficiencies and
infectious illness [12].
Careful observations showed a correlation between nutritional status and morbid-
ity and mortality largely due to infections [13]. It was shown that the risk of death
increased from ∼0.1% in the well-nourished to as much as 18% in severely mal-
nourished infants. The number of episodes of diarrhea increased by 40%, and the
duration of each episode increased by more than twofold. The effect of malnutrition
on different infections is variable. For some organisms, for example, measles, tuber-
culosis, and Pneumocystis carinii, there is little doubt that nutritional deficiencies
enhance susceptibility and worsen prognosis. For others, such as yellow fever and
poliomyelitis, nutrition does not appear to have a major influence on natural history
and outcome.
Protein-energy malnutrition causes widespread atrophy of lymphoid tissues, espe-
cially in children. The thymus, spleen, tonsils, and lymph nodes are all affected,
with histological evidence of atrophy being greatest in the T-lymphocyte areas of

these tissues. Lymphocytes and eosinophils show lowered blood counts. NK cells
show reduced activity [39]. Cultured blood lymphocytes react poorly to mitogens.
Production of thymic hormones is reduced, as is a patient’s ability to fend off and
recover from infectious illnesses. These closely linked events can initiate a downhill
spiral or a vicious cycle that leads inexorably to death. Protein energy malnutrition
(PEM) causes a marked repression of cell-mediated immunity and the function of
T-lymphocytes. Malnourished children show dermal anergy, with loss of delayed
dermal hypersensitivity (DDH) reactions, a decrease or reversal of the T-helper/
suppressor-cell ratio, and loss of the ability of killer lymphocytes to recognize and
destroy foreign tissues. In contrast, B-lymphocyte numbers and functions gener-
ally appear to be maintained. While existing antibody production is conserved or
even increased during generalized malnutrition, new primary antibody responses to
T-cell-dependent antigens and antibody affinity are impaired. Research conducted in
military personnel has shown immune dysregulation caused by stress that is similar
to the immune dysregulation noted in the elderly (e.g., anergy and decreased prolif-
erative response) [40].
IMMUNE FUNCTION IN OBESITY

As abdominal fat expands in response to positive energy balance, there is a need for
new blood- vessel formation. However, these new vessels can often not keep up with
the metabolic needs of expanding abdominal fat, leading to the death of adipocytes.
As these adipocytes die, they release cellular debris, which results in the activation
of macrophages which release cytokines resulting in insulin resistance in fat cells as
well as a systemic inflammation. The macrophages collect in circular collections and
engulf the fat released from dead adipose cells in both abdominal fat and subcutane-
ous fat tissue [14].
This systemic inflammation leads to an increase in innate immune function and
defects in adaptive immunity. These defects have been identified in mice fed high-fat
diets to induce obesity. Mice fed a 70% fat diet develop obesity and specific defects
in the ability of dendritic cells to present antigens to the immune system leading to
defects in adaptive immunity [41].
There are a number of chronic diseases which are impacted by the enhanced
innate immune function and inflammation. Since inflammation is a common mecha-
nism across many different chronic diseases of aging, obesity-associated changes
in immune function are critical in mediating the obesity-associated increased risks
of heart disease, diabetes, common forms of cancer, asthma, and connective tissue
diseases.
MACROPHAGE RECEPTORS FOR OMEGA-3 FATTY ACIDS

As noted earlier, chronic activation of inflammatory pathways plays an important
role in the pathogenesis of insulin resistance, and the macrophage/adipocyte nexus
provides a key mechanism underlying many common chronic diseases associated
with excess body fat [42]. Migration of macrophages to adipose tissue (including
intramuscular fat depots) and liver with subsequent activation of macrophage proin-
flammatory pathways and cytokine secretion is the critical link between overnutri-
tion and inflammation.
Omega-3 fatty acids (ω-3 FAs), DHA and EPA, exert anti-inflammatory effects,
but the mechanisms are poorly understood. Recently, it was discovered that the
G protein–coupled receptor 120 (GPR120) functions as an ω-3 FA receptor/sensor
[43]. Stimulation of GPR120 with ω-3 FAs or a chemical agonist caused broad anti-
inflammatory effects in monocytes (RAW 264.7 cells) and in macrophages obtained
from the intraperitoneal fluid. All of these effects were abrogated by GPR120 knock-
down, demonstrating that the GPR120 membrane protein functions as an ω-3 FA
receptor/sensor in proinflammatory macrophages and mature adipocytes. Moreover,
GPR120 is highly expressed in proinflammatory macrophages and functions as an
ω-3 FA receptor, mediating the anti-inflammatory effects of this class of FAs to
inhibit both the TLR2/3/4 and the TNF-α response pathways and cause systemic
insulin sensitization. Therefore, the in vivo anti-inflammatory and insulin-sensitizing
effects of ω-3 FAs are dependent on expression of GPR120, as demonstrated in stud-
ies of obese GPR120 KO animals and WT littermates.
The worldwide diversity of dietary intakes of n-6 and n-3 FAs influences tissue
compositions of n-3 long-chain FAs (LCFAs: eicosapentaenoic, docosapentaenoic,
and docosahexaenoic acids) and risks of cardiovascular and mental illnesses [44] via
inflammatory mechanisms mediated by eicosanoids synthesized from arachidonic acid
and other long-chain omega-6 FAs. By increasing ω-3 FA intake from fish and fish oil
or algae oil supplements and decreasing ω-6 FA consumption from vegetable oils and
processed foods, it is possible to change tissue and plasma FA balance. More research
is needed to connect these changes to changes in immune function, but there is evi-
dence from epidemiological studies [45] that increases in the ratio of ω-6: ω-3 poly-
unsaturated fatty acid (PUFA) are associated with increases in chronic inflammatory
diseases such as nonalcoholic fatty liver disease (NAFLD), cardiovascular disease,
obesity, inflammatory bowel disease (IBD), rheumatoid arthritis, and Alzheimer’s dis-
ease (AD). By decreasing the ratio of ω-6:ω-3 PUFA in the Western diet, reductions
may be achieved in the incidence of these chronic inflammatory diseases.
IMMUNE FUNCTION AND VITAMIN AND MINERAL BALANCE

Vitamin A deficiency has long been known to be associated with increased suscep-
tibility to viral infections such as mumps. While it has long been recognized that
vitamin A and its metabolites have immune-regulatory roles, the mechanisms of
action have not been known. Recently, there has been a significant progress in elu-
cidating the functions of retinoic acid in the regulation of immune cell development
[46]. Retinoic acid (all-trans and 9-cis retinoic acid) is produced from the cells of the
intestine such as dendritic cells and provides an intestine-specific environmental cue
to differentiating immune cells. When T-cells and B-cells are activated in the intes-
tine and associated lymphoid tissues, gut-homing receptors are induced on the cells
in a retinoic acid and antigen-dependent manner. Retinoic acid, produced by gut den-
dritic cells, is also an important signal that induces IgA-producing B-cells. The gut-
homing T-cells and B-cells play essential roles in protecting the digestive tract from
pathogens. Retinoic acid is required also for production of mature phagocytes in

bone marrow. On the other hand, retinoic acid induces a subset of FoxP3+regulatory
T-cells, which is important for maintaining immune tolerance in the gut. Therefore,
retinoids provide both positive and negative regulatory signals to fine-control the
mucosal immune system.
Although the best-known actions of vitamin D involve its regulation of bone
mineral homeostasis, vitamin D exerts its influence on many physiological processes.
One of these processes is the immune system. Both the adaptive and innate immune
systems are impacted by the active metabolite of vitamin D, 1,25(OH)[2]D. These
observations have been proposed as potential mechanisms mediating the predisposi-
tion of individuals with vitamin D deficiency to infectious diseases such as tubercu-
losis, as well as to autoimmune diseases such as type 1 diabetes mellitus and multiple
sclerosis [47].
Selenium (Se) is an essential trace element needed for the biosynthesis of a
small number of mammalian selenoproteins. Se intake and personal Se status are
implicated in widespread human pathologies including cancer, cardiovascular dis-
ease, and neurodegeneration [48]. Positive effects of Se supplementation have been
observed in a number of clinical trials with patients suffering from sepsis, HIV
infection, or autoimmune thyroid disease [49]. Most importantly, the lower the Se
status during critical illness, the more likely that the patients will not survive [50].
Supplementation with trace elements in some individuals with nutritional deficiency
to correct a selenium deficiency enhanced antibody titers to influenza vaccination
and showed a trend toward fewer subjects with respiratory tract infections. Another
study reported better immune responsiveness and fewer infection-related illnesses
with a multivitamin supplement than with a placebo in the apparently healthy, inde-
pendent-living elderly [51]. Other researchers have supplemented the diets of seniors
with poor eating habits and found no immunological benefit or reduction in acute
respiratory tract infections. The reason for the variability of results is likely related
to the adaptation to starvation which can maintain immune function until late in the
process as long as multivitamin and mineral intake are normal. Some, but not all,
clinical trials have proven effective in improving the outcome of critically ill patients
by Se supplementation, but the best application regimen, most suitable Se compound,
and the mechanisms of action are not yet established.
A randomized, double-blind, placebo-controlled trial was carried out to investi-
gate the effects of micronutrient supplementation on immunity and the incidence of
common infections in type 2 diabetic outpatients [52]. A total of 196 type 2 diabetic
outpatients were randomized to receive tablets of micronutrients (n = 97) or placebo
(n = 99) for six months. Individualized dietary energy intake and daily physical activ-
ity were recommended. Anthropometric measurements, blood biochemical variables,
and the incidence of common infections were measured at baseline and at 6 months.
Data on diet, exercise, and infection (upper respiratory tract infection, skin infection,
urinary and genital tract infections, other infections) were recorded 1 month before
the study and every month during the study. Blood concentrations of total protein,
iron (Fe), folic acid, and hemoglobin increased, and unsaturated iron-binding capac-
ity (UIBC) levels decreased in the micronutrient supplementation group compared to
the placebo group at six months. Moreover, at six months, compared to the placebo
group, the blood concentrations of IgE, CD4+, CD4+/CD8+, WBC, lymphocyte

counts, basophilic leukocyte increased, and CD8+ count decreased in the supple-
mentation group, and the levels of IgA, IgM, IgG, and complements C3 and C4
did not differ. The incidence of upper respiratory infection, vaginitis, urinary tract
infection, gingivitis, and dental ulcer were lower, and body temperature and duration
of fever greatly improved in the supplementation over the placebo group. These data
suggest that supplementation of micronutrients might increase immune function and
reduce the incidence of common infections in type 2 diabetic outpatients.
In addition, deficiencies of vitamins B6 and folate are associated with reduced
immunocompetence [51]. Trace elements modulate immune responses through
their critical role in enzyme activity. Although dietary requirements of most of
these elements are met by a balanced diet, there are certain population groups
and specific disease states which are likely to be associated with deficiency of
one or more of these essential elements. The role of trace elements in mainte-
nance of immune function and their causal role in secondary immunodeficiency
are increasingly being recognized. There is growing research concerning the role
of zinc, copper, selenium, and other elements in immunity and the mechanisms
that underlie such roles. The problem of interaction of trace elements and immu-
nity is a complex one because of the frequently associated other nutritional
deficiencies, the presence of clinical or subclinical infections, which in themselves
have a significant effect on immunity, and finally the altered metabolism due to the
underlying disease [51].
PRACTICAL CONSIDERATIONS FOR

MODULATING IMMUNE FUNCTION
The practical applications of the interaction of immune function and nutrition are
important in dealing with both malnourished patients and the overweight or obese
individuals at risk or suffering from age-related chronic diseases.
In overweight and obese individuals, it is important to achieve and maintain opti-
mal intra-abdominal fat depots that do not trigger chronic inflammation systemi-
cally. The growing global epidemic of obesity and type 2 diabetes associated with
the adoption of Western diets and lifestyles has significant implications for immune
function but in an opposite direction to that associated with malnutrition. Activation
of innate immunity and inhibition of adaptive immune function may occur with
overnutrition.
Recent evidence has suggested that changes in the microbial flora of the intestines
can also modulate immune function through changes that occur as the result of high-
fat/high-sugar diets. The effects of prebiotic and probiotic interventions to modulate
bacterial populations in the gastrointestinal tract and the impact of fruits, vegetables,
nuts, and grains on microbial populations and secondary metabolites of phytochemi-
cals are active areas of ongoing research.
It is clear that malnutrition, especially protein-energy malnutrition associated
with kwashiorkor-like findings in children in the developed world or in hospitalized
patients, impairs immune function, especially delayed hypersensitivity. Therefore,
recognizing and treating malnutrition is a critical component of maintaining normal
immune function in hospitalized patients, the elderly, and military personnel work-
ing under stressed conditions.
Finally, in addition to calorie and protein balance, micronutrients and lipid bal-
ance of omega-3 and omega-6 are critical. These needs can be met through a balanced
diet, but in at-risk groups including the elderly, individuals consuming unbalanced
diets, and military personnel under stress, it may be advisable to include a multi-
vitamin/multimineral dietary supplement to support healthy immune function. For
balancing omega-3 and omega-6 FAs in cells, it is important to both increase the
intake of DHA and EPA from fish or supplements while also reducing the intake of
omega-6 FAs from foods containing vegetable oils.
REFERENCES
1. Hoffmann JA, Kafatos FC, Janeway CA, Ezekowitz RA. 1999. Phylogenetic perspec-
tives in innate immunity. Science 284:1313–1318.
2. Nakamoto M, Moy RH, Xu J, Bambina S, Yasunaga A, Shelly SS et al. 2012. Virus rec-
ognition by Toll-7 activates antiviral autophagy in Drosophila. Immunity 36:658–667.
3. Leulier F, Lemaitre B. 2008. Toll-like receptors—Taking an evolutionary approach. Nat
Rev Genet 9:165–178.
4. Oltz EM. 2001. Regulation of antigen receptor gene assembly in lymphocytes. Immunol
Res 23:121–133.
5. Purchiaroni F, Tortora A, Gabrielli M, Bertucci F, Gigante G, Ianiro G et al. 2013. The
role of intestinal microbiota and the immune system. Eur Rev Med Pharmacol Sci
17:323–333.
6. Maekawa T, Kracher B, Vernaldi S, Ver Loren van Themaat E, Schulze-Lefert P. 2012.
Conservation of NLR-triggered immunity across plant lineages. Proc Natl Acad Sci
USA 109:20119–20123.
7. Montague CT, Farooqi IS, Whitehead JP, Soos MA, Rau H, Wareham NJ et al. 1997.
Congenital leptin deficiency is associated with severe early-onset obesity in humans.
Nature 387:903–908.
8. Amirkalali B, Sharifi F, Fakhrzadeh H, Mirarefein M, Ghaderpanahi M, Badamchizadeh
Z et al. 2010. Low serum leptin serves as a biomarker of malnutrition in elderly patients.
Nutr Res 30:314–319.
9. Faggioni, R, Feingold KR, Grunfeld C. 2001. Leptin regulation of the immune response
and the immunodeficiency of malnutrition. FASEB J 15:2565–2571.
10. Thea DM, Porat R, Khondi N, Matela B, St. Louis ME, Kaplan G et al. 1996.
Relationship of cytokine and cytokine antagonist plasma levels to disease progression in
African women with HIV-1 infection. Ann Int Med 124:757–762.
11. Scrimshaw NS, Taylor CE, Gordon JE. 1959. Interactions of nutrition and infection. Am
J Med Sci 237:367–372.
12. Scrimshaw NS, Taylor CE, Gordon JE. 1968. Interactions of nutrition and infection.
Monogr Ser World Health Organ 57:3–329.
13. O’Neill SM, Fitzgerald A, Briend A, Van den Broeck J. 2012. Child mortality as pre-
dicted by nutritional status and recent weight velocity in children under two in rural
Africa. J Nutr 142:520–525.
14. Lê KA, Mahurkar S, Alderete TL, Hasson RE, Adam TC, Kim JS et al. 2011.
Subcutaneous adipose tissue macrophage infiltration is associated with hepatic and
visceral fat deposition, hyperinsulinemia, and stimulation of NF-κB stress pathway.
Diabetes 60:2802–2809.
15. Janeway CA Jr. 1988. Frontiers of the immune system. Nature 333:804–806.
16. Lemaitre B, Reichhart JM, Hoffmann JA. 1997. Drosophila host defense: Differential
induction of antimicrobial peptide genes after infection by various classes of microor-
ganisms. Proc Natl Acad Sci USA 94:14614–14619.
17. Bulet P, Hetru C, Dimarcq JL, Hoffmann D. 1999. Antimicrobial peptides in insects;
structure and function. Dev Comp Immunol 23:329–344.
18. Hoffmann JA, Reichhart JM. 2002. Drosophila innate immunity: An evolutionary per-
spective. Nat Immunol 3:121–126.
19. Buchanan BB, Gruissem W, Jones RL. 2000. Biochemistry & Molecular Biology of
Plants, pp. 1152–1154. American Society of Plant Physiologists, Rockville, MD.
20. Pozo MJ, Van Loon LC, Pieterse CMJ. 2004. Jasmonates—Signals in plant-microbe
interactions. J Plant Growth Regul 23:211–222.
21. Van Loon LC, Geraats BPJ, Linthorst HJM. 2006. Ethylene as a modulator of disease
resistance in plants. Trends Plant Sci 11:184–191.
22. Loake G, Grant M. 2007. Salicylic acid in plant defence—The players and protagonists.
Curr Opin Plant Biol 10:466–472.
23. Howe GA. 2004. Jasmonates as signals in the wound response. J Plant Growth Regul
23:223–237.
24. Von Dahl CC, Baldwin IT. 2007. Deciphering the role of ethylene in plant-herbivore
interactions. J Plant Growth Regul 26:201–209.
25. Asselbergh B, De Vleesschauwer D, Höfte M. 2008. Global switches and fine-tuning—
ABA modulates plant pathogen defense. Mol Plant Microbe Interact 21:709–719.
26. Mauch-Mani B, Mauch F. 2005. The role of abscisic acid in plant-pathogen interactions.
Curr Opin Plant Biol 8:409–414.
27. Navarro L, Dunoyer P, Jay F, Arnold B, Dharmasiri N, Estelle M et al. 2006. A plant
miRNA contributes to antibacterial resistance by repressing auxin signaling. Science
312:436–439.
28. Wang D, Pajerowska-Mukhtar K, Hendrickson Culler A, Dong X. 2007. Salicylic acid
inhibits pathogen growth in plants through repression of the auxin signaling pathway.
Curr Biol 17:1784–1790.
29. Navarro L, Bari R, Achard P, Lison P, Nemri A, Harberd NP et al. 2008. DELLAs control
plant immune responses by modulating the balance of jasmonic acid and salicylic acid
signaling. Curr Biol 18:650–655.
30. Walters DR, McRoberts N. 2006. Plants and biotrophs: A pivotal role for cytokinins?
Trends Plant Sci 11:581–586.
31. Siemens J, Keller I, Sarx J, Kunz S, Schuller A, Nagel W et al. 2006. Transcriptome
analysis of Arabidopsis clubroots indicate a key role for cytokinins in disease develop-
ment. Mol Plant Microbe Interact 19:480–494.
32. Nakashita H, Yasuda M, Nitta T, Asami T, Fujioka S, Arai Y et al. 2003. Brassino
steroid functions in a broad range of disease resistance in tobacco and rice. Plant J
33:887–898.
33. Matzinger P. 2002. An innate sense of danger. Ann N Y Acad Sci 961:341–342.
34. Matzinger P. 2002. The danger model: A renewed sense of self. Science 296:301–305.
35. Ingersoll MA, Platt AM, Potteaux S, Randolph GJ. 2011. Monocyte trafficking in acute
and chronic inflammation. Trends Immunol 32:470–477.
36. Serhan CN. 2007. Resolution phase of inflammation: Novel endogenous anti-
inflammatory and proresolving lipid mediators and pathways. Annu Rev Immunol
25:101–137.
37. Medzhitov R, Janeway CA Jr. 1999. Innate immune induction of the adaptive immune
response. Cold Spring Harb Symp Quant Biol 64:429–435.
38. Bhaskaram P. 2002. Micronutrient malnutrition, infection, and immunity: An overview.
Nutr Rev 60:S40–S45.
39. Salimonu LS. 1992. Acute phase proteins in “small for dates” babies. II. Haptoglobin,
transferrin, alpha-1-feto protein, alpha-1-acid glycoprotein and caeruloplasmin levels.
Afr J Med Med Sci 21:55–59.
40. Castell LM, Thake CD, Ensign W. 2010. Biochemical markers of possible immunode-
pression in military training in harsh environments. Mil Med 175:158–165.
41. Smith AG, Sheridan PA, Harp JB, Beck MA. 2007. Diet-induced obese mice have
increased mortality and altered immune responses when infected with influenza virus.
J Nutr 137:1236–1243.
42. Schenk S, Saberi M, Olefsky JM. 2008. Insulin sensitivity: Modulation by nutrients and
inflammation. J Clin Invest 118:2992–3002.
43. Oh DY, Talukdar S, Bae EJ, Imamura T, Morinaga H, Fan W et al. 2010. GPR120 is an
omega-3 fatty acid receptor mediating potent anti-inflammatory and insulin-sensitizing
effects. Cell 142:687–698.
44. Hibbeln JR, Nieminen LR, Blasbalg TL, Riggs JA, Lands WE. 2006. Healthy intakes
of n-3 and n-6 fatty acids: Estimations considering worldwide diversity. Am J Clin Nutr
83:1483S–1493S.
45. Patterson E, Wall R, Fitzgerald GF, Ross RP, Stanton C. 2012. Health implications of
high dietary omega-6 polyunsaturated fatty acids. J Nutr Metab 2012:539426.
46. Kim CH. 2008. Roles of retinoic acid in induction of immunity and immune tolerance.
Endocr Metab Immune Disord Drug Targets 8:289–294.
47. Bikle DD. 2011. Vitamin D regulation of immune function. Vitam Horm 86:1–21.
48. Papp LV, Lu J, Holmgren A, Khanna KK. 2007. From selenium to selenoproteins:
Synthesis, identity, and their role in human health. Antioxid Redox Signal 9:775–806.
49. Broome CS, McArdle F, Kyle JA, Andrews F, Lowe NM, Hart CA et al. 2004. An
increase in selenium intake improves immune function and poliovirus handling in adults
with marginal selenium status. Am J Clin Nutr 80:154–162.
50. Manzanares W, Biestro A, Galusso F, Torre MH, Manay N, Pittini G et al. 2009. Serum
selenium and glutathione peroxidase-3 activity: Biomarkers of systemic inflammation
in the critically ill? Intensive Care Med 35:882–889.
51. Chandra S, Chandra RK. 1986. Nutrition, immune response, and outcome. Prog Food
Nutr Sci 10:1–65.
52. Liu Y, Jing H, Wang J, Zhang R, Zhang Y, Zhang Y et al. 2011. Micronutrients decrease
incidence of common infections in type 2 diabetic outpatients. Asia Pac J Clin Nutr
20:375–382.
53. Janeway CA Jr, Travers P, Walport M, Shlomchik MJ. 2005. Immunobiology: The
Immune System in Health and Disease, 6th edn., pp. 52–53. Garland Science, New York.
2 Cellular Mechanisms of
Cytokine Activation
CONTENTS
Introduction............................................................................................................... 19
Transcription Factors and Mitogen-Activated Kinase Pathways..............................20
Nuclear Factor-Kappa B.......................................................................................20
Nuclear Factor of Activated T-Cells..................................................................... 21
Activating Protein-1............................................................................................. 21
Extracellular Signal–Regulated Kinases.............................................................. 22
c-Jun N-Terminal Kinases.................................................................................... 22
p38........................................................................................................................ 22
Reactive Oxygen Species and Immune Function..................................................... 23
Lipid Peroxidation Products.....................................................................................25
Cytokine Activation Pathways..................................................................................25
Inflammasome, Cytokines, and Pyroptosis............................................................... 30
Conclusion................................................................................................................ 31
References................................................................................................................. 31
INTRODUCTION
Inflammation begins with the activation and recruitment of immune effector cells
and the secretion of cytokines, which are essential for the host defense system. In
this chapter, the cellular mechanisms of cytokine activation will be reviewed. As dis-
cussed further in later chapters, chronic inflammation persists even after elimination
of pathogen(s). Chronic low-grade inflammation has been associated with cancer
[1,2], inflammatory bowel disease, ulcerative colitis [3,4], atherosclerosis, rheuma-
toid arthritis [5], asthma, and Alzheimer’s disease [6]. The damaging responses sec-
ondary to chronic inflammation in different organ systems reviewed throughout this
chapter are mediated by cytokines activated through similar molecular mechanisms
within the immune system, in adipocytes, and in other specialized cells. Cytokine
regulation and dysregulation play a significant role in the pathogenesis of various
chronic inflammatory diseases [7,8], and understanding the molecular basis of cyto-
kine activation is critical to knowing about the role of nutrients and phytochemicals
in immune function presented throughout this chapter.
19
TRANSCRIPTION FACTORS AND MITOGEN-ACTIVATED

KINASE PATHWAYS
Transcription factors (NF-κB, NF-AT, and AP1) along with mitogen-activated pro-
tein (MAP) kinases (ERK, JNK, and p38) are all known to regulate these inflam-
matory cytokines and enzymes and have been studied in various models of chronic
inflammation [9–15].
Cellular behavior in response to extracellular stimuli is mediated through intra-
cellular signaling pathways such as the MAP kinase pathways. MAP kinases are
members of discrete signaling cascades and serve as focal points in response to
a variety of extracellular stimuli. Four distinct subgroups within the MAP kinase
family have been described: (a) extracellular signal–regulated kinases (ERKs),
(b) c-jun N-terminal or stress-activated protein kinases (JNK/SAPK), (c) ERK/big
MAP kinase 1 (BMK1), and (d) the p38 group of protein kinases. There are also
several reports showing the involvement of reactive oxygen species (ROS) and gluta-
thione (GSH) in modulating these immunologically important kinases and transcrip-
tion factors and resulting in altered immune responses [16,17]. The interactions and
actions of these cytokines in response to internal and external signaling will be the
focus of this chapter.
Nuclear Factor-K appa B

The transcription factor nuclear factor-kappa B (NF-κB) is crucial in a series of cel-
lular processes including inflammation, immunity, cell proliferation, and apoptosis.
It includes a group of five proteins, namely NF-κB1 (p50 and its precursor p105),
NFκ-B2 (p52 and its precursor p100), p65/RelA, c-Rel, and RelB [18]. In the resting
state, NF-κB is sequestered in the cytoplasm of the cell through its tight association
with inhibitory proteins called IκBs, comprising IκBa, IκBb, IκBg, IκBe, Bcl-3, p100,
and p105. Upon cell stimulation, cytokine activation occurs when the IκB proteins
are rapidly phosphorylated and degraded by the proteasome, and the freed NF-κB
translocates into the nucleus to regulate the expression of multiple target genes [18].
The activation of NF-κB in various immune cells, including T cells, B cells, mac-
rophages, dendritic cells, and neutrophils, leads to expression of proinflammatory
cytokines. Although M1-type macrophages, activated by IFN-γ, promote the adap-
tive immune response through the secretion of proinflammatory cytokines, M2-type
macrophages activated by IL-4 and IL-13 have been linked to anti-inflammatory
signaling and wound healing. NF-κB thus plays an important role in the immune
response regardless of the specific macrophage type.
Among all the transcription factors discussed in this chapter, no transcription
factor has been examined more extensively than NF-κB. NF-κB controls the expres-
sion of more than 500 different gene products that have been closely linked to
inflammation, cellular transformation, tumor-cell survival, proliferation, invasion,
angiogenesis, and metastasis [19]. In addition, this transcription factor is activated
in response to a wide variety of stimuli that are shown to be lifestyle risk factors,
such as stress (physical, psychological, mechanical, or chemical), tobacco, radiation,
Cellular Mechanisms of Cytokine Activation 21
asbestos, dietary agents, environmental pollutants, obesity, and various infectious

agents closely linked to cancer.
Nuclear Factor of Activated T-Cells

Nuclear factor of activated T-cells (NFAT) is a general name applied to a family of
transcription factors shown to be important in immune function. One or more mem-
bers of the NFAT family are expressed in most cells of the immune system. NFAT
is also involved in the development of cardiac, skeletal muscle, and nervous systems.
The NFAT transcription factor family consists of five members: NFATc1, NFATc2,
NFATc3, NFATc4, and NFAT5 [20]. NFATc1 through NFATc4 are regulated by cal-
cium signaling. Calcium signaling is critical to NFAT activation because calmodulin
(CaM), a well-known calcium sensor protein, activates the serine/threonine phospha-
tase calcineurin (CN). Activated CN rapidly dephosphorylates the serine-rich region
(SRR) and SP-repeats in the amino termini of NFAT proteins, resulting in a confor-
mational change that exposes a nuclear localization signal, which in turn results in
NFAT nuclear import. Nuclear import of NFAT proteins is opposed by maintenance
kinases in the cytoplasm and export kinases in the nucleus. Export kinases, such
as PKA and GSK-3β, must be inactivated for NFAT nuclear retention. NFAT pro-
teins have weak DNA-binding capacity. Therefore, to effectively bind DNA, NFAT
proteins must cooperate with other nuclear resident transcription factors generically
referred to as NFATn [21]. This important feature of NFAT transcription factors
enables integration and coincidence detection of calcium signals with other signal-
ing pathways such as Ras-MAPK or PKC. In addition, this signaling integration is
involved in tissue-specific gene expression during development.
Activating Protein-1
AP-1 is not a single factor but rather refers to a group of transcription factors including
Jun, Fos, or ATF (activating transcription factor) subunits that form dimers and bind
to a common DNA site, the AP-1-binding site [22]. This common site was first iden-
tified by its role in human metallothionein IIA gene regulation [23]. Following its
discovery, AP-1 activity was found to be induced by many stimuli, including growth
factors, cytokines, T-cell activators, neurotransmitters, and UV irradiation [22].
The signaling pathways leading to NF-κB and AP-1 activation are overlapping,
where both are involved in the induction and regulation of cytokines/chemokines.
NF-κB is activated in response to stress, such as oxidative stress, bacterial toxins,
viruses, and UV light [24], and is essential for differentiation, proliferation, and sur-
vival of many cell types including T-lymphocytes [25]. AP-1 activation requires Fos
(c-Fos, FosB, Fra-1, Fra-2) and Jun (c-Jun, v-Jun, JunB, JunD) through the formation
of homo- and heterodimers [26,27] and regulates transcription of a broad range of
genes involved in immune responses [28–31]. Both AP-1 and NF-κB binding sites
have been identified in the promoter region of IL-6 and CXCL8 [32]; however, the
mechanism by which these interleukins are regulated in T-cells is still not clear.
CXCL8 is a C-X-C chemokine with properties enabling it to recruit T-cells and
basophils and to activate neutrophils and monocytes [33]. IL-6 is a cytokine that
possesses both pro- and anti-inflammatory characteristics and that plays a key role
in hematopoiesis and acute-phase responses [34,35].
Extracellular Signal–Regulated Kinases

Two similar protein kinases with 85% homology are extracellular signal–regulated
kinases called ERK1 and ERK2 [36]. To further complicate the nomenclature in the
literature, mitogen-activated protein kinase 3 (MAPK3) is also known as ERK1, and
MAPK1 is also known as ERK2. The ERKs were found during a search for protein
kinases that are rapidly phosphorylated after activation of tyrosine kinases in the
cell membrane by growth factors such as epidermal growth factor and many other
extracellular protein growth factors and hormones following binding to their specific
membrane receptors. Phosphorylation of ERKs activates them to be kinases so that
further phosphorylation of intracellular proteins can carry out intracellular signaling
leading to induction of cytokine release. The molecular events linking membrane
receptors to activation of ERKs are complex. GTP-binding proteins are involved in
the activation of ERKs [37]. Raf-1 is a protein kinase that phosphorylates a MAPK
kinase, thus making this a MAPK kinase kinase [38]. The name for this MAPK
kinase is MAPK/ERK kinase, or MEK [39].
Other receptor-linked tyrosine kinases Raf, MEK, and MAPK are part of a
signaling cascade called the MAPK/ERK pathway. There are over 5000 different
phosphate-related proteins in the cell pointing out the importance of phosphoryla-
tion and dephosphorylation in cell biology. Transgenic mice lacking MAPK1 have
major defects in development [40]. Transgenic mice lacking ERK1 are viable, and it
is thought that ERK2 can fulfill most ERK1 functions in most cells [41]. The main
exception is T lymphocytes. Mice lacking ERK 1 have reduced T-cell development
past the CD4+CD8+ stage.
c-Jun N-Terminal Kinases

c-Jun N-terminal kinases were originally identified due to their phosphorylation of the
oncogene c-Jun [42]. The site of the phosphorylation is with the transcriptional activa-
tion domain of the protein. JNKs belong to the MAP kinase family and are activated
in response to stress stimuli including inflammatory signaling through cytokines, UV
radiation, heat shock, and osmotic shock. JNKs also play a role in the differentiation
of T lymphocytes. The activation of JNK is carried out by two MAP kinases (MKK4
and MKK7), and JNK is inactivated by specific phosphatases. This signaling pathway
has been implicated in inflammatory responses in mammals and insects.
p38
p38 gets its name from the fact that it was first isolated as a 38 kDa protein rap-
idly tyrosine-phosphorylated in response to lipopolysaccharide stimulation [43,44].
Four splice variants of the p38 family have been identified: p38α, p38β [45], p38γ
(ERK6, SAPK3) [46,47], and p38δ (SAPK4) [48,49]. Of these, p38 and p38β are
ubiquitously expressed while p3γ8 and p38δ are differentially expressed depending
on tissue type. Sequence comparisons have revealed that each p38 isoform shares
∼60% identity within the p38 group but only 40%–45% to the other three MAP
kinase family members.
In common with other MAP kinases, p38 kinases are activated by dual kinases
termed the MAP kinase kinases (MKKs). There are two main MAPKKs that are
known to activate p38, MKK3 and MKK6. Also, it has been shown that MKK4,
an upstream kinase of JNK, can aid in the activation of p38α and p38δ in specific
cell types [48]. These data suggest, then, that activation of p38 isoforms can be spe-
cifically controlled through different regulators and coactivated by various combi-
nations of upstream regulators. The cellular specificity and specific activation and
inactivation of MAP kinases described earlier provide a flexible and responsive sys-
tem that can be activated in response to specific inflammatory stimuli and inacti-
vated. The activation of the p38 pathway plays essential roles in the production of
proinflammatory cytokines (IL-1 β, tumor necrosis factor alpha (TNF-α), and IL-6)
[50]; induction of enzymes such as COX-2, which controls connective-tissue remod-
eling in pathological conditions [51]; expression of intracellular enzymes such as
iNOS, a regulator of oxidation [52,53]; and induction of VCAM-1 and other adherent
proteins along with other inflammatory-related molecules [54]. In addition, a regula-
tory role for p38 in the proliferation and differentiation of immune system cells such
as GM-CSF, EPO, CSF, and CD-40 has been established [55,56]. A strong link has
been established between the p38 pathway and inflammation.
REACTIVE OXYGEN SPECIES AND IMMUNE FUNCTION

The air we breathe is about 20% oxygen, and life for humans and other mammals is
dependent on the utilization of this oxygen for the production of high-energy phos-
phate bonds in ATP to power life processes. This is accomplished by accessing the
energy of the oxygen bond between two atoms of oxygen in the oxygen molecule.
A single unpaired electron is present in the outer ring of the oxygen atom, and this
electron, when unpaired, is highly reactive as it seeks a partner to return to a paired
condition. Intracellular conversions of oxygen into compounds with unpaired elec-
trons are carefully controlled reactions, such as the electron transport chain in the
mitochondria or certain intracellular signaling pathways. However, random produc-
tion of oxygen radicals as a by-product of ongoing metabolic processes or as the
result of external radiation also occurs.
The various substances incorporating an oxygen with an unpaired electron are col-
lectively called ROS and include hydrogen peroxide (H2O2), superoxide anion (O2•−),
and hydroxyl radical (OH•) [57,58]. ROS are generated through multiple sources in
the cell in addition to the electron-transport chain in mitochondria already men-
tioned earlier. Ionizing radiation [59,60] and various enzymes such as phagocytic
and nonphagocytic NADPH oxidases [61–63], lipoxygenases [64], and cycloxygen-
ases [65] produce superoxide anion. Hypochlorous acid (HOCl) is produced by the
myeloperoxidase enzyme in neutrophils [66]. Another ROS is singlet oxygen, which
is generated upon photosensitization and UVA irradiation [67]. Given that ROS are
cytotoxic, cells have developed antioxidant defenses such as enzymes that dismutate
O2 into H2O2 (SOD-1, -2, and -3) or degrade H2O2 (catalase, glutathione peroxidases,
and peroxiredoxins) [68,69].
When cellular production of ROS overwhelms its antioxidant capacity, a state of
oxidative stress is reached leading to serious cellular injuries and contributing to the
pathogenesis of several diseases. Nevertheless, if not generated in too high concen-
tration, ROS act as second messengers in signal transduction and gene regulation in a
variety of cell types and under several biological conditions such as cytokine, growth
factor and hormone actions, ion transport, transcription, neuromodulation, and apop-
tosis [70,71]. It is now well established that H2O2 is the main ROS mediating cellular
signaling because of its capacity to inhibit tyrosine phosphatases through oxida-
tion of cysteine residues in their catalytic domain, which in turn activates tyrosine
kinases and downstream signaling [72,73]. Depending on the level of ROS, different
redox-sensitive transcription factors are activated and coordinate distinct biological
responses. A low oxidative stress induces Nrf2, a transcription factor implicated in
the transactivation of gene coding for antioxidant enzymes [74].
An intermediate amount of ROS triggers an inflammatory response through the
activation of NF-κB and AP-1, and a high level of oxidative stress induces perturba-
tion of the mitochondrial pores and disruption of electron transfer, thereby resulting
in apoptosis or necrosis of cells (Figure 2.1) [74]. NF-κB was the first transcription
factor shown to be redox-regulated [75,76].
Cellular redox status may play a key role in the regulation of immune responses
mediated by other transcription factors in addition to NF-κB. Addition of anti-
oxidants has been shown to modulate T-cell responses as measured in terms of
proliferation and cytokine secretion implicating the importance of ROS in antigen-
mediated T-cell activation [16]. Cytokines secreted by different cells participating
in the immune response are known to play a critical role in successful pathogen
Level of oxidative stress
Low Intermediate High

Nrf-2 NF-κB Mitochondrial
Signaling pathway AP-1 PT pore
Outcome Antioxidant Inflammation Apoptosis

enzymes proteins proteins
FIGURE 2.1 (See color insert.) Hierarchical oxidative stress model. A low oxidative stress
induces Nrf2, a transcription factor implicated in the transactivation of gene coding for
antioxidant enzymes. An intermediate amount of ROS triggers an inflammatory response
through the activation of NF-κB and AP-1, and a high amount of oxidative stress induces
perturbation of the mitochondrial PT pore and disruption of the electron transfer, thereby
resulting in apoptosis or necrosis. (Adapted from Williams, M.S. and Kwon, J., J Free Radic.
Biol. Med., 37, 1144, 2004.)
clearance. Any alteration in this highly regulated network of cytokines by external

or internal factors may result in dysregulation leading to chronic low-grade inflam-
mation. For example, IL-2, TNF-α, and IFN-γ are secreted by Th1-type cells and can
activate macrophages and promote cell-mediated immune responses against invasive
intracellular pathogens. Th2 (IL-4, IL-5, IL-6, IL-10, and IL-13) cytokines promote
humoral immune responses against extracellular pathogens [77]. For complete T-cell
activation and cytokine secretion, the cooperative binding of NFAT and AP-1 to
composite NFAT/AP-1 binding sites is necessary [29,78,79].
LIPID PEROXIDATION PRODUCTS

Lipid peroxidation occurs both external to the body during cooking of foods and as
a result of internal generation of peroxidation products by enzymes in the stomach.
Potentially toxic lipid aldehyde species such as 4-hydroxy-trans-2-nonenal (HNE),
acrolein, and malondialdehyde (MDA) are formed, and these lipid aldehydes can
activate various signaling intermediates that can lead to chronic inflammation. These
lipid aldehyde species also result in modification of proteins and DNA. Lipid alde-
hydes have been implicated in a number of oxidative stress–induced diseases linked
to chronic low-grade inflammation, including diabetes, heart disease, common forms
of cancer, age-related macular degeneration, and aging [80–91]. Malondialdehyde
(MDA), 4-hydroxy-2-nonenal (HNE), and acrolein are generated upon degradation
of lipid peroxides in a free radical–induced oxidation of membrane-lipid double
bonds found in polyunsaturated fatty acids within the structure of cell membranes
[92,93]. Once formed, these lipid peroxides are more stable than other ROS and can
initiate chain reactions in the lipid membrane resulting in an amplification of the
oxidant-stress–induced formation of lipid aldehydes [92]. MDA is perhaps the best-
known lipid aldehyde and has been used frequently as a biomarker of lipid peroxida-
tion in both urine and plasma samples in human studies [93]. Both HNE and MDA
are the aldehydes formed in greatest quantity as the result of lipid peroxidation, but
acrolein is chemically most reactive. HNE generated from the peroxidation of ara-
chidonic acid is highly toxic and the most abundant LDA in the living tissue, reach-
ing a reported concentration of up to 10 nmol/g tissue [92]. Its in situ concentration
plays a key role in the cell growth, death, and differentiation.
Lipid aldehydes such as HNE have been shown to interact with a number of
nucleophilic substances within the cell, leading to cellular damage [94]. They react
with proteins and nucleic acids through thiols and amine groups leading to the accu-
mulation of denatured macromolecules [95,96]. In various disease states, conjugates
of lipid aldehydes with proteins and nucleic acids have been identified. For example,
HNE-protein adducts have been detected in brain tumor tissue [97]. Lipid aldehydes
can also propagate other redox signaling pathways leading to cellular and tissue
injury [98–100].
CYTOKINE ACTIVATION PATHWAYS

Interleukin-1 beta (IL-1β) is a potent proinflammatory cytokine that binds to its
specific receptor (IL1-R1) on cell membranes and then induces the recruitment to
the receptor cytoplasmic tail of specific adaptor and effector proteins, including
IL-1RacP, MyD88, and Tollip [101–103]. MyD88 then mediates the recruitment of
the interleukin-1 receptor- associated kinase (IRAK) family members to the IL-1R
[104], which in turn recruit TRAF6 [105]. Then, TRAF6 recruits TAK1, which
mediates the phosphorylation of the IKK complex, a crucial step already reviewed
earlier in NF-κB activation [106].
TNF-α is another potent proinflammatory cytokine that plays a crucial role in
apoptosis, cell proliferation, differentiation, and septic shock [107]. TNF-α binds
to its cellular TNFR1 receptor, which triggers an intracellular pathway leading to
activation of NF-κB and AP-1 transcription factors. The signaling pathway that
leads to NF-κB activation is well understood [18,108]. After binding to TNFR1 by
TNF-α, aggregation of the receptor and dissociation of silencer of death domain
(SODD) follows. SODD is an inhibitor of TNFR1 activity, and dissociation of
SODD allows binding of TNFR-associated death domain protein (TRADD pro-
tein) [109]. TRADD then recruits downstream adapters like TNF–receptor associ-
ated factor (TRAF proteins) [110]. Although many members of the TRAF family
have been implicated in TNF signaling, TRAF2 and TRAF5 have been shown
to have a role in NF-κB activation by TNF-α [111]. Receptor-interacting protein
(RIP1) is also involved in NF-κB activation by TNF-α [112]. RIP1 acts as a scaf-
fold protein permitting the recruitment of the IKK complex, which is critical to
NF-κB activation following TNF-α binding to the cell membrane [113].
Cytokines such as TNF-α and IL-1β activate the classical NF-κB pathway
[114,115]. In addition, the innate immune system activation by Toll-like receptors
(TLRs) [116] and antigen receptors (TCR, BCR) trigger this pathway [117–119].
After signaling through their different receptors, these various stimuli all result in
the activation of IκB-kinase (IKK) complex, which includes the scaffold protein
NF-κB essential modulator (NEMO, also called IKKγ) [120], IKKα, and IKKβ
kinases [121]. Once activated by phosphorylation, the IKK complex phosphorylates
IκBα. The complex is then ubiquitinated and degraded via the proteasome pathway.
The freed NF-κB subunits p50 and p65 can then move into the nucleus where they
activate the transcription of genes for cytokines, chemokines, adhesion molecules,
and inhibitors of apoptosis (Figure 2.2) [118].
In addition to this classical pathway, there is an alternate pathway that does not
involve NEMO (IKKγ). This alternate NF-κB pathway is involved in secondary
lymphoid organ development and in adaptive immunity. This pathway is induced
by B-cell activating factor (BAFF) [122], lymphotoxin b (LTb) [123], CD40 ligand
[124], and human T-cell leukemia (HTLV) and Epstein–Barr (EBV) virus [125,126].
It enhances NF-κB-inducing kinase (NIK)- and IKKα-dependent processing of p100
into p52, which binds DNA in association with its partners, like RelB. These stimuli
also activate the classical pathway (Figure 2.3).
Lipopolysaccharide (LPS) is found in the outer membrane of gram-negative
bacteria and activates host innate immunity by stimulating phagocytic cells,
monocytes, macrophages, and neutrophils to produce inflammatory cytokines,
including IL-1, IL-6, and TNF-α [127]. LPS is recognized by TLR4 membrane
receptors that mediate the innate immunity inflammatory response. After LPS
binds to TLR4, the portion of the TLR4 receptor protein in the cytoplasm recruits
TNF-α
TNFR1
TRADD TRAF2
RING
RIP Ubc13
γ γ Uev1A
P P TRAF2
IKK
complex α β β α
MEKK3 Ub 53
P P
Ub 53
Ub 63
Ub
P
P IκBα
p50 p65
s
leu
c
Nu
p50 p65 Target gene
FIGURE 2.2 (See color insert.) TNFR1 signaling. In this model of TNFR1 signaling,
the IKK complex is activated while associated with the receptor. The IKK complex is
recruited to the receptor in a TNF- α-dependent manner. This recruitment requires
TRAF2 and may also involve the interaction between IKKγ and RIP. TRAF2 is thought
to activate the IKK complex via a ubiquitin-dependent signaling pathway. The TRAF2/
ubiquitin signaling complex may lead to the activation of MEKK3, although this has yet
to be demonstrated. RIP is also likely to play a role in activation of the IKK complex,
possibly by interacting with MEKK3 (From Yang, J. et al., Nat. Immunol. 2, 620, 2001.)
Once activated, the IKK complex phosphorylates IκBα on serines 32 and 36, leading to
its proteasome-mediated degradation. (Reprinted with permission from Silverman, N. and
Maniatis, T., Genes Dev., 15, 2321. Copyright 2001 by Cold Spring Harbor Laboratory
Press, www.genesdev.org 2321.)
MyD88, which links TLR4 to IRAK and TRAF6 and leads to NF-κB activation
[128,129]. CD14, which is expressed on the surface and in the cytoplasm (sCD14)
of monocytes, macrophages, and neutrophils, interacts with LPS-binding protein
(LBP), which binds to a lipid region of LPS and promotes LPS docking at the
TLR4 receptor [130–132] (Figure 2.4).
28
TNFR1, IL-1R1, TLRs, BCR, TCR LTβR, BAFFR, CD40, HTLV, EBV
Classical pathway Alternative pathway
NIK
NEMO NEMO
Ub
Ub
Ub P P
IKKα IKKβ
Ub
P P
Ub
IκBα P IKKα IKKα
P
p50 p65
Ub
P P Ub
IκBα Ub
Ub P P
p50 p65 Ub p100
RelB p52 RelB

p50 p65
Inflammatory proteins Lymphoid organ development and

p50 p65 p52 RelB homeostasis proteins
FIGURE 2.3 (See color insert.) Classical and alternative pathways of NF-κB activation. Ligation of TNFR1, IL-1/TLR, TCR, and BCR induces
IKK-dependent IkBα phosphorylation on S32 and 36, which induces ubiquitination and degradation of the inhibitory protein, thus allowing NF-κB
to migrate into the nucleus and transactivate inflammatory genes (classical pathway). Upon ligation of LTbR, BAFFR or CD40 or infection by HTLV
or EBV, the alternate pathway is induced. It enhances NF-κB inducing kinase (NIK)- and IKKα-dependent processing of p100 into p52, which binds
DNA in association with its partners and stimulates genes implicated in lymphoid organ development and organogenesis. These stimuli also activate
Immunonutrition: Interactions of Diet, Genetics, and Inflammation
the classical pathway.

LPS TLR4
CD14 MD-2
TAB2
MyD88
DD
TIR
IRAK
IRAK
Ubc13
TRAF6 Uev1A
RING
63 Ub TRAF6
Ub
63
63 Ub TAB2 TAK1
Ub
TAB1
γ γ
P P
IKK
complex α β β α
P P
β- T r P
P
P IκBα U b i q uC
iti n
p50 p65 Prote a s o m e
s
leu
c
Nu
p50 p65 Target gene
FIGURE 2.4 (See color insert.) LPS signaling pathway in mammals. In this model, LPS
is recognized by a complex of three proteins: CD14, MD-2, and TLR4. TLR4 activates the
intracellular signaling cascade by recruiting MyD88 and IRAK to the membrane. IRAK
associates with the receptor complex transiently; once released IRAK can associate with and
activate TRAF6. The TRAF6 RING finger, in combination with Ubc13 and Uev1A, mediates
the K63-extended polyubiquitination of TRAF6 itself. The TAK1/TAB1/TAB2 complex is
activated by its association with ubiquitinated TRAF6. Interestingly, the TAK1-associated
protein TAB2 translocates from the membrane fraction to the cytoplasmic fraction upon
treatment with IL-1. Once activated, the TAK1 complex phosphorylates and activates the
IKK complex. The activated IKK complex then phosphorylates IκBα, leading to its ubiqui-
tination and degradation by the proteasome.
INFLAMMASOME, CYTOKINES, AND PYROPTOSIS

Innate immunity is mediated via cells that have membrane proteins called pattern
recognition receptors (PRRs). These PRRs interact with exogenous ligands on the
surface of bacteria called microbe-associated molecular patterns (MAMPs). These
MAMPs, on binding to the PRRs, permit sensing of invading organisms [133].
Another type of ligand that binds to the PRRs is called damage-associated molecular
patterns (DAMPs). Unlike MAMPs, which signal an invading bacteria, the DAMPs
are triggered by substances released from cells due to cellular stress or abnormal
tissue or organ homeostasis. Visceral obesity is an example of a source of DAMPs
from dying adipocytes deprived of nutrients at the periphery of fat tissue poorly
vascularized by the new blood vessels generated during rapid growth of this fat
depot during weight gain. Activation of PRRs is part of the TLR system of innate
immunity, and the binding of the PRR leads to proinflammatory gene expression in
the pathways discussed earlier. Additionally, PRR engagement sets off cascades that
lead to the activation of inflammatory caspases. Caspases are cysteine proteases that
have multiple roles in cell biology, including apoptosis, necrosis, and inflammation.
The cleavage and activation of caspase-1 is initiated with the formation of large mul-
tiprotein signaling platforms, called inflammasomes.
Activated inflammasomes lead to cleavage of the proenzyme of caspase-1, result-
ing in the activation of caspase-1. The subunits formed in this cleavage reaction,
called p10 and p20, build tetramers to form the active cysteine protease. Pro-IL-1β
is expressed in small amounts intracellularly but can be induced following stress
or pathogens via the TLR signaling axis [134], which converts the inactive IL-1β
precursor to the active form of the cytokine. The active form of the cytokine IL-1β
promotes inflammation causing vasodilation, hyperthermia, and extravasation of
immune cells. IL-1β is also involved in the generation of Th17 cells [134]. The sec-
ond well-characterized caspase-1 substrate, IL-18, is also expressed as a procytokine.
The caspase-1-cleaved C-terminal and secretable part of IL-18 promote, together
with IL-12, the production of IFN-γ in Th1, natural killer (NK), and cytotoxic T cells
[134]. Apart from its degradative actions, caspase-1 is required for the release of
IL-1β and various other target proteins from cells via a specialized secretory path-
way [135]. Finally, activity of caspase-1 in myeloid cells results in a special type of
cell death, known as pyroptosis, which is described later.
Pyroptotic cell death is a form of programmed cell death mediated by caspase-1,
which is proinflammatory, in contrast to apoptosis, which is not inflammatory.
Apoptosis does not depend on caspase-1. However, apoptosis does operate through
other caspases, including caspase-3, caspase-6, and caspase-7 [136,137]. Apoptotic
cell death further results in regulated degradation and clearance of cellular contents
through an internal dissolution of the cell contents. However, during pyroptosis,
the cellular matter is released to the extracellular space where it can induce inflam-
mation via the innate immune system [136]. There is support for the notion that
pyroptosis supplements host defense mechanisms by engaging the immune system
to clear intracellular pathogens [138]. Even though the molecular events resulting in
inflammasome activation have become clearer in recent years for certain sensors,
the precise mechanisms of activation remain largely unknown for the majority of
inflammasome complexes. Due to its prominent role in the innate immune response
against microbial pathogens and its role in metabolic diseases and autoinflamma-
tory disorders, elucidating the mechanisms of inflammasome activation will be of
great interest.
CONCLUSION
The cellular and intracellular processes reviewed in this chapter mediate the immune
response to external and internal stresses including oxidant stress. These pathways
are complex and interacting, which provides added security to cells and organs
against invasion, loss of nutrients or blood flow, or destruction by trauma. When
these defense mechanisms work well in a controlled manner, they are vital to human
survival. However, when low-grade chronic activation of these pathways occurs, it
results in cellular and DNA damage. In the extreme, these changes can lead to com-
mon forms of cancer. Much of what has been learned about these mechanisms and
pathways comes from cancer research. In cancer cells, many of these pathways are
permanently in the on position, enabling scientists to manipulate the pathways in
order to understand them. As the science implicating inflammation in many different
organ systems is examined in further chapters, we will revisit these same pathways
as markers of inflammation and as signposts in basic research.
REFERENCES
1. Dalgleish AG, O’Byrne KJ. 2002. Chronic immune activation and inflammation in the
pathogenesis of AIDS and cancer. Adv Cancer Res 84: 231–276.
2. Coussens LM, Werb Z. 2002. Inflammation and cancer. Nature 420: 860–867.
3. Kaser A, Zeissig S, Blumberg RS. 2010. Inflammatory bowel disease. Annu Rev
Immunol 28: 573–621.
4. Ishiguro Y. 1999. Mucosal proinflammatory cytokine production correlates with endo-
scopic activity of ulcerative colitis. J Gastroenterol 34: 66–74.
5. Scott DL, Wolfe F, Huizinga TW. 2010. Rheumatoid arthritis. Lancet 376: 1094–1108.
6. Lee YJ, Han SB, Nam SY, Oh KW, Hong JT. 2010. Inflammation and Alzheimer’s dis-
ease. Arch Pharm Res 33: 1539–1556.
7. O’Shea JJ, Ma A, Lipsky P. 2002. Cytokines and autoimmunity. Nat Rev Immunol 2:
37–45.
8. Ricci M, Matucci A, Rossi O. 1997. IL-4 as a key factor influencing the development of
allergen-specific Th2-like cells in atopic individuals. J Investig Allergol Clin Immunol
7: 144–150.
9. Lewis DB. 2002. Allergy immunotherapy and inhibition of Th2 immune responses: A
sufficient strategy? Curr Opin Immunol 14: 644–651.
10. Tsai EY, Yie J, Thanos D, Goldfeld AE. 1996. Cell-type-specific regulation of the human
tumor necrosis factor alpha gene in B cells and T cells by NFATp and ATF-2/JUN. Mol
Cell Biol 16: 5232–5244.
11. Sica A, Dorman L, Viggiano V, Cippitelli M, Ghosh P, Rice N et al. 1997. Interaction
of NF-kappaB and NFAT with the interferon-gamma promoter. J Biol Chem 272:
30412–30420.
12. Falvo JV, Uglialoro AM, Brinkman BM, Merika M, Parekh BS, Tsai EY et al. 2000.
Stimulus-specific assembly of enhancer complexes on the tumor necrosis factor alpha
gene promoter. Mol Cell Biol 20: 2239–2247.
13. Macian F, Garcia-Rodriguez C, Rao A. 2000. Gene expression elicited by NFAT

in the presence or absence of cooperative recruitment of Fos and Jun. EMBO J 19:
4783–4795.
14. Gautam R, Jachak SM. 2009. Recent developments in anti-inflammatory natural prod-
ucts. Med Res Rev 29: 767–820.
15. Wilankar C, Sharma D, Checker R, Khan NM, Patwardhan R, Patil A et al. 2011. Role
of immunoregulatory transcription factors in differential immunomodulatory effects of
tocotrienols. J Free Radic Biol Med 51: 129–143.
16. Williams MS, Kwon J. 2004. T cell receptor stimulation, reactive oxygen species, and
cell signaling. J Free Radic Biol Med 37: 1144–1151.
17. Larbi A, Kempf J, Pawelec G. 2007. Oxidative stress modulation and T cell activation.
Exp Gerontol 42: 852–858.
18. Hayden MS, Ghosh S. 2004. Signaling to NF-kappaB. Genes Dev 18: 2195–2224.
19. Aggarwal BB. 2004. Nuclear factor-kappaB: The enemy within. Cancer Cell 6:
203–208.
20. Crabtree GR, Olson EN. 2002. NFAT signaling: Choreographing the social lives of
cells. Cell 109(Suppl): S67–S79.
21. Macian F. 2005. NFAT proteins: Key regulators of T-cell development and function. Nat
Rev Immunol 5: 472–484.
22. Karin M, Shaulian E. 2001. AP-1: Linking hydrogen peroxide and oxidative stress to the
control of cell proliferation and death. IUBMB Life 52: 17–24.
23. Shaulian E, Karin M. 2002. AP-1 as a regulator of cell life and death. Nat Cell Biol 4:
E131–E136.
24. Ginn-Pease ME, Whisler RL. 1996. Optimal NF kappa B mediated transcriptional
responses in Jurkat T cells exposed to oxidative stress are dependent on intracellular
glutathione and costimulatory signals. Biochem Biophys Res Commun 226: 695–702.
25. Aifantis I, Gounari F, Scorrano L, Borowski C, von Boehmer H. 2001. Constitutive pre-
TCR signaling promotes differentiation through Ca2+ mobilization and activation of
NF-kappaB and NFAT. Nat Immunol 2: 403–409.
26. Bassuk AG, Leiden JM. 1995. A direct physical association between ETS and AP-1
transcription factors in normal human T cells. Immunity 3: 223–237.
27. Chang JH, Pratt JC, Sawasdikosol S, Kapeller R, Burakoff SJ. 1998. The small GTP
binding protein Rho potentiates AP-1 transcription in T cells. Mol Cell Biol 18:
4986–4993.
28. Boise LH, Petryniak B, Mao X, June CH, Wang CY, Lindsten T et al. 1993. The NFAT-1
DNA binding complex in activated T cells contains Fra-1 and JunB. Mol Cell Biol 13:
1911–1919.
29. Rooney JW, Hoey T, Glimcher LH. 1995. Coordinate and cooperative roles for NF-AT
and AP-1 in the regulation of the murine IL-4 gene. Immunity 2: 473–483.
30. Tsai EY, Jain J, Pesavento PA, Rao A, Goldfeld AE. 1996. Tumor necrosis factor alpha
gene regulation in activated T cells involves ATF-2/Jun and NFATp. Mol Cell Biol 16:
459–467.
31. Vacca A, Felli MP, Farina AR, Martinotti S, Maroder M, Screpanti I et al. 1992.
Glucocorticoid receptor-mediated suppression of the interleukin 2 gene expression
through impairment of the cooperativity between nuclear factor of activated T cells and
AP-1 enhancer elements. J Exp Med 175: 637–646.
32. Yang Y, Pares-Matos EI, Tesmer VM, Dai C, Ashworth S, Huai J et al. 2002.
Organization of the promoter region of the human NF-IL6 gene. Biochim Biophys Acta
1577: 102–108.
33. Standiford TJ, Kunkel SL, Strieter RM. 1993. Interleukin-8: A major mediator of acute
pulmonary inflammation. Reg Immunol 5: 134–141.
34. Heinrich PC, Behrmann I, Haan S, Hermanns HM, Muller-Newen G, Schaper F. 2003.
Principles of interleukin (IL)-6-type cytokine signalling and its regulation. Biochem J
374: 1–20.
35. Tilg H, Trehu E, Atkins MB, Dinarello CA, Mier JW. 1994. Interleukin-6 (IL-6) as
an anti-inflammatory cytokine: Induction of circulating IL-1 receptor antagonist and
soluble tumor necrosis factor receptor p55. Blood 83: 113–118.
36. Boulton TG, Cobb MH. 1991. Identification of multiple extracellular signal-regulated
kinases (ERKs) with antipeptide antibodies. Cell Regul 2: 357–371.
37. Leevers SJ, Marshall CJ. 1992. Activation of extracellular signal-regulated kinase,
ERK2, by p21ras oncoprotein. EMBO J 11: 569–574.
38. Kyriakis JM, App H, Zhang XF, Banerjee P, Brautigan DL, Rapp UR et al. 1992. Raf-1
activates MAP kinase-kinase. Nature 358: 417–421.
39. Crews CM, Erikson RL. 1992. Purification of a murine protein-tyrosine-threonine
kinase that phosphorylates and activates the Erk-1 gene product: Relationship to the
fission yeast byr1 gene product. Proc Natl Acad Sci USA 89: 8205–8209.
40. Yao Y, Li W, Wu J, Germann UA, Su MSS, Kuida K et al. 2003. Extracellular signal-
regulated kinase 2 is necessary for mesoderm differentiation. Proc Natl Acad Sci USA
100: 12759–12764.
41. Pagès G, Guérin S, Grall D, Bonino F, Smith A, Anjuere F et al. 1999. Defective thymocyte
maturation in p44 MAP kinase (Erk1) knockout mice. Science 286: 1374–1377.
42. Ip YT, Davis RJ. 1998. Signal transduction by the c-Jun N-terminal kinase (JNK)—
From inflammation to development. Curr Opin Cell Biol 10: 205–219.
43. Han J, Lee JD, Tobias PS, Ulevitch RJ. 1993. Endotoxin induces rapid protein tyrosine
phosphorylation in 70Z/3 cells expressing CD14. J Biol Chem 268: 25009–25014.
44. Han J, Lee JD, Bibbs L, Ulevitch RJ. 1994. A MAP kinase targeted by endotoxin and
hyperosmolarity in mammalian cells. Science 265: 808–811.
45. Jiang Y, Chen C, Li Z, Guo W, Gegner JA, Lin S et al. 1996. Characterization of the
structure and function of a new mitogen-activated protein kinase (p38β). J Biol Chem
271: 17920–17926.
46. Lechner C, Zahalka MA, Giot JF, Moler NPH, Ullrich A. 1996. ERK6, a mitogen-
activated protein kinase involved in C2C12 myoblast differentiation. Proc Natl Acad Sci
USA 93: 4355–4359.
47. Li Z, Jiang Y, Ulevitch RJ, Han J. 1996. The primary structure of p38 gamma: A new
member of p38 group of MAP kinase. Biochem Biophys Res Commun 228: 334–340.
48. Jiang Y, Gram H, Zhao M, New L, Gu J, Feng L et al. 1997. Characterization of the
structure and function of the fourth member of p38 group mitogen-activated protein
kinases, p38delta. J Biol Chem 272: 30122–30128.
49. Kumar S, McDonnell PC, Gum RJ, Hand AT, Lee JC, Young PR et al. 1997. Novel
homologues of CSBP/p38 MAP kinase: Activation, substrate specificity and sen-
sitivity to inhibition by pyridinyl imidazoles. Biochem Biophys Res Commun 235:
533–538.
50. Guan Z, Buckman SY, Pentland AP, Templeton DJ, Morrison AR. 1998. Induction
of cyclooxygenase-2 by the activated MEKK1—> SEK1/MKK4—> p38 mitogen-
activated protein kinase pathway. J Biol Chem 273: 12901–12908.
51. Badger AM, Cook MN, Lark MW, Newman-Tarr TM, Swift BA, Nelson AH et al. 1998.
SB 203580 inhibits p38 mitogen-activated protein kinase, nitric oxide production, and
inducible nitric oxide synthase in bovine cartilage-derived chondrocytes. J Immunol
161: 467–473.
52. Da Silva J, Pierrat B, Mary JL, Lesslauer W. 1997. Blockade of p38 mitogen-activated
protein kinase pathway inhibits inducible nitric-oxide synthase expression in mouse
astrocytes. J Biol Chem 272: 28373–28380.
53. Craxton A, Shu G, Graves JD, Saklatvala J, Krebs EG, Clark EA et al. 1998. p38 MAPK
is required for CD40-induced gene expression and proliferation in B lymphocytes.
J Immunol 161: 3225–3236.
54. Pietersma A, Tilly BC, Gaestel M, de Jong N, Lee JC, Koster JF et al. 1997. p38
mitogen activated protein kinase regulates endothelial VCAM-1 expression at the post-
transcriptional level. Biochem Biophys Res Commun 230: 44–48.
55. Lee JC, Laydon JT, McDonnell PC, Gallagher TF, Kumar S, Green D et al. 1994.
Identification and characterization of a novel protein kinase involved in the regulation of
inflammatory cytokine biosynthesis. Nature 372: 739–746.
56. Kummer JL, Rao PK, Heidenreich KA. 1997. Apoptosis induced by withdrawal of tro-
phic factors is mediated by p38 mitogen-activated protein kinase. J Biol Chem 272:
20490–20494.
57. Adler V, Yin Z, Tew KD, Ronai Z. 1999. Role of redox potential and reactive oxygen
species in stress signalling. Oncogene 18: 6104–6111.
58. Haddad JJ. 2002. Antioxidant and prooxidant mechanisms in the regulation of redox(y)-
sensitive transcription factors. Cell Signal 14: 879–897.
59. Ogawa Y, Kobayashi T, Nishioka A, Kariya S, Hamasato S, Seguchi H et al. 2003.
Radiation-induced oxidative DNA damage, 8-oxoguanine, in human peripheral T cells.
Int J Mol Med 11: 27–32.
60. Genova ML, Pich MM, Bernacchia A, Bianchi C, Biondi A, Bovina C et al. 2004. The
mitochondrial production of reactive oxygen species in relation to aging and pathology.
Ann N Y Acad Sci 1011:86–100.
61. Babior BM. 1999. NADPH oxidase: An update. Blood 93: 1464–1476.
62. Griendling KK, Harrison DG. 1999. Dual role of reactive oxygen species in vascular
growth. Circ Res 85: 562–563.
63. Van Heerebeek L, Meischl C, Stooker W, Meijer CJ, Niessen HW, Roos D. 2002.
NADPH oxidase(s): New source(s) of reactive oxygen species in the vascular system?
J Clin Pathol 55: 561–568.
64. Kuhn H, Thiele BJ. 1999. The diversity of the lipoxygenase family. Many sequence data
but little information on biological significance. FEBS Lett 449: 7–11.
65. Kuehl Jr FA, Egan RW. 1980. Prostaglandins, arachidonic acid, and inflammation.
Science 210: 978–984.
66. Hampton MB, Kettle AJ, Winterbourn CC. 1998. Inside the neutrophil phagosome:
Oxidants, myeloperoxidase, and bacterial killing. Blood 92: 3007–3017.
67. Volanti C, Matroule JY, Piette J. 2002. Involvement of oxidative stress in NF-kappaB
activation in endothelial cells treated by photodynamic therapy. Photochem Photobiol
75: 36–45.
68. Engelhardt JF. 1999. Redox-mediated gene therapies for environmental injury:
Approaches and concepts. Antioxid Redox Signal 1: 5–27.
69. Rhee SG, Chang TS, Bae YS, Lee SR, Kang SW. 2003. Cellular regulation by hydrogen
peroxide. J Am Soc Nephrol 14: S211–S215.
70. Hensley K, Robinson KA, Gabbita SP, Salsman S, Floyd RA. 2000. Reactive oxygen
species, cell signaling, and cell injury. Free Rad Biol Med 28: 1456–1462.
71. Lander HM. 1997. An essential role for free radicals and derived species in signal trans-
duction. FASEB J 11: 118–124.
72. Tonks NK. 2005. Redox redux: Revisiting PTPs and the control of cell signalling. Cell
121: 667–670.
73. Aslan M, Ozben T. 2003. Oxidants in receptor tyrosine kinase signal transduction path-
ways. Antioxid Redox Signal 5: 781–788.
74. Halliwell B, Gutteridge J. 1999. Free Radicals in Biology and Medicine. Oxford, U.K.:
Oxford University Press.
75. Schreck R, Rieber P, Baeuerle PA. 1991. Reactive oxygen intermediates as apparently
widely used messengers in the activation of the NF-kappa B transcription factor and
HIV-1. EMBO J 10: 2247–2258.
76. Legrand-Poels S, Vaira D, Pincemail J, van de Vorst A, Piette J. 1990. Activation of
human immunodeficiency virus type 1 by oxidative stress. AIDS Res Hum Retroviruses
6: 1389–1397.
77. Opal SM, DePalo VA. 2000. Anti-inflammatory cytokines. Chest 117: 1162–1172.
78. Macian F, Lopez-Rodriguez C, Rao A. 2001. Partners in transcription: NFAT and AP-1.
Oncogene 20: 2476–2489.
79. Rooney JW, Sun YL, Glimcher LH, Hoey T. 1995. Novel NFAT sites that mediate acti-
vation of the interleukin-2 promoter in response to T-cell receptor stimulation. Mol Cell
Biol 15: 6299–6310.
80. Pillon NJ, Croze ML, Vella RE, Soul`ere L, Lagarde M, Soulage CO. 2012. The lipid
peroxidation by-product 4-hydroxy-2-nonenal (4-HNE) induces insulin resistance
in skeletal muscle through both carbonyl and oxidative stress. Endocrinology 153:
2099–2111.
81. Mattson MP. 2009. Roles of the lipid peroxidation product 4-hydroxynonenal in obesity,
the metabolic syndrome, and associated vascular and neurodegenerative disorders. Exp
Gerontol 44: 625–633.
82. Kang CS, Kim HW, Kim BK Kwack SJ, Ahn IY, Bae JY et al. 2011. Hepatotoxicity
and nephrotoxicity produced by 4-hydroxy-2-nonenal (4-HNE) following 4-week oral
administration to Sprague-Dawley rats. J Toxicol Environ Health A 74: 779–789.
83. Karihtala P, Kauppila S, Puistola U, Jukkola-Vuorinen A. 2011. Divergent behaviour
of oxidative stress markers 8-hydroxydeoxyguanosine (8-OHdG) and 4-hydroxy-2-
nonenal (HNE) in breast carcinogenesis. Histopathology 58: 854–862.
84. Uno K, Kato K, Kusaka G, Asano N, Iijima K, Shimosegawa T. 2011. The balance
between 4-hydroxynonenal and intrinsic glutathione/glutathione S-transferase A4 sys-
tem may be critical for the epidermal growth factor receptor phosphorylation of human
esophageal squamous cell carcinomas. Mol Carcinog 50: 781–790.
85. Vatsyayan R, Chaudhary P, Sharma A, Sharma R, Rao Lelsani PC, Awasthi S et al. 2011.
Role of 4-hydroxynonenal in epidermal growth factor receptor-mediated signaling in
retinal pigment epithelial cells. Exp Eye Res 92: 147–154.
86. Qin S, Rodrigues GA. 2010. Differential roles of AMPKα and AMPKβ in regulating
4-HNE-induced RPE cell death and permeability. Exp Eye Res 91: 818–824.
87. Singh FM, Dang TM, Arseneault M, Ramassamy C. 2010. Role of by-products of lipid
oxidation in Alzheimer’s disease brain: A focus on acrolein. J Alzheimer’s Dis 21:
741–756.
88. Butterfield DA, Bader Lange ML, Sultana R. 2010. Involvements of the lipid peroxida-
tion product, HNE, in the pathogenesis and progression of Alzheimer’s disease. Biochim
Biophys Acta 1801: 924–929.
89. Volkel W, Sicilia T, Pahler A, Gsell W, Tatschner T, Jellinger K et al. 2006. Increased
brain levels of 4-hydroxy-2-nonenal glutathione conjugates in severe Alzheimer’s dis-
ease. Neurochem Int 48: 679–686.
90. Fukai M, Hayashi T, Yokota R, Shimamura T, Suzuki T, Taniguchi M et al. 2005. Lipid
peroxidation during ischemia depends on ischemia time in warm ischemia and reperfu-
sion of rat liver. J Free Radic Biol Med 38: 1372–1381.
91. McCracken E, Graham DI, Nilsen M, Stewart J, Nicoll JA, Horsburgh K. 2001.
4-Hydroxynonenal immunoreactivity is increased in human hippocampus after global
ischemia. Brain Pathol 11: 414–421.
92. Esterbauer H, Schaur RJ, Zollner H. 1991. Chemistry and biochemistry of 4 hydroxynon-
enal, malonaldehyde and related aldehydes. J Free Radic Biol Med 11: 81–128.
93. Li Z, Henning SM, Zhang Y, Zerlin A, Li L, Gao K et al. 2010. Antioxidant-rich spice
added to hamburger meat during cooking results in reduced meat, plasma, and urine
malondialdehyde concentrations. Am J Clin Nutr 91: 1180–1184.
94. Loidl-Stahlhofen A, Hannemann K, Spiteller G. 1994. Generation of α hydroxyal-
dehydic compounds in the course of lipid peroxidation. Biochim Biophys Acta 1213:
140–148.
95. LoPachin RM, Gavin T, Petersen DR, Barber DS. 2009. Molecular mechanisms of
4-hydroxy-2-nonenal and acrolein toxicity: Nucleophilic targets and adduct formation.
Chem Res Toxicol 22: 1499–1508.
96. Jacobs AT, Marnett LJ. 2010. Systems analysis of protein modification and cellular
responses induced by electrophile stress. Acc Chem Res 43: 673–683.
97. Juric-Sekhar G, Zarkovic K, Waeg G, Cipak A, Zarkovic N. 2009. Distribution of
4-hydroxynonenal-protein conjugates as a marker of lipid peroxidation and param-
eter of malignancy in astrocytic and ependymal tumors of the brain. Tumori 95:
762–768.
98. Yadav UCS, Ramana KV, Awasthi YC, Srivastava SK. 2008. Glutathione level regulates
HNE-induced genotoxicity in human erythroleukemia cells. Toxicol Appl Pharmacol
227: 257–264.
99. Zarkovic N, Zarkovic K, Schaur RJ, Stolc S, Schlag G, Redl H et al. 1999.
4-Hydroxynonenal as a second messenger of free radicals and growth modifying factor.
Life Sci 65: 1901–1904.
100. Thompson CA, Burcham PC. 2008. Genome-wide transcriptional responses to acrolein.
Chem Res Toxicol 21: 2245–2256.
101. Muzio M, Ni J, Feng P, Dixit VM. 1997. IRAK (Pelle) family member IRAK-2 and
MyD88 as proximal mediators of IL-1 signaling. Science 278: 1612–1615.
102. Huang J, Gao X, Li S, Cao Z. 1997. Recruitment of IRAK to the interleukin 1 receptor
complex requires interleukin 1 receptor accessory protein. Proc Natl Acad Sci USA 94:
12829–12832.
103. Burns K, Clatworthy J, Martin L, Martinon F, Plumpton C, Maschera B et al. 2000.
Tollip a new component of the IL-1RI pathway, links IRAK to the IL-1 receptor. Nat
Cell Biol 2: 346–351.
104. Wesche H, Henzel WJ, Shillinglaw W, Li S, Cao Z. 1997. MyD88: An adapter that
recruits IRAK to the IL-1 receptor complex. Immunity 7: 837–847.
105. Qian Y, Commane M, Ninomiya-Tsuji J, Matsumoto K, Li X. 2001. IRAK-mediated
translocation of TRAF6 and TAB2 in the interleukin-1-induced activation of NFkappa
B. J Biol Chem 276: 41661–41667.
106. Wang C, Deng L, Hong M, Akkaraju GR, Inoue J, Chen ZJ. 2001. TAK1 is a ubiquitin-
dependent kinase of MKK and IKK. Nature 412: 346–351.
107. Tracey KJ, Cerami A. 1993. Tumor necrosis factor: An updated review of its biology.
Crit Care Med 21(10 Suppl.): S415–S422.
108. Aggarwal BB. 2003. Signalling pathways of the TNF superfamily: A double-edged
sword. Nat Rev Immunol 3: 745–756.
109. Jiang Y, Woronicz JD, Liu W, Goeddel DV. Prevention of constitutive TNF receptor 1
signaling by silencer of death domains. Science 283: 543–546.
110. Dempsey PW, Doyle SE, He JQ, Cheng G. 2003. The signaling adaptors and pathways
activated by TNF superfamily. Cytokine Growth Factor Rev 14: 193–209.
111. Tada K, Okazaki T, Sakon S, Kobarai T, Kurosawa K, Yamaoka S et al. 2001. Critical
roles of TRAF2 and TRAF5 in tumor necrosis factor-induced NF-kappa B activation
and protection from cell death. J Biol Chem 276: 36530–36534.
112. Hsu H, Huang J, Shu HB, Baichwal V, Goeddel DV. 1996. TNF dependent recruit-
ment of the protein kinase RIP to the TNF receptor-1 signaling complex. Immunity 4:
387–396.
113. Zhang SQ, Kovalenko A, Cantarella G, Wallach D. 2000. Recruitment of the IKK sig-
nalosome to the p55 TNF receptor: RIP and A20 bind to NEMO (IKKgamma) upon
receptor stimulation. Immunity 12: 301–311.
114. Martin MU, Wesche H. 2002. Summary and comparison of the signaling mechanisms of
the Toll/interleukin-1 receptor family. Biochim Biophys Acta 1592: 265–280.
115. Devin A, Cook A, Lin Y, Rodriguez Y, Kelliher M, Liu Z. 2000. The distinct roles of
TRAF2 and RIP in IKK activation by TNFR1: TRAF2 recruits IKK to TNF-R1 while
RIP mediates IKK activation. Immunity 12: 419–429.
116. O’Neill LA. 2006. How Toll-like receptors signal: What we know and what we don’t
know. Curr Opin Immunol 18: 3–9.
117. Weil R, Israel A. 2004. T-cell-receptor- and B-cell-receptor mediated activation of
NF-kappaB in lymphocytes. Curr Opin Immunol 16: 374–381.
118. Bonizzi G, Karin M. 2004. The two NF-kappaB activation pathways and their role in
innate and adaptive immunity. Trends Immunol 25: 280–288.
119. Weil R, Israel A. 2006. Deciphering the pathway from the TCR to NF-kappaB. Cell
Death Differ 13: 826–833.
120. Yamaoka S, Courtois G, Bessia C, Whiteside ST, Weil R, Agou F et al. 1998.
Complementation cloning of NEMO, a component of the IkappaB kinase complex
essential for NF-kappaB activation. Cell 93: 1231–1240.
121. Zandi E, Rothwarf DM, Delhase M, Hayakawa M, Karin M. 1997. The IkappaB kinase
complex (IKK) contains two kinase subunits, IKKalpha and IKKbeta, necessary for
IkappaB phosphorylation and NF-kappaB activation. Cell 91: 243–252.
122. Claudio E, Brown K, Park S, Wang H, Siebenlist U. 2002. BAFF induced NEMO-
independent processing of NF-kappa B2 in maturing B cells. Nat Immunol 3: 958–965.
123. Dejardin E, Droin NM, Delhase M, Haas E, Cao Y, Makris C et al. 2002. The lympho-
toxin-beta receptor induces different patterns of gene expression via two NF-kappaB
pathways. Immunity 17: 525–535.
124. Coope HJ, Atkinson PG, Huhse B, Belich M, Janzen J, Holman MJ et al. 2002. CD40
regulates the processing of NFkappaB2 p100 to p52. EMBO J 21: 5375–5385.
125. Eliopoulos AG, Caamano JH, Flavell J, Reynolds GM, Murray PG, Poyet JL et al. 2003.
Epstein-Barr virus-encoded latent infection membrane protein 1 regulates the process-
ing of p100 NF-kappaB2 to p52 via an IKKgamma/NEMO-independent signalling path-
way. Oncogene 22: 7557–7569.
126. Xiao G, Cvijic ME, Fong A, Harhaj EW, Uhlik MT, Waterfield M et al. 2001. Retroviral
oncoprotein tax induces processing of NF-kappaB2/p100 in T cells: Evidence for the
involvement of IKKalpha. EMBO J 20: 6805–6815.
127. Medvedev AE, Kopydlowski KM, Vogel SN. 2000. Inhibition of lipopolysaccharide-
induced signal transduction in endotoxin-tolerized mouse macrophages: Dysregulation
of cytokine, chemokine, and toll-like receptor 2 and 4 gene expression. J Immunol 164:
5564–5574.
128. Takeda K, Akira S. 2004. TLR signaling pathways. Semin Immunol 16: 3–9.
129. Akira S. 2004. Toll receptor families: Structure and function. Semin Immunol 16: 1–2.
130. Wright SD, Ramos RA, Tobias PS, Ulevitch RJ, Mathison JC. 1990. CD14 a recep-
tor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science 249:
1431–1433.
131. Dentener MA, Bazil V, Von Asmuth EJ, Ceska M, Buurman WA. 1993. Involvement of
CD14 in lipopolysaccharide-induced tumor necrosis factor-alpha IL-6 and IL-8 release
by human monocytes and alveolar macrophages. J Immunol 150: 2885–2891.
132. Schumann RR, Leong SR, Flaggs GW, Gray PW, Wright SD, Mathison JC et al. 1990.
Structure and function of lipopolysaccharide binding protein. Science 249: 1429–1431.
133. Medzhitov R. 2008. Origin and physiological roles of inflammation. Nature 454:
428–435.
134. Dinarello CA. 2009. Immunological and inflammatory functions of the interleukin-1
family. Annu Rev Immunol 27: 519–550.
135. Keller M, Ruegg A, Werner S, Beer HD. 2008. Active caspase-1 is a regulator of uncon-
ventional protein secretion. Cell 132: 818–831.
136. Bergsbaken T, Fink SL, Cookson BT. 2009. Pyroptosis: Host cell death and inflamma-
tion. Nat Rev Microbiol 7: 99–109.
137. Kuida K, Lippke JA, Ku G, Harding MW, Livingston DJ, Su MS, Flavell RA. 1995.
Altered cytokine export and apoptosis in mice deficient in interleukin-1 beta converting
enzyme. Science 267: 2000–2003.
138. Miao EA, Leaf IA, Treuting PM, Mao DP, Dors M, Sarkar A et al. 2010. Caspase-
1-induced pyroptosis is an innate immune effector mechanism against intracellular
bacteria. Nat Immunol 11: 1136–1142.
139. Silverman N, and Maniatis T. 2001. NF-kappaB signaling pathways in mammalian and
insect innate immunity. Genes Dev 15: 2321–2342.
3 Cellular Lipids and
Inflammation
CONTENTS
Introduction............................................................................................................... 39
Fat as an Essential Macronutrient.............................................................................40
Relationship of Excess Fat Calories and Visceral Fat to Inflammation.................... 41
Subcellular Signaling Pathways Linking Inflammation and Metabolism................. 43
Eicosanoids...............................................................................................................44
Lipid Rafts and Cellular Signaling........................................................................... 45
Conclusion................................................................................................................ 48
References................................................................................................................. 49
INTRODUCTION
Lipids play a key role in the body both as stores of energy and as cellular signals
for a number of essential physiological processes. Dietary fats and the fats stored
in the body are predominantly triglycerides consisting of a three-carbon backbone
(glycerol) and three fatty acids linked to these carbons. In the process of digestion,
the fatty acids are removed from the backbone and then reattached in the cells of
the body. This complex process enables the body to modulate the biology of the fats
ingested to avoid starvation and infection, the two major threats to the survival of
ancient mankind. From an evolutionary standpoint, lipids represent the most effec-
tive store of portable energy, carrying 9 Cal/g, but the cells adapted to store this lipid
evolved in an environment where storage of energy was critical to survival. There
was no evolutionary pressure for ridding the body of excess dietary fat and calories.
The modern environment, where the increased availability of foods and a sedentary
lifestyle have led to the common occurrence of positive calorie balance, is a rela-
tively recent development in human evolution. It is now recognized that this posi-
tive energy balance has led to an activation of innate immune responses evolved in
ancient times to protect the body from infection. However, when persistent activation
of inflammation occurs even at a low level, as it does in visceral obesity, it is associ-
ated with the many diseases affected by excess inflammation that are associated with
obesity and discussed elsewhere in this text.
The relevance of the lipids discussed in this chapter relates to the ability to modify
dietary fat intake both in quantity, which affects calorie balance, and in the chemi-
cal composition of dietary fats, which affects the degree to which specific immune
39
functions are activated at a cellular and molecular level. Unlike coldwater fish that
can elongate and desaturate the 18-carbon fatty acids to the metabolites with more
than 20 carbons, such as arachidonic acid and eicosapentanoic acid, which compete
for sites on enzymes leading to the production of the eicosanoids, which mediate
inflammation at a cellular level, humans are highly inefficient at this enzymatic junc-
ture and so must alter the dietary intake of these two competing fats by reducing
their intake of n-6-rich fats and increasing the intake of n-3-rich fats from fish and
algae in order to modulate immune functions at a cellular level.
The term perfect storm is used in meteorology when two or more active storm
systems come together to cause more damage and flooding than when a single storm
is present. The modern Western diet has resulted in an excess of calories relative to
energy expenditure due to the addition of hidden fats in the diet over the last several
decades. This has promoted growth of fatty tissue, which triggers systemic inflam-
mation at some critical level of adipose tissue accumulation. The second storm is
related to the nature of the fat which has been added. The predominance of omega-6
and saturated fats has led to an imbalance of signaling systems at a cellular and
molecular level, which promotes inflammation. Inflammation, in turn, results in
oxidant stress, which enhances the inflammatory responses by forming oxidized
phospholipids and triggering inflammation secondary to the accumulation of toxic
products. The targets of this inflammatory response at a cellular and tissue level
include the heart where inflammation is integral to atherosclerosis, the liver where
inflammation can lead to liver failure, the brain where inflammation can lead to
dementia, and the muscles where inflammation can result in sarcopenia. Finally,
inflammation is integral to promoting carcinogenesis as discussed elsewhere in this
text and may account in part for the association of obesity with many common forms
of cancer.
FAT AS AN ESSENTIAL MACRONUTRIENT

Animals and humans cannot survive without dietary fat due to a minimal need for
the biological activities of two fatty acids derived from plants and animals called
linoleic and linolenic acid. These fats are made from carbon and hydrogen with vary-
ing numbers and locations of double bonds, which affect their susceptibility to oxi-
dation, their physicochemical properties at different temperatures, their metabolism
into bioactive substances, and the recent discovery that they can bind to membrane
receptors and trigger intracellular pathways that affect immune function [1].
Plants contain these two essential fatty acids, linolenic acid (18:3, n-3) and lin-
oleic acid (18:2, n-6), in low amounts of about 10% of total calories. Many modern
snack foods contain vegetable oils with linoleate concentrations in the oils of up to
60% of the total fatty acids. Despite the excess of linoleate in the modern diet, both
linolenic and linoleic acids are called essential fatty acids (EFA), since they were
noted to be essential to survival much like a vitamin in the era of vitamin discovery
[2]. They are metabolized to longer forms with over 20 carbons, which have been
called highly unsaturated fatty acids, or HUFAs, by Dr. William Lands, a pioneer
in the study of the impact of imbalances of dietary fatty acids on atherosclerosis and
inflammation [3–5]. The chemical notations in parentheses are standard chemical
Cellular Lipids and Inflammation 41
notations indicating the number of carbons (i.e., 18 carbons), the number of double
bonds (i.e., 3), and the location of the first double bond from one end of the molecule
as a number of carbons from the end, with n-6 indicating six carbons from the end
and n-3 indicating three carbons from the end. This difference in chemical structure
affects the flexibility of the molecule so that n-3 fatty acids are more flexible at low
temperatures, which accounts for their enrichment in the tissues of coldwater fish
and aqueous plants and algae. They also have different effects on immune function.
The n-6 fats are more proinflammatory and in some instances help the body mount
an inflammatory response in the face of a threatening pathogen. However, when
found in large excess as in the modern diet, the n-6 fat imbalance versus n-3 fats
results in a chronic inflammatory state commonly found in individuals with excess
abdominal visceral fat and obesity.
These two fats are essential at very low levels in the diet—up to 5% of calories—
in order to maintain normal cell function and support normal growth. Essential fatty
acid deficiency leads to death in animals that develop fatal dermatitis. However,
essential fatty acid deficiency is unknown in humans with an intact gastrointestinal
tract. Individuals dependent on parenteral nutrition due to the loss of gastrointestinal
function are dependent on essential fats for survival.
The practical importance of understanding the differences between n-3 and n-6
fats derives from the fact that there is a great deal of global diversity in these fats.
Hibbeln, Lands, and co-workers have calculated the proportion of long-chain fatty
acids greater than 20 carbons in the diets of many countries and found a variation of
the n-6 proportion from 32% to 87% [6]. Moreover, the dietary diversity in these pro-
portions is reflected in blood samples obtained by fingerstick. It has also been shown
that it is possible to change the blood levels of fatty acids to alter the proportions in a
beneficial direction. This thesis [7] is described in much greater detail by Dr. Lands
in a subsequent chapter of this book.
RELATIONSHIP OF EXCESS FAT CALORIES AND

VISCERAL FAT TO INFLAMMATION
Ad libitum intake of hidden fats in foods combined with a sedentary lifestyle leads
to positive calorie balance and a stimulus that results in growth of intra-abdominal
fat and fat in the liver, pancreas, pericardium and muscle, periprostatic fat, and fat
in the outer quadrant of the breasts and other so-called ectopic sites. In addition,
there is an association between inflammation of excess visceral fat and inflamma-
tion in the brain. The low-grade chronic inflammation associated with excess intra-
abdominal fat, unlike the acute inflammatory response to pathogens or injury, does
not resolve spontaneously. It persists as a tonic low-grade activation of the innate
immune system that affects metabolism. In addition to this chronic inflammation,
there are recurrent acute episodes of nutrition-related immune activation secondary
to high-fat/high-carbohydrate meals, which are associated with oxidant stress and
formation of oxidized phospholipids [8–10].
Inflammation is now accepted as an integral component in both the etiology of obe-
sity and in its associated complications including type 2 diabetes mellitus, heart dis-
ease, and common forms of cancer. Adipose tissue in obese individuals demonstrates
increased expression of genes that are found in inflammation and are characteristic
of activated macrophages [11,12]. Moreover, there are increases in circulating proin-
flammatory cytokines [13] and decreases in anti-inflammatory adipocytokines (e.g.,
adiponectin) [14]. Abdominal visceral fat is a limited storage depot for fat as it carries
out its function as a portable storage of fat calories. As long as the amount of fat is in
the physiological range and is not growing rapidly, the adipose tissue macrophages
(ATM) remain in the unstimulated or M2 alternatively activated state. However,
following rapid growth of intra-abdominal fat where there is evidence of dead fat
cells missing their perilipin protein, there is activation of M1 classically activated
macrophages, which impair insulin signaling and adipogenesis [15] as well as some
T cells [16]. Insulin sensitivity is also impaired in the liver in parallel secondary to
the effects of interactions of immune cells and hepatocytes [17,18].
Increasing abdominal-fat deposition and growth of this fatty tissue involves
angiogenesis, which forms neovascularization analogous to what occurs with tumor
growth. As with tumors, the tissue is poorly oxygenated, and some fat cells ultimately
die, sending the necessary signal to mobilize the ingress of immune cells as well as
a shift in the inflammatory profile of macrophages from M2 to M1, while the M2
state is linked to the activity of peroxisome proliferator-activated receptors (PPARs)
gamma and delta [19]. Adipose tissue also contains potent tolerogenic CD4+ Tregs
that are downregulated by obesity, another potential initiating event in inflamma-
tion [16,20]. On the other hand, visceral obesity induces an increase in expression
of GPR120, an omega-3 fatty acid receptor that can reduce M1 macrophage activa-
tion while increasing M2 gene expression. As discussed further in the following,
this adaptation limits inflammation. There are as many as 30 million macrophages
in each kilogram of excess fat, so that the mass of inflammatory cells is markedly
increased in visceral obesity [21].
In addition, there is apoptotic cell death and tissue hypoxia [22–24] occurring at
the same time as changes in macrophage expression profile toward a more inflamma-
tory state [22,25]. While there are some studies in animals which attempt to describe
a logical temporal evolution of the inflammatory state associated with abdominal
visceral fat accumulation, there may be parallel events which make such a theoreti-
cal construct impossible to test. In addition to fat in the abdominal visceral depot,
there is fat that occurs outside this area, and it is called ectopic fat.
Ectopic fat in the liver can be an important marker of metabolic syndrome. Fat is
normally stored in the liver, but when inflammation is present, this is called nonalco-
holic steatosis hepatis (NASH). While it is estimated that 80% of obese individuals
have excess liver fat with the best marker being elevated triglycerides in the blood,
only a small fraction have NASH. A small fraction of those go on to liver failure or
cirrhosis. However, a small portion of a very large number is a large number, and
fatty liver and NASH are the third leading cause of liver transplantation after viral
hepatitis and alcoholism. NASH is forecast to increase markedly in incidence and
eclipse these other causes in the next few decades. NASH is marked by an increase
in total and M1 macrophages consistent with what has been observed in abdominal
fat [26–28]. Kupffer cells, which are immune cells normally found in the liver, may
be involved, or it may simply be the immune cells entering as the result of processes
coincident with the accumulation of visceral fat [29].
Muscle can be infiltrated with fat, and there is increased cytokine production
in muscle with obesity and insulin resistance. Muscle cells can become inflamed
directly via an innate immune response through TLR4 receptors [30] or from infil-
trating M1 macrophages [31,32]. As discussed in the chapter on muscle and immune
function (see Chapter 15), muscle cells also release anti-inflammatory myokines, but
only when they contract. Therefore, the fat deposition with proinflammatory cyto-
kines may be counteracted by exercising muscles. The exact roles of the myokines
and fat-cell adipokines have yet to be established.
SUBCELLULAR SIGNALING PATHWAYS LINKING

INFLAMMATION AND METABOLISM
The interface between inflammation and the metabolic abnormalities linked to
abdominal visceral fat and obesity has established a central role for immune func-
tion in mediating many of the disorders associated with obesity. Pattern recognition
receptors (PRRs) are cellular sensors of the innate immune system. When pathogen-
associated molecular patterns (PAMP) are detected by PRR, they initiate an immune
response. For example, TLR4 can be activated by free fatty acids to generate a pro-
inflammatory response via the NF-κB pathway [33]. Adipose tissue expresses nearly
all known TLR receptors, and in mice genetically modified to lack TLR-2, their
response to diet-induced obesity with insulin resistance is reduced [34,35]. TLRs
also sense gut microbes in a way that affects metabolism. TLRs act via NF-κB
dependent activation of inflammatory gene transcription [36]. In vivo imaging has
localized NF-κB activation during diet-induced obesity in mice to the adipose tissue
and associated macrophages [37]. The NOD-like receptor (NLR) family are another
group of PRRs, which can sense danger signals from stressed or dying cells and
mobilize leukocytes toward these stimuli to constrain tissue damage [38,39]. Within
the macrophage, NLR activation stimulates the inflammasome to induce IL-1β and
IL-18 from their prehormone forms through the action of caspase-1. This pathway
has been implicated in the loss of beta cells in the pancreas during the progression
of type 2 diabetes mellitus [40].
Another link between metabolism and inflammation may be the balance
between other intracellular lipid species including ceramides and sphingolipids
[41,42]. Inhibition of ceramide production blocks the ability of saturated FAs to
induce insulin resistance [43]. The induction of ceramide synthesis by LPS and
saturated FA is dependent upon TLR4 in many cells including those in the hypo-
thalamus and muscle, where ceramide production inhibits insulin signaling through
Akt [44]. Adiponectin, an anti-inflammatory cytokine from fat cells, stimulates
ceramidase activity and modulates the balance between ceramides and sphingo-
sine-1-phosphate [45].
Obesity can also activate JNK in insulin-responsive tissues, probably through
upstream pathways shared by IKK/NF-κB when stress signals such as fatty acids,
insulin, hyperglycemia, and inflammatory cytokines are detected [46]. ER stress and
the downstream activation of the molecular pathways mediating the unfolded protein
response are linked to both JNK1 and IKK/NF-κB activation pathways in adipose
and other tissues [47].
EICOSANOIDS
Changing the intake of n-3 fatty acid–rich fat relative to n-6 fatty acid–rich fat can
be achieved both by lowering the total intake of fat, since most processed foods con-
tain a large excess of omega-6, and increasing the intake of coldwater fish or fish-oil
supplements. Reducing total fat intake lowers the amount of arachidonic acid (AA)
made from linoleic acid. Fish oil, algal oil, and fish intake can increase eicosapen-
taenoic acid (EPA) and docosahexaenoic acid (DHA) measured in blood, red cell
membranes, and ultimately tissues. Numerous studies have demonstrated a multi-
plicity of functional effects of n-3 fatty acids in human physiology, human diseases,
and animal models [48–52]. These include effects on plasma lipids and lipoproteins
[53], eicosanoid metabolism, platelet–vessel wall interactions, blood viscosity, arte-
rial blood pressure, coagulation, cytokines, and growth factors.
The mediators of the immune responses triggered by the fatty acids found in tri-
glycerides in dietary fat are mediated by metabolic products called eicosanoids [54].
The key to understanding the effects of different types of dietary fats is to realize
that at key points in the enzymatic pathways leading to the production of eicosanoids,
these different fatty acids compete as substrates for the same enzymes. So, the end
products of the metabolism of fatty acids, namely eicosanoids, are influenced by
this competition. There are four types of eicosanoids—prostaglandins, prostacy-
clins, thromboxanes, and leukotrienes. For each type, there are two or three separate
series, derived either from an n-3 or n-6 fatty acid. Products coming from the metab-
olism of arachidonic acid (20:4 n-6) influence over 20 eicosanoid-mediated signaling
pathways in the cell including impacts on inflammation and immune function.
The eicosanoids are discussed in greater detail in Chapter 18 on balancing n-3
and n-6 dietary fats.
In addition, dietary fats can alter the composition of the cell membrane including
the composition of lipid rafts [55]. Lipid rafts are domains in the plasma membrane
that contain high concentrations of cholesterol and glycosphingolipids. They exist as
distinct liquid-ordered regions of the membrane that are resistant to extraction with
nonionic detergents. Rafts appear to be small in size, but may constitute a relatively
large fraction of the plasma membrane. While rafts have a distinctive protein and
lipid composition, all rafts do not appear to be identical in terms of either the proteins
or the lipids that they contain. A variety of proteins, especially those involved in cell
signaling, have been shown to partition into lipid rafts. As a result, lipid rafts are
thought to be involved in the regulation of signal transduction. For example, andro-
gen receptors in lipid rafts in the prostate-cancer cell membrane can activate tyrosine
kinase, leading to a nonnuclear action of androgens in these cells. Experimental evi-
dence suggests that there are probably several different mechanisms through which
rafts control cell signaling. For example, rafts may contain incomplete signaling
pathways that are activated when a receptor or other required molecule is recruited
into the raft. Rafts may also be important in limiting signaling, either by physical
sequestration of signaling components to block nonspecific interactions, or by sup-
pressing the intrinsic activity of signaling proteins present within rafts.
Dietary fats can also affect cytokine synthesis and directly affect gene expression
[56]. Figure 3.1 illustrates the various n-3 and n-6 enzymatic pathways, along with
n-3 Fatty acids: ALA SDA ETA EPA PGE3
δ-6 δ-5
–
LA GLA DGLA AA LTB4

n-6 Fatty acids:
PGE2
FIGURE 3.1 Metabolism of n-6 and n-3 PUFAs. δ-5 and δ-6 desaturase enzymes (ovals)
are active in both n-3 and n-6 fatty acid metabolism, converting intermediate-chain n-3
ALA to long-chain n-3 EPA and n-6 linoleic acid (LA) to n-6 arachidonic acid (AA). EPA
is converted to prostaglandin E3 (PGE3), an eicosanoid with potential anti-inflammatory and
antithrombotic effects, whereas AA is converted to PGE2 and leukotriene B4 (LTB4), both
proinflammatory eicosanoids. Thus, n-3 and n-6 fatty acids compete for common metabolic
enzymes, and relative intake of these fatty acids has been hypothesized to determine potential
proinflammatory versus anti-inflammatory, thrombotic, and aggregatory effects. Metabolites
in these pathways also exert feedback inhibition (black arrows); for example, long-chain n-3
EPA inhibits an important step in the elongation of intermediate-chain n-3 ALA. SDA indi-
cates stearidonic acid (octadecatetranoic acid); ETA, eicosatetraenoic acid; GLA, γ-linolenic
acid; and DGLA, dihomo-γ-linolenic acid. (From Mozaffarian, D. et al., Circulation, 111(2),
157, January 18, 2005. With permission.)
the major eicosanoids from AA, EPA, and DGLA. DGLA and EPA compete with
AA for the actions of the lipoxygenase and cyclooxygenase enzymes. Competing
with arachidonic acid is eicosapentanoic acid (20:5, n-3) and, to a lesser extent, diho-
mogammalinoleic acid (20:3 n-6). Low dietary intake of these fatty acids, especially
the n-3 series, has been implicated in the progression of heart disease and in brain
health. There is emerging science suggesting that dietary n-3 fatty acids may be use-
ful in psychiatric disorders.
LIPID RAFTS AND CELLULAR SIGNALING

There are several different mechanisms through which rafts can control cell sig-
naling. Lipid rafts may contain incomplete signaling pathways that are activated
when a receptor or other required molecule is recruited into the raft. On the other
hand, lipid rafts may also be important in limiting signaling, either by physically
containing the receptor and other signaling components to block nonspecific inter-
actions or by inhibiting the activity of signaling proteins present within the rafts.
Lipid rafts may modulate cell function and be affected by lipids and nutrients
leading to alterations in lipid metabolism, since important membrane-signaling
proteins are located within the raft regions of the membrane, and alterations in raft
structure can alter activity of these signaling proteins. Therefore, lipid rafts which
are lipid based have a composition, structure, and function that are susceptible to
manipulation by dietary components such as omega-3 polyunsaturated fatty acids

and by cholesterol depletion.
Many receptor tyrosine kinases, including the EGF receptor, the PDGF receptor,
and the insulin receptor, have been shown to be localized to lipid rafts [56–60]. EGF
receptors rapidly move out of lipid rafts upon activation by ligands [61], a behavior
that is unique among receptor tyrosine kinases. In cells that contain caveolae, insulin
receptors are constitutively sequestered in caveolae [62]. However, in cells that lack
caveolae, insulin receptors are recruited into rafts by the addition of insulin. The
localization of PDGF and nerve growth factor (NGF) receptors to rafts appears to be
relatively unaffected by ligands [56,63]. The functional implications of such changes
in receptor compartmentalization is unclear; however, a rough correlation between
the effect of ligands on receptor localization and the effect of cholesterol depletion
on receptor-mediated signaling suggests that those receptors that remain in, or are
recruited to, rafts following ligand binding are much more dependent on raft integ-
rity for function than are receptors that exit rafts upon ligand binding.
A large number of G protein–coupled receptors have been shown to be enriched in
lipid rafts or caveolae. This includes beta-adrenergic receptors, adenosine A1 recep-
tors, angiotensin II type 1 receptors, EDG-1 receptors, endothelin receptors, musca-
rinic cholinergic receptors [64], rhodopsin [65], and bradykinin receptors [66–73].
Like the receptor tyrosine kinases, the localization of G protein–coupled receptors
to lipid rafts is modulated by ligands. For the beta-adrenergic receptor [74,75] and
the adenosine A1 receptor [70], treatment with agonists causes translocation of the
receptor out of lipid rafts or caveolae. By contrast, the angiotensin II type 1 recep-
tor [75], the muscarinic receptor [64], the EDG-1 receptor [71], and the bradykinin
receptor [72,73,76] are targeted to rafts upon activation by agonists. The localiza-
tion of the endothelin receptor is apparently unaffected by agonists [66]. Cholesterol
depletion generally impairs G protein–mediated signaling.
Oh et al. characterized the tissue expression patterns of GPR120, with an expres-
sion profile that correlated well with a potential role in regulating metabolism [1].
GPR120 is an orphan receptor for which no endogenous ligands are known. It was
found that GPR120 was expressed in macrophages found in adipose tissue, in adi-
pocytes, and in Kupffer cells. When mice were fed a high-fat diet, the expression of
this receptor was increased, consistent with the hypothesis that GPR120 could be
controlled by inflammatory signals. The omega-3 fatty acids DHA, EPA, and pal-
mitoleate were agonists of GPR120. In addition, when DHA activated the GPR120
receptor, it antagonized the inflammatory effects of TNF-alpha and lipopolysaccha-
ride in a macrophage cell line. DHA not only blocked the NF-κB and JNK pathways
but also prevented expression of inflammatory cytokines by the macrophages.
GPR120 is known to couple with a family of G proteins. After the omega-3
ligand binds and a specific G protein (Gq/11) is released, the G protein receptor
kinases phosphorylate the receptor. The phosphorylation results in the appearance
of binding sites for beta-arrestins, which mediate internalization and downregulation
of the receptors. However, beta-arrestins also interact with downstream signaling
molecules [77] mediating the anti-inflammatory effects of omega-3 fatty acids in
macrophages. In fact, beta-arrestin inhibits both the JNK and NF-κB intracellular
signaling pathways by sequestering specifically the TAK1-binding protein TAB1.
The inhibition of TAB1 prevents phosphorylation and thus activation of IκB kinase
upstream of NFκB and MKK4 (mitogen-activated protein kinase kinase 4) upstream
of JNK [78]. This work has demonstrated the cellular and molecular mechanisms
underlying the anti-inflammatory effects of omega-3 fatty acids on macrophages.
Clearly, these are not the only metabolic effects as Dr. William Lands will expand
upon the biochemical effects of competition between omega-3 and omega-6 fatty
acids in the production of eicosanoids.
However, studies in mice fed a high-fat diet with low levels of omega-3 fatty acids
resulted in activation of the immune system, insulin resistance, and hepatic steatosis,
all of which was prevented when this diet was supplemented with omega-3 fatty
acids (DHA and EPA).
Transgenic animals who were lacking a functioning GPR120 receptor and fed a
high-fat diet did not benefit from supplementation with omega-3 fatty acids. These
experiments elegantly demonstrated that GPR120 plays an important role in the met-
abolic benefits of DHA and EPA. Mice receiving bone marrow transplants from the
GPR120-deficient mice with macrophages lacking the GPR 120 receptor were also
resistant to the beneficial properties of DHA and EPA. Thus, the omega-3 fatty acids
appear to act primarily through macrophages.
Taken together with previous data, these findings support a model in which
dietary fatty acids control the inflammatory properties of macrophages in adipose
tissue by regulating the activity and expression of opposing receptors. With a normal
diet containing a balanced ratio of saturated and omega-3 unsaturated fatty acids,
anti-inflammatory M2 macrophages protect adipose cells by dampening excess
inflammation and maintaining insulin sensitivity of fat cells [78]. When mice are
given a high-fat diet with excess calories and little omega-3 fatty acids, TLR4 is
left unchecked in fat cells (see Figure 3.2). The activated receptor induces expres-
sion and release of chemokines, such as MCP-1 (monocyte chemotactic protein-1),
which then recruit proinflammatory M1 macrophages into adipose tissue [78]. These
cells produce cytokines, such as TNF-alpha, which further activate the macrophages
and attenuate insulin action in adipocyte, ultimately leading to insulin resistance.
Activated M1 macrophages express elevated levels of GPR120. Thus, addition of
omega-3 fatty acids to the diet stimulates GPR120 and initiates a signaling pathway
through b-arrestin2, which blocks the effects of TLR4 and inflammatory cytokine
receptors. This reduces the inflammatory state of these cells and simultaneously
promotes the return of anti-inflammatory M2 macrophages to adipose tissue, which
leads to the restoration of insulin sensitivity.
Despite these elegant findings in animal models, more research is needed to
determine whether omega-3 dietary supplements and increased consumption of
omega-3-rich fish can provide high enough concentrations of circulating omega-3
fatty acids to promote GPR120 activation. Nevertheless, the new insights presented
by these studies into the anti-inflammatory mechanisms of omega-3 fatty acids pro-
vide a platform for investigating these important questions. The identification of the
GPR120 receptor provides a biomarker for use in clinical investigations of the effects
of omega-3 fatty acids in chronic diseases of aging including type 2 diabetes mel-
litus, cardiovascular disease, and common forms of cancer. Our group is currently
studying these receptors in a clinical trial in prostate cancer patients.
High-fat diet High-fat diet

Low-fat diet Low ω-3 fatty acids High ω-3 fatty acids
Interleukin-10
Interleukin-10 Arginase
Polyunsaturated
Arginase Saturated ω-3 fatty acids
fatty acids TNFα
DHA, EPA
Insulin sensitive MCP1

Insulin resistant Insulin sensitive
ω-3 M1 macrophage
M2 macrophage Saturated
GPR120 fatty acids
(anti-inflammatory) fatty acids
M1 macrophage NFκB
(proinflammatory) JNK
Adipose cell TLR4 β-Arrestin Cytokines

TNFα
FIGURE 3.2 (See color insert.) A high-fat diet with a disproportionate ratio of saturated
fatty acids to ω-3 fatty acids triggers activation of Toll-like receptor 4 (TLR4) in adipocytes
and circulating immune cells. This launches an inflammatory cascade that results in the
recruitment of proinflammatory M1 macrophages, increased secretion of TNFα, and insulin
resistance in adipocytes. The addition of ω-3 fatty acids to the diet activates the G protein-
coupled receptor GPR120 on proinflammatory M1 macrophages (Oh et al., 2010), which in
turn attenuates the inflammatory response and recruits anti-inflammatory M2 macrophages
to adipose tissue. Eventually, these M2 macrophages restore secretion of interleukin-10 and
improve insulin sensitivity. (Courtesy of A.R. Saltiel, Life Sciences Institute, Departments
of Internal Medicine and Molecular and Integrative Physiology, University of Michigan
Medical School, Ann Arbor, MI. With permission.)
CONCLUSION
This chapter has summarized the role of lipids in triggering inflammation at a cel-
lular and molecular level. Much of the evidence for the role of lipids at a cellular level
comes from animal studies demonstrating that when excess lipids are consumed,
there is growth of abdominal adipocytes and deposition of triglycerides in the liver.
These events trigger a hormonal response in the body as the result of insulin resis-
tance and a secondary inflammatory response in adipose tissue both in the abdomi-
nal visceral fat and in other fat depots in the pancreas, muscle, and hypothalamus.
At a cellular level, the accumulation of lipids and specific fatty acids can affect the
physiological function of adipocyte tissue macrophages, which originate in the bone
marrow and enter the abdominal visceral fat from the circulation in order to phago-
cytize dead fat cells which have outgrown their blood supply from neovasculature
formed during the growth of adipose tissue, as discussed in separate chapters on
abdominal adiposity in diabetes and obesity. At a molecular level, the role of specific
types of fatty acids in these tissues has been demonstrated in cell culture and animal
studies. Saturated fatty acids, omega-3 fatty acids, and other polyunsaturated fatty
acids as well as monounsaturated fatty acids have all been studied and specific sig-
naling pathways identified at a cellular level. Among the most well recognized of
these are the pathways mediated by saturated fatty acids and those mediated by the
competition between omega-3 and omega-6 fatty acids, which results in a balance
of proinflammatory or anti-inflammatory eicosanoids in cells which in turn activate
inflammatory responses in cells.
REFERENCES
1. Oh DY, Talukdar S, Bae EJ et al. 2010. GPR120 is an omega-3 fatty acid receptor medi-
ating potent anti-inflammatory and insulin-sensitizing effects. Cell 142:687–698.
2. Burr GO, Burr MM, Miller ES. 1932. On the fatty acids essential in nutrition. J Biol
Chem 97:1–9.
3. Thomasson HJ. 1962. Essential fatty acids. Nature 194:973.
4. Hansen AE, Wiese HF, Boelsch AN et al. 1963. Role of linoleic acid in infant nutrition.
Clinical and chemical study of 428 infants fed on milk mixtures varying in kind and
amount of fat. Pediatrics 31:171–192.
5. Lands WEM. 2005. Fish, Omega-3 and Human Health, 2nd edn., AOCS Press:
Champaign, IL.
6. Hibbeln JR, Nieminen LR, Blasbalg TL, Riggs JA, Lands WEM. 2006. Healthy intakes
83:1483S–1493S.
7. Freeman MP, Hibbeln JR, Wisner KL et al. 2006. Omega-3 fatty acids: Evidence basis
for treatment and future research in psychiatry. J Clin Psychiatry 67:1954–1967.
8. Kosteli A, Sugaru E, Haemmerle G et al. 2010. Weight loss and lipolysis promote a
dynamic immune response in murine adipose tissue. J Clin Invest 120:3466–3479.
9. Alipour A, Elte JW, van Zaanen HC, Rietveld AP, Cabezas MC. 2007. Postprandial
inflammation and endothelial dysfunction. Biochem Soc Trans 35:466–469.
10. Blackburn P, Després JP, Lamarche B et al. 2006. Postprandial variations of plasma
inflammatory markers in abdominally obese men. Obesity (Silver Spring) 14:1747–1754.
11. Chen Y, Zhu J, Lum PY et al. 2008. Variations in DNA elucidate molecular networks
that cause disease. Nature 452:429–435.
12. Emilsson V, Thorleifsson G, Zhang B et al. 2008. Genetics of gene expression and its
effect on disease. Nature 452:423–428.
13. Hotamisligil GS, Arner P, Caro JF, Atkinson RL, Spiegelman BM. 1995. Increased
adipose tissue expression of tumor necrosis factor-alpha in human obesity and insulin
resistance. J Clin Invest 95:2409–2415.
14. Hotta K, Funahashi T, Arita Y et al. 2000. Plasma concentrations of a novel, adipose-
specific protein, adiponectin, in type 2 diabetic patients. Arterioscler Thromb Vasc Biol
20:1595–1999.
15. Lumeng CN, Del Proposto JB, Westcott DJ, Saltiel AR. 2008. Phenotypic switching of
adipose tissue macrophages with obesity is generated by spatiotemporal differences in
macrophage subtypes. Diabetes 57:3239–3246.
16. Feuerer M, Herrero L, Cipolletta D et al. 2009. Lean, but not obese, fat is enriched for
a unique population of regulatory T cells that affect metabolic parameters. Nat Med
15:930–939.
17. Varma V, Yao-Borengasser A, Rasouli N et al. 2009. Muscle inflammatory response and
insulin resistance: Synergistic interaction between macrophages and fatty acids leads to
impaired insulin action. Am J Physiol Endocrinol Metab 296:E1300–E1310.
18. Huang W, Metlakunta A, Dedousis N et al. 2010. Depletion of liver Kupffer cells
prevents the development of diet-induced hepatic steatosis and insulin resistance.
Diabetes 59:347–357.
19. Odegaard JI, Chawla A. 2011. Alternative macrophage activation and metabolism. Annu
Rev Pathol 6:275–297.
20. Winer S, Chan Y, Paltser G et al. 2009. Normalization of obesity-associated insulin
resistance through immunotherapy: CD4+ T cells control glucose homeostasis. Nat Med
15:921–929.
21. O’Rourke RW, Metcalf MD, White AD et al. 2009. Depot-specific differences in inflam-
matory mediators and a role for NK cells and IFN-gamma in inflammation in human
adipose tissue. Int J Obes (Lond) 33:978–990.
22. Strissel KJ, Stancheva Z, Miyoshi H et al. 2007. Adipocyte death, adipose tissue
remodeling, and obesity complications. Diabetes 56:2910–2918.
23. Hosogai N, Fukuhara A, Oshima K et al. 2007. Adipose tissue hypoxia in obesity and its
impact on adipocytokine dysregulation. Diabetes 56:901–911.
24. Ozcan U, Cao Q, Yilmaz E et al. 2004. Endoplasmic reticulum stress links obesity,
insulin action, and type 2 diabetes. Science 306(5695):457–461.
25. Nishimura S, Manabe I, Nagasaki M et al. 2009. CD8+ effector T cells contribute to mac-
rophage recruitment and adipose tissue inflammation in obesity. Nat Med 15:914–920.
26. Li Z, Soloski MJ, Diehl AM. 2005. Dietary factors alter hepatic innate immune system
in mice with nonalcoholic fatty liver disease. Hepatology 42(4):880–885.
27. Kremer M, Hines IN, Milton RJ, Wheeler MD. 2006. Favored T helper 1 response in a
mouse model of hepatosteatosis is associated with enhanced T cell-mediated hepatitis.
Hepatology 44:216–227.
28. Baffy G. 2009. Kupffer cells in non-alcoholic fatty liver disease: The emerging view.
J Hepatol 51:212–223.
29. Obstfeld AE, Sugaru E, Thearle M et al. 2010. C-C chemokine receptor 2 (CCR2)
regulates the hepatic recruitment of myeloid cells that promote obesity-induced hepatic
steatosis. Diabetes 59:916–925.
30. Frisard MI, McMillan RP, Marchand J et al. 2010. Toll-like receptor 4 modulates skeletal
muscle substrate metabolism. Am J Physiol Endocrinol Metab 298:E988–E998.
31. Patsouris D, Li PP, Thapar D, Chapman J, Olefsky JM, Neels JG. 2008. Ablation of
CD11c-positive cells normalizes insulin sensitivity in obese insulin resistant animals.
Cell Metab 8:301–309.
32. Hong EG, Ko HJ, Cho YR et al. 2009. Interleukin-10 prevents diet-induced insulin resis-
tance by attenuating macrophage and cytokine response in skeletal muscle. Diabetes
58:2525–2535.
33. Fessler MB, Rudel LL, Brown JM. 2009. Toll-like receptor signaling links dietary fatty
acids to the metabolic syndrome. Curr Opin Lipidol 20:379–385.
34. Poulain-Godefroy O, Le Bacquer O, Plancq P et al. 2010. Inflammatory role of Toll-like
receptors in human and murine adipose tissue. Mediators Inflamm 2010:823486.
35. Himes RW, Smith CW. 2010. Tlr2 is critical for diet-induced metabolic syndrome in a
murine model. FASEB J 24:731–739.
36. Baker RG, Hayden MS, Ghosh S. 2011. NF-kappaB, inflammation, and metabolic
disease. Cell Metab 13:11–22.
37. Chiang S-H, Bazuine M, Lumeng CN et al. 2009. The protein kinase IKKepsilon
regulates energy balance in obese mice. Cell 138:961–975.
38. Chen G, Shaw MH, Kim YG, Nunez G. 2009. NOD-like receptors: Role in innate
immunity and inflammatory disease. Annu Rev Pathol 4:365–398.
39. Schroder K, Zhou R, Tschopp J. 2010. The NLRP3 inflammasome: A sensor for
metabolic danger? Science 327:296–300.
40. Zhou R, Tardivel A, Thorens B, Choi I, Tschopp J. 2010. Thioredoxin-interacting

protein links oxidative stress to inflammasome activation. Nat Immunol 11:136–140.
41. Hannun YA, Obeid LM. 2008. Principles of bioactive lipid signalling: Lessons from
sphingolipids. Nat Rev Mol Cell Biol 9:139–150.
42. Summers SA. 2010. Sphingolipids and insulin resistance: The five Ws. Curr Opin
Lipidol 21:128–135.
43. Holland WL, Brozinick JT, Wang LP et al. 2007. Inhibition of ceramide synthesis ame-
liorates glucocorticoid-, saturated-fat-, and obesity-induced insulin resistance. Cell
Metab 5(3):167–179.
44. Holland WL, Bikman BT, Wang LP et al. 2011. Lipid-induced insulin resistance medi-
ated by the proinflammatory receptor TLR4 requires saturated fatty acid–induced
ceramide biosynthesis in mice. J Clin Invest 121:1858–1870.
45. Holland WL, Miller RA, Wang ZV et al. 2011. Receptor-mediated activation of cerami-
dase activity initiates the pleiotropic actions of adiponectin. Nat Med 17(1):55–63.
46. Solinas G, Karin M. 2010. JNK1 and IKKbeta: Molecular links between obesity and
metabolic dysfunction. FASEB J 24:2596–2611.
47. Hotamisligil GS. 2010. Endoplasmic reticulum stress and the inflammatory basis of
metabolic disease. Cell 140:900–917.
48. Lands WEM. 1986. Fish and Human Health. Orlando, FL: Academic Press.
49. Kinsella JE. 1987. Seafoods and fish oils in human health and disease. In: Kinsella JE,
ed., Food Science and Technology, Vol. 23. New York: Marcel Dekker.
50. Simopoulos AP, Kifer RR, Martin RE, Barlow SM, eds. 1991. Health effects of ω3 poly-
unsaturated fatty acids in seafoods. In: World Review of Nutrition and Dietetics, Vol. 66.
Basel, Switzerland: Karger.
51. De Caterina R, Kristensen SD, Schmidt EB, eds. 1992. Fish oil and vascular disease. In:
Current Topics in Cardiovascular Disease. Verona, Italy: Bi & Gi Publishers.
52. De Caterina R, Endres S, Kristensen SD, Schmidt EB, eds. 1993. n-3 fatty acids and
vascular disease. In: Current Topics in Cardiovascular Disease. Verona, Italy: Bi & Gi
Publishers.
53. Harris WS. 1989. Fish oils and plasma lipid and lipoprotein metabolism in humans:
A critical review. J Lipid Res 30:785–807.
54. Funk CD. 2001. Prostaglandins and leukotrienes: Advances in eicosanoid biology.
Science 294:1871–1875.
55. Sonnino S, Prinetti A. 2013. Membrane domains and the “lipid raft” concept. Curr Med
Chem 20(1):4–21.
56. Liu P, Ying Y, Ko Y, Anderson RGW. 1996. Localization of platelet-derived growth
factor-stimulated phosphorylation cascade to caveolae. J Biol Chem 271:10299–10303.
57. Mineo C, James GL, Smart EJ, Anderson RGW. 1996. Localization of epidermal
growth factor-stimulated Ras/Raf-1 interaction to caveolae membrane. J Biol Chem
271:11930–11935.
58. Gustavsson J, Parpal S, Karlsson M et al. 1999. Localization of the insulin receptor in
caveolae of adipocyte plasma membrane. FASEB J 13:1961–1971.
59. Wu C, Butz S, Ying Y, Anderson RGW. 1997. Tyrosine kinase receptors concentrated in
caveolae-like domains from neuronal plasma membrane. J Biol Chem 272:3554–3559.
60. Bilderback TR, Grigsby RJ, Dobrowsky RT. 1997. Association of p75NTR with cave-
olin and localization of neurotrophin induced sphingomyelin hydrolysis to caveolae.
J Biol Chem 272:10922–10927.
61. Mineo C, Gill GN, Anderson RGW. 1999. Regulated migration of epidermal growth
factor receptor from caveolae. J Biol Chem 274:30636–30643.
62. Vainio S, Heino S, Mansson J-E et al. 2002. Dynamic association of human insulin
receptor with lipid rafts in cells lacking caveolae. EMBO Rep 3:95–100.
63. Huang C-S, Zhou J, Feng AK et al. 1999. NGF signaling in caveolae-like domains at the
plasma membrane. J Biol Chem 274:36707–36714.
64. Feron O, Smith TW, Michel T, Kelly RA. 1997. Dynamic targeting of the agonist-
stimulated m2 muscarinic acetylcholine receptor to caveolae in cardiac myocytes. J Biol
Chem 272:17744–17748.
65. Seno K, Kishimoto M, Abe M et al. 2001. Light- and guanosine 5-3-O-(thio)triphosphate-
sensitive localization of a G protein and its effector on detergent-resistant membrane
rafts in rod photoreceptor outer segments. J Biol Chem 276:20813–20816.
66. Chu M, Liyanage UK, Lisanti MP, Lodish HF. 1994. Signal transduction of a G protein-
coupled receptor in caveolae: Colocalization of endothelin and its receptor with caveo-
lin. Proc Natl Acad Sci USA 91:11728–11732.
67. Rybin VO, Xu X, Lisanti MP, Steinberg SF. 2000. Differential targeting of ß-adrenergic
receptor subtypes and adenylyl cyclase to cardiomyocyte caveolae. J Biol Chem
275:41447–41457.
68. Xiang Y, Rybin VO, Steinberg SF, Kobilka BK. 2002. Caveolar localization dictates
physiologic signaling of β2-adrenoceptors in neonatal cardiac myocytes. J Biol Chem
277:34280–34286.
69. Ostrom RS, Gregorian C, Drenan RM et al. 2001. Receptor number and caveolar co-
localization determine receptor coupling efficiency to adenylyl cyclase. J Biol Chem
276:42063–42069.
70. Lasley RD, Narayan P, Uittenbogaard A, Smart EJ. 2000. Activated cardiac adenosine
A1 receptors translocate out of caveolae. J Biol Chem 275:4417–4421.
71. Igarashi J, Michel T. 2000. Agonist-modulated targeting of the EDG-1 receptor to plas-
malemmal caveolae. J Biol Chem 275:32363–32370.
72. de Weerd WFC, Leeb-Lundberg LMF. 1997. Bradykinin sequesters B2 bradykinin
receptors and the receptor-coupled Gα subunits Gαq and Gαi in caveolae in DDT1 MF-2
smooth muscle cells. J Biol Chem 272:17858–17866.
73. Haaseman M, Cartaud J, Muller-Esterl W, Dunia I. 1998. Agonist-induced redistribution
of bradykinin B2 receptor in caveolae. J Cell Sci 111:917–928.
74. Dessy C, Kelly R, Balligand JL, Feron O. 2000. Dynamin mediates caveolar seques-
tration of muscarinic cholinergic receptors and alteration in NO signaling. EMBO J
19:4272–4280.
75. Ishizaka N, Griendling K, Lassegue B, Alexander RW. 1998. Angiotensin II Type 1
receptor: Relationship with caveolae and caveolin after initial agonist stimulation.
Hypertension 32:459–466.
76. Sabourin T, Bastien L, Bachvarov DR, Marceau F. 2002. Agonist-induced translocation
of the kinin B(1) receptor to caveolae-related rafts. Mol Pharmacol 61:546–553.
77. Rajagopal S, Kim J, Ahn S et al. 2010. Beta-arrestin-but not G protein-mediated
signaling by the “decoy” receptor CXCR7. Proc Natl Acad Sci USA 107:628–632.
78. Lumeng CN, Bodzin JL, Saltiel AR. 2007. Obesity induces a phenotypic switch in
adipose tissue macrophage polarization. J Clin Invest 117:175–184.
79. Mozaffarian D, Ascherio A, Hu FB et al. 2005. Interplay between different polyunsatu-
rated fatty acids and risk of coronary heart disease in men. Circulation 111(2):157–164.
4 Biomarkers of
Inflammation and
the Western Diet
CONTENTS
Introduction............................................................................................................... 53
Mechanisms Underlying Chronic Low-Grade Inflammation................................... 54
C-Reactive Protein and Other Acute-Phase Reactants.............................................. 54
Proinflammatory Cytokines...................................................................................... 55
Chemokines............................................................................................................... 56
Telomere Length....................................................................................................... 56
Adiponectin............................................................................................................... 57
Vitamin D and Inflammation.................................................................................... 58
Conclusion................................................................................................................ 58
References................................................................................................................. 59
INTRODUCTION
Contemporary research in nutritional sciences and immunology has led to a new
understanding of the role of inflammation in obesity- and age-related chronic
diseases. This realization has also led to a convergence of the fields of immunology
and nutrient physiology and the understanding that they are closely linked [1,2]. This
chapter will review numerous ways to evaluate the impact of inflammation on chronic
diseases utilizing biomarker measurements. Recent studies forecast that by the year
2030 a doubling to tripling of obesity-associated diseases such as diabetes, heart
disease, and common forms of cancer will occur [3,4]. While the human genome
has changed very little in the last 50,000 years, our diets and lifestyles have changed
greatly in just the last 500 years with the introduction of sugars, fats, and salt at levels
never seen in the history of mankind. The discovery of agriculture some 10,000 years
ago led to increased intakes of dairy products and grains. This change in diets was
accelerated during the last 200 years by the Industrial Revolution, leading some
individuals to argue that we should imitate aspects of the diet that was prevalent in
the Paleolithic era [5–7]. The results of these changes in diet and lifestyle have been
linked to inflammation as reviewed in this chapter.
53
MECHANISMS UNDERLYING CHRONIC

LOW-GRADE INFLAMMATION
Scientists have begun to unravel disease mechanisms in complex metabolic sys-
tems using modern tools such as genomics, proteomics, and systems biology in the
interdisciplinary field of immunonutrition outlined in this text. For example, gene
expression networks in adipose tissue identify a pattern of increased expression
of recognized inflammatory genes associated with obesity and metabolic disease
[8,9]. Multiple inflammatory inputs contribute to metabolic dysfunction, including
increases in circulating cytokines [10], decreases in protective factors [11], and
interactions of inflammatory cells with adipocytes and myocytes. For example, M1
macrophages impair insulin signaling and adipogenesis in adipocytes, while M2
alternatively activated macrophages do not [12]. Interferon-gamma (IFN-γ) from
activated T-lymphocytes has similar effects on adipocytes while macrophages can
impact both myocytes and hepatocytes, underlining the cellular connections between
inflammation and ectopic fat deposition in these organs [13–15]. New and more sen-
sitive assays for biomarkers of inflammation have demonstrated an increased risk of
all-cause mortality among persons who were previously thought to have circulating
(plasma/serum) values within the normal range. Systemic low-level inflammation
is defined as two- to fourfold elevation in circulating levels of proinflammatory and
anti-inflammatory cytokines, naturally occurring cytokine antagonists, and acute-
phase proteins, as well as minor increases in counts of neutrophils and natural killer
cells. Although these increases are far below the levels observed during acute, severe
infections, systemic low-level inflammation is strongly associated with increasing
age, lifestyle factors such as smoking, obesity, and dietary patterns, together with
increased risk of cardiovascular disease (CVD), type 2 diabetes mellitus (T2DM),
cognitive decline, and sarcopenia. Moreover, systemic low-level inflammation is a
strong, consistent, and independent predictor of all-cause mortality and CVD-cause
mortality in elderly populations as described later.
C-REACTIVE PROTEIN AND OTHER ACUTE-PHASE REACTANTS

C-reactive protein (CRP) is synthesized by the liver in response to factors released by
macrophages and adipocytes. CRP binds to phosphocholine on microbes and assists
in complement binding to foreign and damaged cells and enhances opsonin-mediated
phagocytosis by macrophages that express a receptor for CRP. It is also believed to
play a role in innate immunity as an early defense system against infections. CRP
rises up to 50,000-fold in acute inflammation, such as infection. It rises above normal
limits within 6 h and peaks at 48 h. Its half-life is constant, and therefore its level is
mainly determined by the rate of production (and hence the severity of the precipi-
tating cause). Serum amyloid A (SAA) is a related acute-phase marker that responds
rapidly in similar circumstances. CRP, SAA, and vascular adhesion molecules such
as soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cell
adhesion molecule-1 (sVCAM-1) reflect low-grade inflammation when present in
low concentrations [16,17], whereas high concentrations of CRP (>10 mg/L) reflect
acute inflammation or infection [18–20]. Widely available analytical methods have
Biomarkers of Inflammation and the Western Diet 55
enabled the analysis of single biomarkers of low-grade inflammation in one run using
enzyme-linked immunoassays. However, obtaining multiple biomarkers based on
many single-biomarker measurements is very labor intensive and expensive. These
issues represent a significant challenge to an efficient multiple biomarker approach,
particularly in large observational cohort or clinical trial studies. A solution to these
challenges is the simultaneous measurement of a set of low-grade inflammatory bio-
markers in one run. Such methods have recently become available with the use of
multiarray platforms (e.g., Luminex® and Meso Scale Discovery® [MSD]). However,
it remains to be established to what extent biomarker concentrations, as measured
with these multiarray platforms, are comparable to well-established single-biomarker
measurements. Although some cross-validation studies have been performed, most
have not focused on biomarkers of low-grade inflammation [21–25], and the two
studies that did so pointed out the problem of different measured concentrations,
which may lead to bias in epidemiological associations [25].
CRP is the most studied biomarker of chronic low-grade inflammation associated
with obesity, diabetes, and CVD risk. Numerous studies have been published on the
use of CRP measurement to improve assessments of cardiovascular risk for patients
in primary prevention programs. More than 20 prospective studies of distinct
cohorts demonstrated that elevated levels of CRP are associated with an elevated
risk of future coronary events after adjustment for at least four traditional risk fac-
tors, including Framingham risk factors and/or diabetes and obesity [26–45]. This
association applied both to men and to women across a wide age range (e.g., from
middle-aged to elderly). Some studies stratified groups of patients by their highly
sensitive CRP (hsCRP) test results—hsCRP levels of less than 1 mg/L, 1–3 mg/L,
and greater than 3 mg/L—and showed that these cutoffs correspond with low-risk,
moderate-risk, and high-risk groups, respectively, although actual levels of risk were
fairly linear across a wide range of CRP levels. A high-sensitivity CRP (hsCRP)
test measures low levels of CRP using laser nephelometry. The test gives results in
25 min with a sensitivity down to 0.04 mg/L.
PROINFLAMMATORY CYTOKINES
Proinflammatory cytokines induce systemic inflammation and include chemokines
and cytokines. Some cytokines (such as interleukin-6 [IL-6]) circulate in picomolar
(10 −12 M) concentrations that can increase up to 1000-fold during trauma or infec-
tion. The widespread distribution of cellular sources for cytokines may be a feature
that differentiates them from hormones. Virtually all nucleated cells, but especially
endothelial cells, epithelial cells, and resident macrophages, are potent producers of
IL-1, IL-6, and TNFα. IL-6 is responsible for stimulating acute-phase protein syn-
thesis, as well as the production of neutrophils in the bone marrow. It supports the
growth of B-lymphocytes and is antagonistic to regulatory T cells. IL-6 is the most
studied of the cytokines that use gp130, also known as CD 130 or IL-6 signal trans-
ducer (IL6ST), in their signaling complexes. Other cytokines that signal through
receptors containing gp130 are IL-11, IL-27, ciliary neurotrophic factor, leukemia
inhibitory factor, and oncostatin M. In addition to the membrane-bound receptor,
a soluble form of IL-6R (sIL-6R) has been purified from human serum and urine.
Inflammatory reactions depend on a cluster of cytokines rather than on any single

cytokine. Patterns of cytokine production differ with different inflammatory condi-
tions, and cytokines are components of a large complex signaling network [46–48].
The influence of cytokines on lipid metabolism may be important in mediating the
effects of a combined elevation of different cytokines. For example, both IL-6 and
IL-1β act on the liver to produce the characteristic dyslipidemia of the metabolic
syndrome, with increased VLDL and decreased HDL [49]. Combined elevation of
IL-6 and IL-1β dramatically increased the expression of the acute-phase proteins,
compared with the effect of each cytokine alone [46]. Another potential molecular
mechanism exemplifying how inflammation may be involved in the pathogenesis
of T2DM has been elucidated in recent elegant studies showing that sensitizing of
insulin signaling by salicylates is induced via inhibition of the activity of IκB kinase
β [50–52]. IL-1β is well known to activate the IκB kinase β and might thereby induce
insulin resistance.
CHEMOKINES
Chemokines derive their name from the Greek root kinos meaning movement, and
they are a family of small protein cytokines secreted by immune cells that induce
directed movement or chemotaxis of nearby responsive cells. Chemokines have been
classified into four main subfamilies: CXC, CC, CX3C, and XC. All of these pro-
teins exert their biological effects by interacting with G-protein-linked transmem-
brane receptors on the surface of their target cells.
With inflammation, chemokines are released from a wide variety of cells in
response to stimuli such as bacterial or viral infections. Their release is also stimu-
lated by proinflammatory cytokines such as IL-1. The chemokines function during
inflammation to attract immune effector cells such as macrophages and other white
blood cells to sites of infection or tissue damage. Inflammatory cytokines include
CXCL-8, CCL2, CCL3, CCL4, CCL5, CCL11, and CXCL10.
Chemokines have been shown to participate in and control the process of a num-
ber of acute and chronic inflammatory conditions by promoting the infiltration and
activation of inflammatory cells into injured or infected tissues [53].
TELOMERE LENGTH
Telomeres are regions of repetitive DNA sequence that prevent the DNA replication
process or damage from degrading the ends of chromosomes, essentially acting as
buffers and protecting the genes closest to the chromosome ends. Russian biologist
Alexei Olovnikov first hypothesized in the early 1970s that chromosomes could not
completely replicate their ends and that such losses could ultimately lead to the end
of cell division. Elizabeth Blackburn and her colleagues published work suggesting
that telomere shortening was linked with aging at the cellular level, affected lifespan,
and could lead to cancer. In 1984, telomerase, the enzyme that replenishes telomeres,
was discovered in Blackburn’s lab leading to the award of the Nobel Prize in 2009
for the discovery of telomeres and telomerase. The rate of telomere shortening
is a biomarker of inflammation and aging [54]. It has been proposed that eating
antioxidant-rich foods might reduce the risk of many age-related chronic diseases by
inhibiting low-grade chronic inflammation [55]. Several studies have demonstrated
an association of telomere length with both cellular senescence and development
of chronic disease associated with physiological aging [56,57]. Although telomere
length may predict clinical outcomes and mortality among humans, cells with short-
ened telomeres remain genetically stable if the telomere maintenance system, which
includes mainly telomerase, is fully functioning [58]. Metabolic factors, such as
abdominal fat and increased circulating glucose levels, are related to shorter telo-
meres and lower telomerase activity [59–61], supporting the role of lifestyle and
environmental factors in telomeres maintenance. Population-based studies and
large-scale clinical trials have provided scientific evidences that diet, especially
those rich in fruits, vegetables, fish, and low-fat dairy products, is associated with
a lower incidence of age-related chronic diseases [62,63]. Telomere length has been
related to dietary factors including a greater intake of antioxidants [64,65], less pro-
cessed meat consumption [65], and intake of fruits and vegetables and less dietary fat
[66,67]. Changes in diet and lifestyle have been shown to influence telomere length
through mechanisms reflecting their role in inflammation, oxidative stress, DNA
integrity, and DNA methylation [55].
The analysis of telomere length is emerging as a commercial biomarker for aging
and disease, as well as a tool in the search for new medications. Several companies
offer tests for telomere length. Despite commercial enthusiasm, interpreting pre-
cisely what an individual’s telomeres mean for their health and longevity remains
challenging. As a result, there is a division within the research community between
those who are pushing ahead with ventures to offer tests to the public and those who
feel that telomere testing is premature given our current state of knowledge.
ADIPONECTIN
Adiponectin is the most abundant adipose tissue derived cytokine. The circu-
lating level of adiponectin ranges from 5 to 30 μg/mL in humans [68], which
represents up to 0.05% of total plasma proteins [69,70]. It has anti-inflammatory,
antidiabetic, and antiatherogenic properties, and low circulating levels are asso-
ciated with central obesity, insulin resistance, metabolic syndrome (MetS), and
T2DM [71]. Serum adiponectin concentrations are highly heritable, and a number
of genome-wide association studies have identified ADIPOQ, the gene encod-
ing adiponectin, as the main locus contributing to variations in serum levels
in European and Asian populations [72–75]. Cross-sectional studies in healthy
and diabetic populations have provided further evidence for the association of
single-nucleotide polymorphisms in ADIPOQ with serum adiponectin concen-
trations [76–80]. Several studies have linked ADIPOQ variants to T2DM and
MetS, although the results to date have been discordant and not replicated across
whole populations [75,78–82]. Adiponectin cellular signaling is mediated by two
adiponectin receptors. The genes for these (ADIPOR1 and ADIPOR2), although
generally not associated with serum adiponectin, have themselves been impli-
cated in insulin resistance and T2DM risk in genetic association studies but also
with inconsistent results [83–87].
VITAMIN D AND INFLAMMATION

Research now indicates that vitamin D has anti-inflammatory activity beyond its
established roles in bone and mineral metabolism including actions that affect insulin
secretion and action [88,89]. Vitamin D deficiency may contribute to the etiology of
a number of age-related chronic diseases including obesity and metabolic syndrome
[90–92]. Population studies and clinical intervention studies have shown that obese
individuals tend to have low vitamin D status that can be corrected with supplemen-
tation [93–95]. Absorption of vitamin D into adipose tissue, less exposure to sunlight,
and low intake of vitamin D in obese individuals may all contribute to observed low
levels of vitamin D in the circulation [95–97]. 25-Hydroxycholecalciferol (25(OH)
D3) is the major circulating form of vitamin D3, which is converted to the active form
1,25-dihydroxycholecalciferol (1,25(OH)2D3). 1,25(OH)2D3 acts as a ligand for the
vitamin D receptor (VDR) that facilitates the transcription of target genes [98,99].
Both VDR and vitamin D–metabolizing enzymes have been identified in human
adipose tissue [100,101]. Therefore, human adipose tissue could be a direct target of
vitamin D, and deficiency may have pathological consequences in this tissue [102].
Therefore, vitamin D is a potential biomarker of inflammation. In obesity, there is a
marked increase in the synthesis and release of proinflammatory factors (e.g., TNFα,
IL-6, IL-8, and MCP-1), which may contribute to the elevated circulating levels seen
as well as to local tissue inflammation [103,104]. Adipose tissue inflammation, exac-
erbated by increased infiltration of macrophages and other immune cells, is a central
pathological process of adipose tissue dysfunction in obesity [105,106]. Macrophage-
derived factors potently stimulate the release of proinflammatory chemokines/
cytokines and a number of proteins involved in extracellular matrix remodeling
from human preadipocytes and adipocytes. All of these factors are known to induce
inflammation, fibrosis, and insulin resistance in adipose tissue, which is associated
with metabolic disorders [107–110]. Evidence has accumulated that vitamin D exerts
potent immunoregulatory effects, such as inhibiting the production of TNFα, IL-6,
and IL-8 by peripheral blood mononuclear cells in humans [111–113]. The effects of
vitamin D may be through targeting the nuclear factor-kappa B (NFκB) and mitogen-
activated protein kinase signaling pathways [114–117]. The emerging role of adipose
tissue in adaptive immunity suggests that vitamin D may provide some protection
against adipose tissue–induced inflammation.
CONCLUSION
The impact of Western diets and lifestyles is leading to an international epidemic
of excess adiposity. With the discovery that adipose cells are an integral component
of immune function and that excess adiposity can activate the innate and adaptive
immune systems in a chronic fashion, immune function and nutrition have become
inextricably linked. There is no single marker of inflammation that can be reliably used
to assess this impact. CRP and selected cytokines have been extensively studied, but
newer data suggest that there is a balance of proinflammatory and anti-inflammatory
cytokines that is beneficial with wound healing but harmful when activated as part
of low-grade chronic inflammation. Hormonal factors, including vitamin D but also
extending to reproductive hormones and glucocorticoids, are known to influence

inflammation, and changes in these are also associated with excess adiposity.
Therefore, we are left with a complex matrix of factors that are biomarkers of
inflammation. Future research using a combination of omic technologies and clinical
correlation holds the promise of unraveling this complex matrix through continued
research into the relationship of diet and lifestyle with inflammation.
REFERENCES
1. Shimabukuro M, Kozuka C, Taira S, Yabiku K, Dagvasumberel M, Ishida M et al. 2013.
Ectopic fat deposition and global cardiometabolic risk: New paradigm in cardiovascular
medicine. J Med Invest 60:1–14.
2. Franceschi S, Wild CP. 2013. Meeting the global demands of epidemiologic transition—
The indispensable role of cancer prevention. Mol Oncol 7:1–13.
3. Lopez AD, Mathers CD. 2006. Measuring the global burden of disease and epidemio-
logical transitions: 2002–2030. Ann Trop Med Parasitol 100:481–499.
4. Centers for Disease Control and Prevention (CDC). 2003. Trends in aging—United
States and worldwide. MMWR Morb Mortal Wkly Rep 52:101–104.
5. Lindeberg S. 2012. Paleolithic diets as a model for prevention and treatment of Western
disease. Am J Hum Biol 24:110–115.
6. Jew S, Abumweis SS, Jones PJ. 2009. Evolution of the human diet: Linking our ancestral
diet to modern functional foods as a means of chronic disease prevention. J Med Food
12:925–934.
7. Cordain L, Eaton SB, Miller JB, Mann N, Hill K. 2002. The paradoxical nature of hunter-
gatherer diets: Meat-based, yet non-atherogenic. Eur J Clin Nutr 56(Suppl 1):S42–S52.
8. Chen Y, Zhu J, Lum PY, Yang X, Pinto S, MacNeil DJ et al. 2008. Variations in DNA
elucidate molecular networks that cause disease. Nature 452:429–435.
9. Emilsson V, Thorleifsson G, Zhang B, Leonardson AS, Zink F, Zhu J et al. 2008.
Genetics of gene expression and its effect on disease. Nature 452(7186):423–428.
10. Hotamisligil GS, Arner P, Caro JF, Atkinson RL, Spiegelman BM. 1995. Increased
adipose tissue expression of tumor necrosis factor-alpha in human obesity and insulin
resistance. J Clin Invest 95:2409–2415.
11. Hotta K, Funahashi T, Arita Y, Takahashi M, Matsuda M, Okamoto Y et al. 2000. Plasma
concentrations of a novel, adipose-specific protein, adiponectin, in type 2 diabetic
patients. Arterioscler Thromb Vasc Biol 20:1595–1599.
12. Lumeng CN, Del Proposto JB, Westcott DJ, Saltiel AR. 2008. Phenotypic switching of
adipose tissue macrophages with obesity is generated by spatiotemporal differences in
macrophage subtypes. Diabetes 57(12):3239–3246.
13. Feuerer M, Herrero L, Cipolletta D, Naaz A, Wong J, Nayer A et al. 2009. Lean, but not
obese, fat is enriched for a unique population of regulatory T cells that affect metabolic
parameters. Nat Med 15:930–939.
14. Varma V, Yao-Borengasser A, Rasouli N, Nolen GT, Phanavanh B, Starks T et al. 2009.
Muscle inflammatory response and insulin resistance: Synergistic interaction between
macrophages and fatty acids leads to impaired insulin action. Am J Physiol Endocrinol
Metab 296:E1300–E1310.
15. Huang W, Metlakunta A, Dedousis N, Zhang P, Sipula I, Dube JJ et al. 2010. Depletion
of liver Kupffer cells prevents the development of diet-induced hepatic steatosis and
insulin resistance. Diabetes 59:347–357.
16. Borissoff JI, Spronk HM, ten Cate H. 2011. The hemostatic system as a modulator of
atherosclerosis. N Engl J Med 364:1746–1760.
17. Ross R. 1999. Atherosclerosis—An inflammatory disease. N Engl J Med 340:115–126.
18. Pearson TA, Mensah GA, Alexander RW, Anderson JL, Cannon RO, 3rd et al. 2003.
Markers of inflammation and cardiovascular disease: Application to clinical and public
health practice: A statement for healthcare professionals from the Centers for Disease
Control and Prevention and the American Heart Association. Circulation 107:499–511.
19. Thanabalasingham G, Shah N, Vaxillaire M, Hansen T, Tuomi T et al. 2010. A large
multi-centre European study validates high-sensitivity C-reactive protein (hsCRP) as a
clinical biomarker for the diagnosis of diabetes subtypes. Diabetologia 54:2801–2810.
20. Myers GL, Rifai N, Tracy RP, Roberts WL, Alexander RW et al. 2004. CDC/AHA work-
shop on markers of inflammation and cardiovascular disease: Application to clinical and
public health practice: Report from the laboratory science discussion group. Circulation
110:e545–e549.
21. Marchese RD, Puchalski D, Miller P, Antonello J, Hammond O et al. 2009. Optimization
and validation of a multiplex, electrochemiluminescence-based detection assay for the
quantitation of immunoglobulin G serotype-specific antipneumococcal antibodies in
human serum. Clin Vaccine Immunol 16:387–396.
22. Oh ES, Mielke MM, Rosenberg PB, Jain A, Fedarko NS et al. 2010. Comparison of con-
ventional ELISA with electrochemiluminescence technology for detection of amyloid-
beta in plasma. J Alzheimers Dis 21:769–773.
23. Prabhakar U, Eirikis E, Davis HM. 2002. Simultaneous quantification of proinflam-
matory cytokines in human plasma using the LabMAP assay. J Immunol Methods
260:207–218.
24. Dupont NC, Wang K, Wadhwa PD, Culhane JF, Nelson EL. 2005. Validation and com-
parison of luminex multiplex cytokine analysis kits with ELISA: Determinations of
a panel of nine cytokines in clinical sample culture supernatants. J Reprod Immunol
66:175–191.
25. de Koning L, Liptak C, Shkreta A, Bradwin G, Hu FB et al. 2012. A multiplex
immunoassay gives different results than singleplex immunoassays which may bias
epidemiologic associations. Clin Biochem 45:848–851.
26. Ridker PM, Rifai N, Clearfield M, Downs JR, Weis SE, Miles JS et al. for the Air Force/
Texas Coronary Atherosclerosis Prevention Study Investigators. 2001. Measurement of
C-reactive protein for the targeting of statin therapy in the primary prevention of acute
coronary events. N Engl J Med 344:1959–1965.
27. Ballantyne CM, Hoogeveen RC, Bang H, Coresh J, Folsom AR, Heiss G et al. 2004.
Lipoprotein-associated phospholipase A2, high-sensitivity C-reactive protein, and risk
for incident coronary heart disease in middle-aged men and women in the Atherosclerosis
Risk in Communities (ARIC) study. Circulation 109:837–842.
28. Danesh J, Whincup P, Walker M, Lennon L, Thomson A, Appleby P et al. 2000. Low
grade inflammation and coronary heart disease: Prospective study and updated meta-
analyses. BMJ 321:199–204.
29. Lowe GD, Sweetnam PM, Yarnell JW, Rumley A, Rumley C, Bainton D et al. 2004.
C-reactive protein, fibrin D-dimer, and risk of ischemic heart disease: The Caerphilly
and Speedwell studies. Arterioscler Thromb Vasc Biol 24:1957–1962.
30. Cushman M et al. 2005. C-reactive protein and the 10-year incidence of coronary
heart disease in older men and women: The cardiovascular health study. Circulation
112:25–31.
31. Tzoulaki I et al. 2007. Relative value of inflammatory, hemostatic, and rheological
factors for incident myocardial infarction and stroke: The Edinburgh Artery Study.
Circulation 115:2119–2127.
32. Boekholdt SM, Murray GD, Lee AJ, Rumley A, Lowe GD, Fowkes FG. 2006. C-reactive
protein levels and coronary artery disease incidence and mortality in apparently
healthy men and women: The EPIC-Norfolk prospective population study 1993–2003.
Atherosclerosis 187:415–422.
33. Wilson PW et al. 2006. Increased CRP and long term risk for cardiovascular events in
middle age men and women [abstract #4070]. Circulation 114(Suppl):11877–11878.
34. Sakkinen P, Abbott RD, Curb JD, Rodriguez BL, Yano K, Tracy RP et al. 2002.
C-reactive protein and myocardial infarction. J Clin Epidemiol 55:445–451.
35. Pai JK, Pischon T, Ma J, Manson JE, Hankinson SE, Joshipura K et al. 2004.
Inflammatory markers and the risk of coronary heart disease in men and women. N Engl
J Med 351:2599–2610.
36. Laaksonen DE, Niskanen L, Nyyssönen K, Punnonen K, Tuomainen TP, Salonen JT
et al. 2005. C-reactive protein in the prediction of cardiovascular and overall mortality
in middle-aged men: A population-based cohort study. Eur Heart J 26:1783–1789.
37. Koenig W, Khuseyinova N, Baumert J, Thorand B, Loewel H, Chambless L et al. 2006.
Increased concentrations of C-reactive protein and IL-6 but not IL-18 are independently
associated with incident coronary events in middle-aged men and women: Results from
the MONICA/KORA Augsburg case-cohort study, 1984–2002. Arterioscler Thromb
Vasc Biol 26:2745–2751.
38. Ridker PM, Cushman M, Stampfer MJ, Tracy RP, Hennekens CH. 1997. Inflammation,
aspirin, and the risk of cardiovascular disease in apparently healthy men. N Engl J Med
336:973–979.
39. Luc G, Bard JM, Juhan-Vague I, Ferrieres J, Evans A, Amouyel P et al. for the
PRIME Study Group. 2003. C-reactive protein, interleukin-6, and fibrinogen as pre-
dictors of coronary heart disease: The PRIME study. Arterioscler Thromb Vasc Biol
23:1255–1261.
40. Sattar N et al. for the PROSPER Study Group. 2007. C-reactive protein and prediction of
coronary heart disease and global vascular events in the Prospective Study of Pravastatin
in the Elderly at Risk (PROSPER). Circulation 115:981–989.
41. Danesh J, Murray HM, McConnachie A, Blauw GJ, Bollen EL, Buckley BM et al. 2004.
C-reactive protein and other circulating markers of inflammation in the prediction of
coronary heart disease. N Engl J Med 350:1387–1397.
42. Tice JA, Browner W, Tracy RP, Cummings SR. 2003. The relation of C-reactive protein
levels to total and cardiovascular mortality in older U.S. women. Am J Med 114:199–205.
43. Pradhan AD, Manson JE, Rossouw JE, Siscovick DS, Mouton CP, Rifai N et al. 2002.
Inflammatory biomarkers, hormone replacement therapy, and incident coronary heart
disease: Prospective analysis from the Women’s Health Initiative observational study.
JAMA 288:980–987.
44. Ridker PM, Rifai N, Rose L, Buring JE, Cook NR. 2002. Comparison of C-reactive pro-
tein and low-density lipoprotein cholesterol levels in the prediction of first cardiovascular
events. N Engl J Med 347:1557–1565.
45. Lowe GD, Rumley A, McMahon AD, Ford I, O’Reilly DS, Packard CJ for the West of
Scotland Coronary Prevention Study Group. 2004. Interleukin-6, fibrin D-dimer, and
coagulation factors VII and XIIa in prediction of coronary heart disease. Arterioscler
Thromb Vasc Biol 24:1529–1534.
46. Gabay C, Kushner I. 1999. Acute-phase proteins and other systemic responses to
inflammation. N Engl J Med 340:448–454.
47. Fattori E, Cappelletti M, Costa P, Sellitto C, Cantoni L, Carelli M et al. 1994. Defective
in inflammatory response in interleukin 6-deficient mice. J Exp Med 180:1243–1250.
48. Zheng H, Fletcher D, Kozak W, Jiang M, Hofmann KJ, Conn CA et al. 1995. Resistance
to fever induction and impaired acute-phase response in interleukin-1 beta-deficient
mice. Immunity 3:9–19.
49. Pickup JC, Crook MA. 1998. Is type II diabetes mellitus a disease of the innate immune
system? Diabetologia 41:1241–1248.
50. Kim JK, Kim YJ, Fillmore JJ, Chen Y, Moore I, Lee J et al. 2001. Prevention of fat-
induced insulin resistance by salicylate. J Clin Invest 108:437–446.
51. Yuan M, Konstantopoulos N, Lee J, Hansen L, Li ZW, Karin M et al. 2001. Reversal
of obesity- and diet-induced insulin resistance with salicylates or targeted disruption of
Ikkbeta. Science 293:1673–1677.
52. Hundal RS, Petersen KF, Mayerson AB, Randhawa PS, Inzucchi S, Shoelson SE et al.
2002. Mechanism by which high-dose aspirin improves glucose metabolism in type 2
diabetes. J Clin Invest 109:1321–1326.
53. Gerard C, Rollins BJ. 2001. Chemokines and disease. Nat Immunol 2:108–115.
54. Kimura M, Barbieri M, Gardner JP, Skurnick J, Cao X et al. 2007. Leukocytes of excep-
tionally old persons display ultra-short telomeres. Am J Physiol Regul Integr Comp
Physiol 293:R2210–R2217.
55. Ligi P. 2011. Diet, nutrition and telomere length. J Nutr Biochem 22:895–901.
56. Donate LE, Blasco MA. 2011. Telomeres in cancer and ageing. Philos Trans R Soc Lond
B Biol Sci 366:76–84.
57. Oeseburg H, de Boer RA, van Gilst WH, van der Harst P. 2010. Telomere biology in
healthy aging and disease. Pflugers Arch 459:259–268.
58. Blackburn EH. 2005. Telomeres and telomerase: Their mechanisms of action and the
effects of altering their functions. FEBS Lett 579:859–862.
59. Epel ES, Lin J, Wilhelm FH, Wolkowitz OM, Cawthon R et al. 2006. Cell aging in relation
to stress arousal and cardiovascular disease risk factors. Psychoneuroendocrinology
31:277–287.
60. Valdes AM, Andrew T, Gardner JP, Kimura M, Oelsner E et al. 2005. Obesity, cigarette
smoking, and telomere length in women. Lancet 366:662–664.
61. Gardner JP, Li S, Srinivasan SR, Chen W, Kimura M et al. 2005. Rise in insulin resistance
is associated with escalated telomere attrition. Circulation 111:2171–2177.
62. Hu FB. 2003. Plant-based foods and prevention of cardiovascular disease: An overview.
Am J Clin Nutr 78:544S–551S.
63. Anderson AL, Harris TB, Tylavsky FA, Perry SE, Houston DK et al. 2011. Health ABC
Study. Dietary patterns and survival of older adults. J Am Diet Assoc 111:84–91.
64. Xu Q, Parks CG, DeRoo LA, Cawthon RM, Sandler DP et al. 2009. Multivitamin use
and telomere length in women. Am J Clin Nutr 89:1857–1863.
65. Nettleton JA, Diez-Roux A, Jenny NS, Fitzpatrick AL, Jacobs DR Jr. 2008. Dietary
patterns, food groups, and telomere length in the Multi-Ethnic Study of Atherosclerosis
(MESA). Am J Clin Nutr 88:1405–1412.
66. Mirabello L, Huang WY, Wong JY, Chatterjee N, Reding D et al. 2009. The association
between leukocyte telomere length and cigarette smoking, dietary and physical vari-
ables, and risk of prostate cancer. Aging Cell 8:405–413.
67. Ornish D, Lin J, Daubenmier J, Weidner G, Epel E et al. 2008. Increased telomerase
activity and comprehensive lifestyle changes: A pilot study. Lancet Oncol 9:1048–1057.
68. Maeda K, Okubo K, Shimomura I, Funahashi T, Matsuzawa Y, Matsubara K. 1996. cDNA
cloning and expression of a novel adipose specific collagen-like factor, apM1 (AdiPose
Most abundant Gene transcript 1). Biochem Biophys Res Commun 221:286–289.
69. Arita Y, Kihara S, Ouchi N, Takahashi M, Maeda K, Miyagawa J et al. 2012. Paradoxical
decrease of an adipose-specific protein, adiponectin, in obesity. Biochem Biophys Res
Commun 425:560–564.
70. Zhu W, Cheng KK, Vanhoutte PM, Lam KS, Xu A. 2008. Vascular effects of adipo-
nectin: Molecular mechanisms and potential therapeutic intervention. Clin Sci (Lond)
114:361–374.
71. Kadowaki T, Yamauchi T, Kubota N, Hara K, Ueki K, Tobe K. 2006. Adiponectin and
adiponectin receptors in insulin resistance, diabetes, and the metabolic syndrome. J Clin
Invest 116:1784–1792.
72. Jee SH, Sull JW, Lee JE, Shin C, Park J, Kimm H et al. 2012. Adiponectin concentra-
tions: A genome-wide association study. Am J Hum Genet 87:545–552.
73. Heid IM, Henneman P, Hicks A, Coassin S, Winkler T, Aulchenko YS et al. 2010.
Clear detection of ADIPOQ locus as the major gene for plasma adiponectin: Results of
genome-wide association analyses including 4659 European individuals. Atherosclerosis
208:412–420.
74. Ling H, Waterworth DM, Stirnadel HA, Pollin TI, Barter PJ, Kesaniemi YA et al. 2009.
Genome-wide linkage and association analyses to identify genes influencing adiponec-
tin levels: The GEMS Study. Obesity (Silver Spring) 17:737–744.
75. Richards JB, Waterworth D, O’Rahilly S, Hivert M-F, Loos RJF, Perry JRB et al. 2009.
A genome-wide association study reveals variants in ARL15 that influence adiponectin
levels. PLoS Genet 5(12):e1000768.
76. Vasseur F, Helbecque N, Dina C, Lobbens S, Delannoy V, Gaget S et al. 2002. Single-
nucleotide polymorphism haplotypes in the both proximal promoter and exon 3 of
the APM1 gene modulate adipocyte-secreted adiponectin hormone levels and con-
tribute to the genetic risk for type 2 diabetes in French Caucasians. Hum Mol Genet
11:2607–2614.
77. Heid IM, Wagner SA, Gohlke H, Iglseder B, Mueller JC, Cip P et al. 2006. Genetic archi-
tecture of the APM1 gene and its influence on adiponectin plasma levels and parameters
of the metabolic syndrome in 1,727 healthy Caucasians. Diabetes 55:375–384.
78. Hivert M-F, Manning AK, McAteer JB, Florez JC, Dupuis J, Fox CS et al. 2008. Common
variants in the adiponectin gene (ADIPOQ) associated with plasma adiponectin levels,
type 2 diabetes, and diabetes-related quantitative traits. Diabetes 57:3353–3359.
79. Menzaghi C, Trischitta V, Doria A. 2007. Genetic influences of adiponectin on insulin
resistance, type 2 diabetes, and cardiovascular disease. Diabetes 56:1198–1209.
80. Henneman P, Aulchenko YS, Frants RR, Zorkoltseva IV, Zillikens MC, Frolich M et al.
2010. Genetic architecture of plasma adiponectin overlaps with the genetics of meta-
bolic syndrome-related traits. Diabetes Care 33:908–913.
81. Vasseur F, Meyre D, Froguel P. 2006. Adiponectin, type 2 diabetes and the metabolic
syndrome: Lessons from human genetic studies. Expert Rev Mol Med 8:1–12.
82. Mackevics V, Heid IM, Wagner SA, Cip P, Doppelmayr H, Lejnieks A et al. 2006. The
adiponectin gene is associated with adiponectin levels but not with characteristics of the
insulin resistance syndrome in healthy Caucasians. Eur J Hum Genet 14:349–356.
83. Stefan N, Machicao F, Staiger H, Machann J, Schick F, Tschritter O et al. 2005.
Polymorphisms in the gene encoding adiponectin receptor 1 are associated with insulin
resistance and high liver fat. Diabetologia 48:2282–2291.
84. Crimmins NA, Martin LJ. 2007. Polymorphisms in adiponectin receptor genes
ADIPOR1 and ADIPOR2 and insulin resistance. Obes Rev 8:419–423.
85. Kim JT, Kim Y, Cho YM, Koo BK, Lee EK, Shin HD et al. 2009. Polymorphisms of
ADIPOR1 and ADIPOR2 are associated with phenotypes of type 2 diabetes in Koreans.
Clin Endocrinol (Oxf) 70:66–74.
86. Hara K, Horikoshi M, Kitazato H, Yamauchi T, Ito C, Noda M et al. 2005. Absence of
an association between the polymorphisms in the genes encoding adiponectin receptors
and type 2 diabetes. Diabetologia 48:1307–1314.
87. Collins SC, Luan J, Thompson AJ, Daly A, Semple RK, O’Rahilly S et al. 2007.
Adiponectin receptor genes: Mutation screening in syndromes of insulin resistance
and association studies for type 2 diabetes and metabolic traits in UK populations.
Diabetologia 50:555–562.
88. Mellanby E. 1976. Nutrition Classics. The Lancet 1:407–412, 1919. An experimental
investigation of rickets. Edward Mellanby. Nutr Rev 34:338–340.
89. Holick MF. 2011. Vitamin D: Evolutionary, physiological and health perspectives. Curr
Drug Targets 12:4–18.
90. Chiu KC, Chu A, Go VL, Saad MF. 2004. Hypovitaminosis D is associated with insulin
resistance and beta cell dysfunction. Am J Clin Nutr 79:820–825.
91. Barchetta I, Angelico F, Del Ben M, Baroni MG, Pozzilli P et al. 2011. Strong asso-
ciation between non alcoholic fatty liver disease (NAFLD) and low 25(OH) vitamin D
levels in an adult population with normal serum liver enzymes. BMC Med 9:85.
92. Olson ML, Maalouf NM, Oden JD, White PC, Hutchison MR. 2012. Vitamin D defi-
ciency in obese children and its relationship to glucose homeostasis. J Clin Endocrinol
Metab 97:279–285.
93. Goldner WS, Stoner JA, Thompson J, Taylor K, Larson L et al. 2008. Prevalence of
vitamin D insufficiency and deficiency in morbidly obese patients: A comparison with
non-obese controls. Obes Surg 18:145–150.
94. Fish E, Beverstein G, Olson D, Reinhardt S, Garren M et al. 2010. Vitamin D status of
morbidly obese bariatric surgery patients. J Surg Res 164:198–202.
95. Brock K, Huang WY, Fraser DR, Ke L, Tseng M et al. 2010. Low vitamin D status is
associated with physical inactivity, obesity and low vitamin D intake in a large US sam-
ple of healthy middle-aged men and women. J Steroid Biochem Mol Biol 121:462–466.
96. Wortsman J, Matsuoka LY, Chen TC, Lu Z, Holick MF. 2000. Decreased bioavailability
of vitamin D in obesity. Am J Clin Nutr 72:690–693.
97. Kull M, Kallikorm R, Lember M. 2009. Body mass index determines sunbathing habits:
Implications on vitamin D levels. Intern Med J 39:256–258.
98. Demay MB. 2006. Mechanism of vitamin D receptor action. Ann N Y Acad Sci
1068:204–213.
99. Zhang Y, Kong J, Deb DK, Chang A, Li YC. 2010. Vitamin D receptor attenuates renal
fibrosis by suppressing the renin-angiotensin system. J Am Soc Nephrol 21:966–973.
100. Wamberg L, Christiansen T, Paulsen SK, Fisker S, Rask P et al. 2012. Expression of
vitamin D-metabolizing enzymes in human adipose tissue-the effect of obesity and diet-
induced weight loss. Int J Obes (Lond) 37:651–657.
101. Ching S, Kashinkunti S, Niehaus MD, Zinser GM. 2011. Mammary adipocytes bioac-
tivate 25-hydroxyvitamin D(3) and signal via vitamin D(3) receptor, modulating mam-
mary epithelial cell growth. J Cell Biochem 112:3393–3405.
102. Ding C, Gao D, Wilding J, Trayhurn P, Bing C. 2012. Vitamin D signalling in adipose
tissue. Br J Nutr 108:1915–1923.
103. Skurk T, Alberti-Huber C, Herder C, Hauner H. 2007. Relationship between adipocyte
size and adipokine expression and secretion. J Clin Endocrinol Metab 92:1023–1033.
104. Fontana L, Eagon JC, Trujillo ME, Scherer PE, Klein S. 2007. Visceral fat adipokine secre-
tion is associated with systemic inflammation in obese humans. Diabetes 56:1010–1013.
105. Bourlier V, Bouloumie A. 2009. Role of macrophage tissue infiltration in obesity and
insulin resistance. Diabetes Metab 35:251–260.
106. Lolmede K, Duffaut C, Zakaroff-Girard A, Bouloumie A. 2011. Immune cells in adipose
tissue: Key players in metabolic disorders. Diabetes Metab 37:283–290.
107. Keophiphath M, Achard V, Henegar C, Rouault C, Clement K et al. 2009. Macrophage-
secreted factors promote a profibrotic phenotype in human preadipocytes. Mol
Endocrinol 23:11–24.
108. Gao D, Bing C. 2011. Macrophage-induced expression and release of matrix metal-
loproteinase 1 and 3 by human preadipocytes is mediated by IL-1B via activation of
MAPK signaling. J Cell Physiol 226:2869–2880.
109. Gao D, Trayhurn P, Bing C. 2010. Macrophage-secreted factors inhibit ZAG expression
and secretion by human adipocytes. Mol Cell Endocrinol 325:135–142.
110. Kos K, Wong S, Tan B, Gummesson A, Jernas M et al. 2009. Regulation of the fibro-
sis and angiogenesis promoter SPARC/osteonectin in human adipose tissue by weight
change, leptin, insulin, and glucose. Diabetes 58:1780–1788.
111. Giulietti A, van Etten E, Overbergh L, Stoffels K, Bouillon R et al. 2007. Monocytes
from type 2 diabetic patients have a pro-inflammatory profile. 1,25-Dihydroxyvitamin
D(3) works as anti-inflammatory. Diabetes Res Clin Pract 77:47–57.
112. Cohen-Lahav M, Douvdevani A, Chaimovitz C, Shany S. 2007. The antiinflammatory

activity of 1,25-dihydroxyvitamin D3 in macrophages. J Steroid Biochem Mol Biol 103:
558–562.
113. Martinesi M, Treves C, d’Albasio G, Bagnoli S, Bonanomi AG et al. 2008. Vitamin D
derivatives induce apoptosis and downregulate ICAM-1 levels in peripheral blood mono-
nuclear cells of inflammatory bowel disease patients. Inflamm Bowel Dis 14:597–604.
114. Stio M, Martinesi M, Bruni S, Treves C, Mathieu C et al. 2007. The Vitamin D analogue
TX 527 blocks NF-kappaB activation in peripheral blood mononuclear cells of patients
with Crohn’s disease. J Steroid Biochem Mol Biol 103:51–60.
115. Suzuki Y, Ichiyama T, Ohsaki A, Hasegawa S, Shiraishi M et al. 2009. Antiinflammatory
effect of 1alpha,25-dihydroxyvitamin D(3) in human coronary arterial endothelial
cells: Implication for the treatment of Kawasaki disease. J Steroid Biochem Mol Biol
113:134–138.
116. Zhang Y, Leung DY, Richers BN, Liu Y, Remigio LK et al. 2012. Vitamin D inhibits
monocyte/macrophage proinflammatory cytokine production by targeting MAPK phos-
phatase-1. J Immunol 188:2127–2135.
117. An H, Ford AL, Vo K, Eldeniz C, Ponisio R et al. 2011. Early changes of tissue perfu-
sion after tissue plasminogen activator in hyperacute ischemic stroke. Stroke 42:65–72.
5 Phytochemicals and
Immune Function
David Heber
CONTENTS
Introduction............................................................................................................... 67
Phytochemical Evolution and Human Immunity...................................................... 69
Classification of Phytochemicals.............................................................................. 70
Phytochemicals and Inhibition of Inflammation....................................................... 74
Tea............................................................................................................................. 74
Berry Polyphenols and Hydrolyzable Tannins from Pomegranate........................... 75
Condensed Tannins from Grape Seed Extract.......................................................... 75
Cocoa Polyphenols from Chocolate.......................................................................... 75
Glucosinolates........................................................................................................... 76
Carotenoids............................................................................................................... 77
Conclusions and Future Directions........................................................................... 78
References................................................................................................................. 78
INTRODUCTION
Epidemiological evidence suggests that diets rich in fruits and vegetables are associ-
ated with a reduced risk of various age-related chronic diseases, including cardio-
vascular disease (CVD), diabetes, certain cancers, and Alzheimer’s disease, where
visceral obesity and low-grade inflammation are common underlying mechanisms
of disease [1]. In the setting of the global epidemic of obesity and age-associated
chronic diseases, the reduction of oxidant stress and low-grade inflammation
associated with increased visceral fat through consumption of colorful fruits and
vegetables may provide one public health nutrition approach [2]. The reduced calorie
density and rich micronutrients characteristic of fruits and vegetables are also likely
to be positive contributors to balanced nutrition when combined with a reduction
in refined carbohydrates, increased protein intake, and adoption of healthy active
lifestyles to restore the balance of energy intake and output undermined by a lack of
physical activity [3].
In fruits and vegetables, polyphenols are the largest group of phytochemicals
and are perceived as being responsible for many of their anti-inflammatory effects.
Polyphenols are able to scavenge free radicals and inactivate other pro-oxidants
but they also have anti-inflammatory actions by inhibiting the activation of nuclear
factor-kappa B (NF-κB) and related cell signaling pathways that trigger systemic
67
inflammation. Alternative molecular mechanisms beyond antioxidation and based

on polyphenol–membrane and polyphenol–protein interactions are possible that are
not directly related to free radical scavenging or metal chelating [4].
In this chapter, the various classes of phytochemicals with known anti-
inflammatory effects will be highlighted. However, other phytochemicals from over
150,000 plant species on Earth may also modulate immune function and suppress
inflammation. Therefore, the number of phytochemicals with these properties is
so great that no review can claim to be comprehensive. Ancient systems of herbal
medicine in Egypt, India, and China were based on the anti-inflammatory effects
of many phytochemicals from plant parts that still remain to be studied. Therefore,
this chapter will provide some well-known examples that illustrate the principles
involved in the evolution of anti-inflammatory phytochemicals from plants to their
function in humans. Some of the data come from cancer cells where inflammatory
pathways are constitutively activated, simplifying the study of mechanisms of action
[5]. These mechanisms are then often studied in immunocompromised animals
bearing human tumor xenografts. These animal studies provide some insights as
do animal studies with transgenic animals that develop cancer or have lesions of
the immune system. Ultimately, limited amounts of human clinical trial data and
information on bioavailability and metabolism can be obtained. The potential anti-
inflammatory actions of a particular phytochemical are surmised by examining data
from a combination of experiments using the aforementioned paradigms, as human
clinical intervention data with disease endpoints do not exist for most phytochemi-
cals with anti-inflammatory potential.
Phytochemicals also interact with the gut microflora since many polyphenols are
only partially absorbed, and the gut microbiota form secondary metabolites such
as phenolic acids and urolithins [6,7]. Studies on the effects of phytochemicals as
prebiotics influencing gut microbial populations are only beginning to emerge at this
time, but this area of investigation promises to contribute significantly to our under-
standing of the anti-inflammatory effects of phytochemicals [8,9].
Another important aspect of phytochemical action is the occurrence of synergis-
tic interactions of related phytochemicals found in fruits and vegetables [10]. For
example, lycopene from tomato is found as part of a family of structurally related
carotenoids including phytoene, phytofluene, α-carotene, and β-carotene [11]. When
tested, there are additive or synergistic effects of these other compounds. The same
can be said of epigallocatechin gallate (EGCG) in green tea, which is found with
a family of catechins [12]. This synergy or additivity of multiple compounds and
different classes of compounds is unique to the action of phytochemicals and is not
characteristic of drugs [13]. It is estimated that two-thirds of all drugs are based on
compounds found in plants. Drugs and phytochemicals are metabolized by the same
enzymes in the liver called drug-metabolizing enzymes. However, since there were no
drugs 50,000 years ago, these are more appropriately called phytochemical-metabo-
lizing enzymes. The activation of cytochrome p450 enzymes by phytochemicals has
been interpreted by some authors as evidence of toxicity. In fact, this activation is part
of the normal course of events when a phytochemical is ingested [14].
In the nutritional range, phytochemicals have numerous beneficial effects,
including balancing oxidant stress, inflammation, and other cellular processes [15].
Phytochemicals and Immune Function 69
Humans, unlike most mammals, do not synthesize vitamin C [16]. It has been pro-
posed that this genetic machinery was inactivated due to the plentiful vitamin C in
the ancient diet based on plant foods rich in vitamin C and other phytochemicals.
Therefore, it may be that our bodies have a need for phytochemicals for optimum
nutrition at levels above the minimum daily requirement to avoid vitamin deficien-
cies. For example, the RDA for vitamin C is 60 mg/day. The human body loses
45 mg/day on average from a store of 1500 mg, but a typical fruit such as an orange
may contain twice the RDA. So amounts up to 500 mg/day are not toxic, but are
simply metabolized by the body and excreted as oxalate in the urine [17]. At very
high doses, oxalate stones are possible, but the safe range of consumption is wide and
well above the RDA. Most phytochemicals including polyphenols have no dietary
recommendation associated with them beyond the recommendation to consume five
servings per day of fruits and vegetables. Today most Americans eat refined carbo-
hydrates removed from their plant sources with lots of calories, the wrong balance of
fats, and too little protein, fiber, vitamins, and minerals. Government surveys show
that 80% of Americans fail to eat the recommended five servings per day of fruits
and vegetables. The translation of the nutrition science of fruits and vegetables to
dietary practice will be discussed in a later chapter. This chapter will consider the
notion that phytochemicals may represent a feasible approach to the inhibition of
low-grade chronic inflammation such as that associated with visceral obesity with-
out the toxicity associated with some anti-inflammatory drugs, and that the preven-
tive potential of phytochemicals in age-related chronic diseases may be due largely
to their anti-inflammatory effects.
PHYTOCHEMICAL EVOLUTION AND HUMAN IMMUNITY

Plants contain secondary metabolites distinguished from their macronutrients and
fibers by being small molecules that have signaling functions in the plant cell. They
are called phytochemicals, but could more appropriately be termed phytonutri-
ents. The term phytochemical has been used to discuss chemical structure and was
adopted by established nutrition authorities who declared that these were nonnutrient
substances in that they did not provide calories to the diet and, as chemicals, could
be toxic (although at levels far beyond the nutritional range). At the time, in the early
2000s, these chemicals were thought to be antioxidants that carried out simply a
radical scavenging function in plants exposed to ultraviolet radiation, heat, and the
reactive oxygen species generated during photosynthesis. However, research over the
last 20 years has provided evidence that these chemicals are actually phytonutrients
carrying out vital nutritional and physiological roles in the body. Furthermore, they
are clearly metabolized by the microflora of the gut and so carry out a nutritional
function in the microbiome where they stimulate growth of the gut microflora, which
may impact immune function. Nonetheless, as a practical matter, the term phyto-
chemicals will be used in this chapter as it is the scientific convention most widely
accepted in the literature.
One theory of why the phytochemicals have a role in human physiology relates to
the notion that plants were here on Earth prior to mankind. Humans evolved on a plant-
based diet digesting the same substances that were innate immune signals in plants.
In plants, the innate immune system functions by recognizing invading bacte-

ria through recognition of antigens called microbe-associated molecular patterns
(MAMPs) and activation of cell surface pattern recognition receptors [18–20]. The
MAMPs are microbial cell envelope components including bacterial lipopolysac-
charide (LPS) [21], an outer membrane glycoconjugate from gram-negative bacteria
that is classically used as an experimental stimulus to human immune cells such as
macrophages and lymphocytes. So both plant and human immune systems react to
these bacterial products. This commonality of plant and human immune cell func-
tions again demonstrates the evolutionary link between innate immunity in plants
and humans. The plant immune response is activated by transcriptional regulators
that reprogram the transcriptome to favor defense responses over routine cellular
requirements such as growth. The activity of these transcriptional regulators is con-
trolled by signaling hormones including salicylic acid, jasmonic acid, and ethylene
[22]. These signaling hormones regulate genes that are induced and/or repressed by
the action of transcriptional regulators. The plant immune system has some similari-
ties to the human system in that it can be triggered by an external factor that results
in cellular signaling pathway activation.
Phytochemicals were produced through evolutionary gene changes in order
to defend plants from stress, plant pathogens, parasites, and herbivores [23–26].
Cruciferous vegetables including broccoli, Brussels sprouts, cabbage, and horserad-
ish are known for their characteristic flavors and odors that repel pests. This defense
mechanism results from the metabolism of inactive substances called glucosinolates
that are activated by enzymes liberated when plant cells are crushed or damaged
by a predator. Glucosinolates have also been studied extensively for their health
benefits in humans [27–30], since when a human chews a cruciferous vegetable the
same enzymes are activated as with mechanical damage, infection, or insect attack.
Several classes of enzymes including myrosinases or thioglucosidases act on stored
glucosinolates [31–36] to produce a wide range of toxic reaction products includ-
ing isothiocyanates, nitriles, epithioalkanes, and thiocyanates [37,38]. Both climate
and agricultural practices influence the content of phytochemicals in plants [39].
Therefore, modern industrial agricultural methods lead to variable contents of glu-
cosinolates in broccoli available in grocery stores depending on the stress under
which plants are grown. Some investigators have studied broccoli sprouts or geneti-
cally modified broccoli in attempts to enhance the effects of glucosinolates in human
clinical research [40].
CLASSIFICATION OF PHYTOCHEMICALS
A brief survey of the classification and nomenclature of phytochemicals is presented
here prior to considering the available literature on some selected phytochemicals
and inflammation. A comprehensive discussion of all phytochemicals is outside the
scope of this chapter and would require a multivolume dedicated text. Phytochemicals
can be broadly classified into three major groups: terpenoids, polyphenols, and alka-
loids. More than 5000 of these compounds have been discovered, and it is expected
that scientists will discover many more. Any one serving of vegetables could provide
over 100 different phytochemicals. For example, while cruciferous vegetables are
classified as a group based on their content of glucosinolates, these vegetables also

contain carotenoids including lutein, zeaxanthin, and β-carotene, as well as tocoph-
erols [41–43]. Of these fat-soluble phytochemicals, α-tocopherol is generally the pre-
dominant compound [44]. Despite occurring together in the same plant foods, the
various classes of phytochemicals are discussed separately in the following text.
The terpenoids—sometimes called isoprenoids—are used extensively for their
aromatic qualities. This large group of phytochemicals includes the carotenoids that
provide certain colors to fruits and vegetables such as red (lycopene), green and
yellow (lutein/zeaxanthin), pink (astaxanthin), and orange (β-carotene). The flavors
of cinnamon, clove, rosemary, salvia, ginger, and other spices are due to terpenoids
[45]. Spices have antibacterial properties based on these phytochemicals and were
used for the preservation of foods prior to the advent of refrigeration [45]. Other
terpenoids include menthol, camphor, cannabinoids found in marijuana, ginkgolides
found in Ginkgo biloba, and the curcuminoids found in turmeric and mustard seed.
Curcumin will be discussed further in a chapter on the effects of spices as there is
a great deal of information available on its anti-inflammatory activities. Isoprenoids
also play other important roles in cellular physiology, which will not be discussed
here, including their role in the regulation of cholesterol synthesis and linking signal-
ing molecules to the cell membrane through isoprenylation [46].
Carotenoids are classified under the terpenoids. Over 600 different carot-
enoids have been identified in plants [47]. In plants, carotenoid pigments function
both in energy production through photosynthesis and in protection from oxidant
stress and ultraviolet radiation. For example, in tomatoes, lycopene is localized
to the chloroplast, which is the energy-producing organelle of the plant cell. The
carotenoids can quench and inactivate various reactive oxygen species formed from
exposure to light in the lipid layers of plant cells. This antioxidant role may also be
associated with antioxidant activity in human organs such as the prostate and the skin
where localization has been demonstrated. Carotenoids can be important sources of
vitamin A especially in populations that do not eat meat or dairy products, which
contain vitamin A. β-Carotene, α-carotene, and β-cryptoxanthin can be metabolized
to vitamin A after ingestion and have been called dietary vitamin A [48]. Orange
vegetables and fruits, including carrots, sweet potatoes, winter squash, pumpkin,
papaya, mango, and cantaloupe, are rich sources of β-carotene. Zeaxanthin and
lutein localize to the macular region of the retina, which is the area that receives the
most ultraviolet radiation from light focused by the lens of the eye. The most com-
mon cause of blindness in individuals over 65 years of age is macular degeneration,
and it is tempting to consider that the localization of these carotenoids to the macula
plays a protective role against ultraviolet radiation and oxidant stress [49]. Lycopene
intake has also been linked to prostate cancer in population studies. Giovannucci et
al. [50], in the era before there was widespread screening for prostate cancer, found
that both increased blood levels of lycopene and the intake of tomato products five or
more times per week were associated with a lower incidence of aggressive prostate
cancer. Lycopene has been shown to modulate immune function in humans [51] and
can be found in the human prostate gland where it has some effects on cellular func-
tion [52]. Tomatoes, watermelons, pink grapefruits, apricots, and pink guavas are the
most common sources of lycopene.
Alkaloids are one of the largest groups of chemical defenses produced by thou-
sands of plant species from hundreds of plant families. Amino acids are the building
blocks for alkaloids, and alkaloids include a very large number of bitter nitrogenous
compounds. Although they existed long before humans, some alkaloids have remark-
able structural similarities with neurotransmitters in the central nervous system of
humans, including dopamine, serotonin, and acetylcholine. The amazing effect of
these alkaloids on humans has led to the development of powerful painkiller medica-
tions and other behavior-altering and addictive drugs [53].
Caffeine, which is one of the most widely studied behavior-altering drugs,
improves all aspects of brain health, including attention, cognition, mood, and
memory [54]. Consumed as a coffee beverage, it comes along with polyphenol anti-
oxidants found in coffee beans. Theobromine, found in chocolate, is accompanied
by antioxidant cocoa flavanols. Therefore, the polyphenols rather than the alkaloid
components of these foods are relevant to this chapter.
This alkaloid class also includes capsaicin, the phytochemical that causes burning
pain through interaction with pain receptors on the tongue when a hot chili pepper is
chewed. Once acclimated to the burning, some individuals find the sensation pleas-
ing, and in ancient cultures, this spice was used as an aphrodisiac. Capsaicin is not
broken down during the digestion process and can cause burning in the lower diges-
tive tract hours after ingestion [55]. Botanists believe that birds are immune to the
burning sensation of capsaicin and may serve to disperse the seeds. Capsaicin may
prevent hungry mammals from devouring the fruits so that they can be eaten by fruit-
eating birds that are attracted to bright red fruits. Passing through the bird’s digestive
tract relatively unharmed, the small seeds are dispersed to other favorable regions.
The pain receptor is found on the tongue while another receptor in the intestine
increases energy expenditure via retrograde neural transmission to the central ner-
vous system and may affect satiety [56]. Capsaicin is also anti-inflammatory as a non-
toxic dose of capsaicin inhibited Helicobacter pylori-induced interleukin-8 (IL-8)
production by gastric epithelial cells through the modulation of the NF-κB and IL-8
pathways [57]. Capsaicin will also be discussed further in the chapter on spices.
Polyphenols are the most important class of dietary antioxidants, and their anti-
inflammatory effects will be discussed in some detail. Polyphenols get their name
from a chemical structure consisting of one or more aromatic rings with one or more
hydroxyl groups (these are the phenols). Within this large class of phytochemicals,
subclasses include the flavonoids, stilbenes, coumarins, and tannins. Polyphenols
are produced in plant cells through secondary metabolism and function in both plant
reproduction and growth. They also act in the plant immune system against patho-
gens, parasites, and predators, as well as contributing to the color of plants. In addi-
tion to their roles in plants, polyphenols may modulate human immune function in
beneficial ways and act as antioxidants. Polyphenols are found in a wide variety of
fruits and vegetables, including pomegranates, strawberries, cherries, apples, cran-
berries, grapes, pineapples, peaches, lemons, oranges, pears, and grapefruits. They
are also found in teas and chocolate as catechins and cocoa flavanols. It is estimated
that flavonoids account for approximately two-thirds of the polyphenols in the diet
with the majority of the remainder from phenolic acids.
Flavonoids are the largest subgroup of polyphenols with more than 5000 different
phytochemical flavonoids identified. The flavonoids include the following structur-
ally different subgroups: (a) flavones (e.g., apigenin, luteolin), which are found in pars-
ley and celery. Hydroxylation on position 3 of the flavone structure gives rise to the
3-hydroxyflavones also known as the (b) flavonols (e.g., kaempferol and quercetin),
which are found in red and yellow onions, broccoli, red grapes, cherries, French
beans, leeks, and apples; (c) isoflavones (e.g., daidzein and genistein), mainly found
in soy and soy products, have a large structural variability, and more than 600 isofla-
vones have been identified to date; (d) flavanones/flavanonols (e.g., hesperetin, narin-
genin/astilbin, and engeletin), which are mainly found in citrus fruits, herbs (oregano),
and wine; (e) flavanols, for example, (+)-catechin, (−)-epicatechin, epigallocatechin,
and EGCG, which are abundant in green tea, red wine, and chocolate. Flavanols are
found both as monomers and oligomers referred to as condensed tannins or proan-
thocyanidins; (f) anthocyanidins (e.g., pelargonidin, cyanidin, and malvidin), whose
sources include red wine and berry fruits. Flavonoids are most frequently found in
nature as conjugates in glycosylated or esterified forms but can occur as aglycones,
especially as a result of the effects of food processing; and (g) hydrolysable tannins
(e.g., ellagitannins) found in pomegranate and walnut. Many different glycosides can
be found in nature. More than 80 different sugars have been discovered conjugated
to flavonoids. Red wine, pomegranate juice, and grape juice also contain significant
levels of flavonoids and anthocyanin pigments. Anthocyanins give the red-purple and
blue colors to berry fruits, fruit juices, and red wine. Flavonoids act as antioxidants
and regenerate vitamin C, inhibit LDL cholesterol oxidation, inhibit platelet aggrega-
tion, and have anti-inflammatory and antitumor actions in experimental models. For
example, quercetin, the major flavonol in the diet, possesses both anticarcinogenic
activity and ability to inhibit LDL oxidation. It also inhibits the methylation of other
flavonoids such as tea catechins increasing their bioactivity [58].
Phenolic acids can be subdivided into two major groups—hydroxybenzoic acids
and hydroxycinnamic acids. Hydroxybenzoic acid derivatives include p-hydroxy-
benzoic, protocatechuic, vannilic, syringic, and gallic acids. They are commonly
present in the bound form and are typically a component of a complex structure
such as lignins and hydrolyzable tannins [59]. They can also be found in the form
of sugar derivatives and organic acids in plant foods. In addition, phenolic acids
are produced from tea catechins and other dietary polyphenols by the gut micro-
flora [60]. Hydroxycinnamic acid derivatives include p-coumaric, caffeic, and ferulic
acids [61]. They are mainly present in the bound form, linked to cell-wall structural
components, such as cellulose, lignin, and proteins through ester bonds [62]. Ferulic
acids occur primarily in the seeds and leaves of plants. Wheat bran is a good source
of ferulic acids, which are esterified to hemicellulose of the cell walls. Food process-
ing, such as thermal processing, pasteurization, fermentation, and freezing, contrib-
utes to the release of the bound phenolic acids from the cell walls. Caffeic, ferulic,
p-coumaric, protocatechuic, and vannilic acids are present in almost all plants.
Chlorogenic acids and curcumin are also major derivatives of hydroxycinnamic
acids present in plants [63]. Chlorogenic acids are the ester of caffeic acids and are
the substrate for enzymatic oxidation leading to browning, particularly in apples
and potatoes. Recent research on chlorogenic acid from green coffee bean extracts
has been featured as dietary supplements for weight loss [64], but chlorogenic acid
can be found in many plants [63].
PHYTOCHEMICALS AND INHIBITION OF INFLAMMATION

Hunter-gatherers living in New Guinea eat over 800 varieties of plant foods, including
roots, leaves, stems, seeds, fruits, flowers, and bark, much as ancient man did 50,000
years ago [65]. Studies show that ancient man ate about 11 lb of plant foods daily,
which were rich in fiber and poor in calories. They had to spend 3–5 h/day gather-
ing foods from their environment just to get the calories they needed to survive [65].
They had to know the botany of the plants and which were safe to eat intimately as it
was their only grocery store. Their bodies had to process the many substances made
by plants, which they ate in such abundance, and their livers developed the proteins
to perform the essential function of detoxifying plant substances and excreting them
from the body.
The major challenges to survival in ancient times were starvation and infection—
if one excludes accidental deaths and deaths in battle. To avoid starvation and infec-
tion, metabolic pathways leading to storage of fat calories and inflammation have
become evident in modern times as part of the global obesity epidemic [66,67]. For
example, about 15–20 million years ago, when apes moved to Europe from Africa,
they were faced with a temperate climate in which fruits grew only in the springtime.
An adaptation in metabolism, marked by a rise in the circulating levels of uric acid
from 3.5 to 5.5 mg/dL, enabled more efficient conversion of fructose from fruits into
stored fat [68]. At the same time, the fat cells that store fat release adipocytokines
that activate inflammation to fight infections [69]. In the modern era of industrial
food production and high-fructose intake from table sugar and high-fructose corn
syrup, the efficient conversion of fructose to fat has contributed to the global epi-
demic of obesity when combined with sedentary lifestyles. Over the last 30 years,
research has found that fat cells secrete a number of proinflammatory cytokines that
activate immune function and may have acted to fight infections and stimulate heal-
ing in ancient times. However, today these proinflammatory cytokines from visceral
fat are linked to a wide array of age-related chronic diseases. Once again, a use-
ful ancient adaptation has the opposite to intended effects when combined with the
modern Western diet and lifestyle in which fruit and vegetable intake is reduced and
there is an increased intake of phytochemical-poor refined carbohydrates that stimu-
late inflammation. The following sections of this chapter will summarize briefly
what is known about some well-studied phytochemicals and their role in inhibiting
the low-grade inflammation associated with overweight and obesity.
TEA
Green tea (Camellia sinensis) is made by stopping the natural process of oxida-
tion in tea leaves by steaming or heating the leaves. Green tea has been a popular
beverage used in traditional Chinese medicine for over 5000 years. Green tea con-
sumption has been associated with decreased risks for obesity [70], diabetes [71,72],
hypertension [73], dyslipidemia [74], and CVD mortality [75] in epidemiological

studies. In clinical trials, green tea has been shown to significantly improve fea-
tures of metabolic syndrome, including decreasing abdominal adiposity indicated
by waist circumference in obese subjects [76–78], reducing blood glucose and hemo-
globin A1C in prediabetic or diabetic patients [79,80], improving postprandial lipid
responses in subjects with mild hypertriglyceridemia [81–84], and increasing flow-
mediated dilation in smokers or subjects with endothelial dysfunction [85,86]. In
common with other polyphenols, green tea catechins inhibit activation of the NF-κB
pathway [87].
BERRY POLYPHENOLS AND HYDROLYZABLE

TANNINS FROM POMEGRANATE
Polyphenols from blueberries and cranberries localize into endothelial cells and
reduce the vulnerability of endothelial cells to increased oxidative stress at both
the membrane and cytosol level. Furthermore, berry polyphenols also reduce
TNFα-induced upregulation of various inflammatory mediators (IL-8, MCP-1, and
ICAM-1) involved in the recruitment of leukocytes to sites of damage or inflamma-
tion along the endothelium. Both blueberry and cranberry polyphenols are able to
afford protection to endothelial cells against stressor-induced upregulation of oxida-
tive and inflammatory insults [88].
Pomegranate extract, which is rich in ellagitannin, the largest polyphenol anti-
oxidant, has been extensively studied for its anti-inflammatory effects using various
cancer models. In breast cancer xenografts, inhibition of angiogenesis, a component
of the inflammatory response in tumors, was inhibited by pomegranate extract [89].
In androgen-independent human prostate cancer xenografts, inhibition of NF-κB
was demonstrated ex vivo in animals administered pomegranate extract orally [90].
CONDENSED TANNINS FROM GRAPE SEED EXTRACT

Grape seed extract (GSE) is rich in dimers, trimers, and other oligomers of flavan-
3-ols named proanthocyanidins, which are strong free radical scavengers and also
known to exert a wide spectrum of biological, pharmacological, and therapeutic
activities [91]. Moreover, GSE has been studied for its chemopreventive and anti-
cancer potential in various cancer models [92–98]. For example, GSE reduces the
incidence of carcinogen-induced mammary tumors in rats, skin tumors in mice,
and growth inhibition of DU145 and HT29 prostate and colon cancer xenografts in
nude mice [93–98], as well as the TRAMP transgenic prostate cancer model [99]. In
human lens epithelial cells, GSE inhibits NF-κB and MAP kinase pathways involved
in oxidant stress and inflammation [100].
COCOA POLYPHENOLS FROM CHOCOLATE

Cocoa contains over 380 phytochemicals, but in raw form, cocoa beans are inedible
due to the extreme bitterness caused by the high polyphenol content. After roast-
ing and processing, final cocoa products such as chocolate have markedly reduced
polyphenol contents depending on the type of manufacturing process used [101].

Much of the research on cocoa polyphenols has been performed with standardized
extracts [102–105]. The bioavailability of cocoa polyphenols is variable. Epicatechin
is well absorbed, with a maximum plasma concentration at around 2 h and with
approximately 20% of consumed epicatechin being excreted in the urine [106].
Procyanidin dimers have also been found [107] in blood samples from healthy adults
who had consumed a cocoa beverage. However, the bioavailability of cocoa polyphe-
nols remains to be established.
The consumption of cocoa/chocolate increases plasma antioxidant capacity,
diminishes platelet function and inflammation, and decreases diastolic and systolic
arterial pressures. Currently available data support the concept that daily consump-
tion of cocoa-rich chocolate or polyphenol-enriched cocoa extracts could reduce the
risk for CVDs [108,109]. Nitric oxide (NO)-dependent, flow-mediated dilation of the
brachial artery and concentrations of nitroso compounds in plasma have been mea-
sured, and it was demonstrated that ingestion of a high-flavanol cocoa drink, but not
a low-flavanol cocoa drink, significantly increased plasma concentrations of nitroso
compounds and flow-mediated dilation of the brachial artery [110]. Therefore,
ingested flavonoids may reverse endothelial dysfunction through enhancement of
NO bioactivity. Oxidative modification of LDL appears to be crucial for athero-
genesis, and one of the mediators is the proinflammatory proatherogenic enzyme
myeloperoxidase. Micromolar concentrations of (−)-epicatechin or other flavonoids
were found to suppress lipid peroxidation in LDL induced by myeloperoxidase in
the presence of physiologically relevant concentrations of nitrite, an NO metabolite.
Therefore, vascular health was improved at the level of the endothelial cell in the
microcirculation in humans.
GLUCOSINOLATES
While the example of glucosinolates from broccoli has already been discussed in
isolation, it is worth revisiting the large class of glucosinolates. About 120 different
glucosinolates are known to occur naturally in plants. They are synthesized from
certain amino acids. The so-called aliphatic glucosinolates are derived mainly from
methionine but also from alanine, isoleucine, leucine, and valine. Glucoraphanin
is derived from dihomomethionine, which is methionine chain-elongated twice.
Aromatic glucosinolates include indolic glucosinolates, such as glucobrassicin,
derived from tryptophan and others from phenylalanine as well as homophenylala-
nine, and sinalbin derived from tyrosine. Plants contain the enzyme myrosinase,
which, in the presence of water, cleaves off the glucose group from a glucosinolate.
The remaining molecule is then converted to either an isothiocyanate, a nitrile, or
a thiocyanate. These are the active substances that serve as defense for the plant.
The plant uses these as a defense when the cell is crushed by a predator, which then
brings together the myrosinase, that is in a separate compartment of the cell from the
glucosinolate, activating this primitive defense mechanism.
Consumption of broccoli sprouts, a rich source of glucoraphanin, has been asso-
ciated with decreased incidence, multiplicity, and tumor growth in animal cancer
models [111–113]. In 1992, Paul Talalay and colleagues at Johns Hopkins University
identified the isothiocyanate, sulforaphane (SFN), a biologically active metabolite of

glucoraphanin, as the compound in broccoli responsible for many of its health ben-
efits [114]. Since that time, more than 500 studies have been conducted on the mech-
anisms and biological activity of SFN and its precursor, glucoraphanin [115]. SFN’s
multiple molecular targets and promising early research have led to 15 clinical trials
currently under way to assess its effects on various cancers, CVD, upper airway
inflammation, radiation dermatitis, and vascular health [115]. Glucoraphanin, also
referred to as SFN glucosinolate, is the most potent naturally occurring inducer of
phase 2 detoxification enzymes and is an indirect long-acting antioxidant [116–118].
SFN has anti-inflammatory activities, as shown by suppressing ligand-induced
and ligand-independent TLR4 activation. It prevents IL-1R-associated kinase-1 deg-
radation, activation of NF-κB and IFN regulatory factor 3, and cyclooxygenase-2
expression induced by LPS or overexpression of TLR4. Receptor oligomerization,
which is one of the initial and critical events of TLR4 activation, is suppressed by
SFN, resulting in the downregulation of NF-κB activation [119]. SFN forms adducts
with cysteine residues in the extracellular domain of TLR4 as confirmed by liquid
chromatography–tandem mass spectrometry analysis, and the inhibitory effects of
SFN on oligomerization and NF-κB activation are reversed by thiol donors (DTT and
N-acetyl-l-cysteine). These results suggest that the reactivity of SFN to sulfhydryl
moiety contributes to its inhibitory activities. Blockade of TLR4 signaling by SFN
resulted in the reduced production of inflammatory cytokines and decreased dermal
inflammation and edema in vivo in experimental inflammatory animal models.
CAROTENOIDS
Lycopene at physiological concentrations is able to attenuate the LPS-mediated
induction of TNFα in RAW 264.7 macrophages, at both the mRNA and protein
levels. The molecular mechanism was studied, and it appeared that the LPS activa-
tion of both JNK and NF-κB signaling pathways was modulated by lycopene [120].
The anti-inflammatory effects of lycopene on macrophages were accompanied by
a decrease in LPS-stimulated macrophage migration in the presence of lycopene.
Furthermore, lycopene decreased macrophage-conditioned medium-induced pro-
inflammatory cytokine, acute phase protein, and chemokine mRNA expression in
3T3-L1 adipocytes.
β-Carotene has shown antioxidant and anti-inflammatory activities. In vitro and
in vivo regulatory functions of β-carotene have been shown in the production of NO
and PGE(2), as well as the expression of inducible NO synthase (iNOS), cyclooxy-
genase-2, TNFα, and IL-1beta. β-Carotene also inhibits the expression and produc-
tion of these inflammatory mediators in both LPS-stimulated RAW264.7 cells and
primary macrophages in a dose-dependent fashion, as well as in LPS-administrated
mice. Furthermore, β-carotene suppressed NF-κB activation and iNOS promoter
activity in RAW264.7 cells stimulated with LPS. β-Carotene blocked nuclear trans-
location of the NF-κB p65 subunit, which correlated with its inhibitory effect on
IκB-α phosphorylation and degradation. These results suggest that β-carotene pos-
sesses anti-inflammatory activity by functioning as a potential inhibitor for redox-
based NF-κB activation, probably due to its antioxidant activity.
CONCLUSIONS AND FUTURE DIRECTIONS

Phytochemicals can modulate inflammation by interactions with the innate immune
system and can inhibit the activation of the NF-κB pathway. There is now a global
epidemic of obesity, and obesity is associated with a low-grade systemic chronic
inflammatory state, characterized by the abnormal production of proinflammatory
adipocytokines. It has been found that macrophages infiltrate adipose tissue and are
responsible for the majority of inflammatory cytokine production in obesity.
Overweight and obesity are now more common than malnutrition globally.
Obesity-induced inflammation is considered a potential mechanism linking obesity
to its related pathologies, such as insulin resistance, CVDs, type-2 diabetes, and
some immune disorders. Therefore, obesity-related inflammation may be inhibited
by phytochemicals from tea, coffee, or fruits and vegetables and prevent or amelio-
rate the development of obesity-related diseases linked to inflammation. The discov-
ery of the impact of the microbiome on immune function discussed in this chapter
and the modulation of microbial flora by both diet and obesity leaves open the ques-
tion of whether phytochemicals, by influencing the microbiome, may indirectly
inhibit inflammation or have other effects on immune function. Considering that
colorful fruits and vegetables have other beneficial effects, research on the impact
of phytochemicals in overweight and obese individuals with increased inflammation
promises to be a rich area of investigation in the future.
At the same time, the impact of phytochemicals should be studied for their ability
to preserve muscle tissue being destroyed by cytokines such as TNFα and IL-6 in
hospitalized malnourished patients, patients with cancer and/or infections including
AIDS.
The same can be said for the need to conduct research on inflammatory bowel
disease, asthma, and rheumatoid arthritis.
Clearly, the impact of phytochemicals on the immune system needs more atten-
tion as it has the potential to enhance human health through actions that balance
excess inflammation.
REFERENCES
1. Heber D. 2004. Phytochemicals beyond antioxidation. J Nutr 134:3175S–3176S.
2. Heber D. 2010. An integrative view of obesity. Am J Clin Nutr 91:280S–283S.
3. Heber D, Bowerman S. 2001. Applying science to changing dietary patterns. J Nutr
131(11 Suppl):3078S–3081S.
4. Fraga CG, Galleano M, Verstraeten SV, Oteiza PI. 2010. Basic biochemical mechanisms
behind the health benefits of polyphenols. Mol Aspects Med 31(6):435–445.
5. Lee KW, Bode AM, Dong Z. 2011. Molecular targets of phytochemicals for cancer
prevention. Nat Rev Cancer 11:211–218.
6. Henning SM, Wang P, Abgaryan N, Vicinanza R, de Oliveira DM, Zhang Y et al. 2013.
Phenolic acid concentrations in plasma and urine from men consuming green or black
tea and potential chemopreventive properties for colon cancer. Mol Nutr Food Res
57:483–493.
7. Seeram NP, Henning SM, Zhang Y, Suchard M, Li Z, Heber D. 2006. Pomegranate juice
ellagitannin metabolites are present in human plasma and some persist in urine for up to
48 hours. J Nutr 136:2481–2485.
8. Jin JS, Touyama M, Hisada T, Benno Y. 2012. Effects of green tea consumption on
human fecal microbiota with special reference to Bifidobacterium species. Microbiol
Immunol 56:729–739.
9. Vodnar DC, Socaciu C. 2012. Green tea increases the survival yield of Bifidobacteria in
simulated gastrointestinal environment and during refrigerated conditions. Chem Cent J
6:61.
10. Efferth T, Koch E. 2011. Complex interactions between phytochemicals. The multi-
target therapeutic concept of phytotherapy. Curr Drug Targets 12:122–132.
11. Heber D, Lu QY. 2002. Overview of mechanisms of action of lycopene. Exp Biol Med
(Maywood) 227:920–923.
12. Henning SM, Fajardo-Lira C, Lee HW, Youssefian AA, Go VL, Heber D. 2003. Catechin
content of 18 teas and a green tea extract supplement correlates with the antioxidant
capacity. Nutr Cancer 45:226–235.
13. Tallarida RJ. 2011. Quantitative methods for assessing drug synergism. Genes Cancer
2:1003–1008.
14. Greenblatt DJ, Derendorf H. 2013. Grapefruit-medication interactions. CMAJ 185:507.
15. Biesalski HK, Erdman JW Jr, Hathcock J, Ellwood K, Beatty S, Johnson E et al. 2013.
Nutrient reference values for bioactives: New approaches needed? A conference report.
Eur J Nutr 52(Suppl 1):1–9.
16. Linster CL, Van Schaftingen E. 2007. Vitamin C. Biosynthesis, recycling and degrada-
tion in mammals. FEBS J 274:1–22.
17. Levine M, Padayatty SJ, Espey MG. 2011. Vitamin C: A concentration-function
approach yields pharmacology and therapeutic discoveries. Adv Nutr 2:78–88.
18. Chisholm ST, Coaker G, Day B, Staskawicz BJ. 2006. Host-microbe interactions:
Shaping the evolution of the plant immune response. Cell 124:803–814.
19. Janeway CA Jr, Medzhitov R. 2002. Innate immune recognition. Annu Rev Immunol
20:197–216.
20. Silipo A, Erbs G, Shinya T, Dow JM, Parrilli M, Lanzetta R et al. 2010. Glyco-conjugates
as elicitors or suppressors of plant innate immunity. Glycobiology 20:406–419.
21. Lian LH, Jin Q, Song SZ, Wu YL, Bai T, Jiang S et al. 2013. Ginsenoside Rh2
downregulates LPS-induced NF-κ B activation through inhibition of TAK1 phos-
phorylation in RAW 264.7 murine macrophage. Evid Based Complement Altern Med
2013:646728.
22. Pieterse CMJ, Leon-Reyes A, Van der Ent S, Van Wees SCM. 2009. Networking by
small-molecule hormones in plant immunity. Nat Chem Biol 5:308–316.
23. Fraenkel GS. 1959. The raison d’etre of secondary plant substances. Science
129:1466–1470.
24. Dong X, Kahmann R. 2009. Battle for survival: Plants and their allies and enemies. Curr
Opin Plant Biol 12:387–389.
25. Rasmann S, Agrawal AA. 2009. Plant defense against herbivory: Progress in identifying
synergism, redundancy, and antagonism between resistance traits. Curr Opin Plant Biol
12:473–478.
26. Reina-Pinto JJ, Yephremov A. 2009. Surface lipids and plant defenses. Plant Physiol
Biochem 47:540–549.
27. Traka M, Mithen R. 2009. Glucosinolates, isothiocyanates and human health. Phytochem
Rev 8:269–282.
28. Verkerk R, Schreiner M, Krumbein A, Ciska E, Holst B, Rowland I et al. 2009.
Glucosinolates in Brassica vegetables: The influence of the food supply chain on intake,
bioavailability and human health. Mol Nutr Food Res 53:219–265.
29. van Horn L, McCoin M, Kris-Etherton PM, Burke F, Carson JA, Champagne CM et al.
2008. The evidence for dietary prevention and treatment of cardiovascular disease. J Am
Diet Assoc 108:287–331.
30. Virgili F, Marino M. 2008. Regulation of cellular signals from nutritional molecules:
A specific role for phytochemicals, beyond antioxidant activity. Free Radic Biol Med
45:1205–1216.
31. Ahuja I, Rohloff J, Bones AM. 2010. Defence mechanisms of Brassicaceae: Implications
for plant–insect interactions and potential for integrated pest management. A review.
Agron Sustain Dev 30:311–348.
32. Bednarek P, Pislewska-Bednarek M, Svatos A, Schneider B, Doubsky J, Mansurova M
et al. 2009. A glucosinolate metabolism pathway in living plant cells mediates broad-
spectrum antifungal defense. Science 323:101–106.
33. Beecher CWW. 1994. Cancer preventive properties of varieties of Brassica oleracea:
A review. Am J Clin Nutr 59:1166–1170.
34. Bones AM, Rossiter JT. 2006. The enzymic and chemically induced decomposition of
glucosinolates. Phytochemistry 67:1053–1067.
35. Clay NK, Adio AM, Denoux C, Jander G, Ausubel FM. 2009. Glucosinolate metabolites
required for an Arabidopsis innate immune response. Science 323:95–101.
36. Kissen R, Bones AM. 2009. Nitrile-specifier proteins involved in glucosinolate hydroly-
sis in Arabidopsis thaliana. J Biol Chem 284:12057–12070.
37. Kissen R, Rossiter J, Bones A. 2009. The ‘mustard oil bomb’: Not so easy to assemble?!
Localization, expression and distribution of the components of the myrosinase enzyme
system. Phytochem Rev 8:69–86.
38. Rask L, Andreasson E, Ekbom B, Eriksson S, Pontoppidan B, Meijer J. 2000.
Myrosinase: Gene family evolution and herbivore defense in Brassicaceae. Plant Mol
Biol 42:93–113.
39. Ahuja I, de Vos RCH, Bones AM, Hall RD. 2010. Plant molecular stress responses face
climate change. Trends Plant Sci 15:664–674.
40. Fahey JW, Wehage SL, Holtzclaw WD, Kensler TW, Egner PA, Shapiro TA et al. 2012.
Protection of humans by plant glucosinolates: Efficiency of conversion of glucosinolates
to isothiocyanates by the gastrointestinal microflora. Cancer Prev Res (Phila) 5:603–611.
41. Olmedilla B, Herrero C, Perez-Sacristan B, Blanco I, Blazquez S. 2006. Bioavailability
of carotenoids and tocopherols from broccoli: In vivo and in vitro assessment. Exp Biol
Med 231:1733–1738.
42. Ibrahim KE, Juvik JA. 2009. Feasibility for improving phytonutrient content in veg-
etable crops using conventional breeding strategies: Case study with carotenoids and
tocopherols in sweet corn and broccoli. J Agric Food Chem 57:4636–4644.
43. Kurilich AC, Tsau GJ, Brown A, Howard L, Klein BP, Jeffery EH et al. 1999. Carotene,
tocopherol, and ascorbate contents in subspecies of Brassica oleracea. J Agric Food
Chem 47:1576–1581.
44. Goffman FD, Mollers C. 2000. Changes in tocopherol and plastochromanol-8 contents
in seeds and oil of oilseed rape (Brassica napus L.) during storage as influenced by
temperature and air oxygen. J Agric Food Chem 48:1605–1609.
45. López P, Sanchez C, Batlle R, Nerín C. 2007. Vapor-phase activities of cinnamon,
thyme, and oregano essential oils and key constituents against foodborne microorgan-
isms. J Agric Food Chem 55:4348–4356.
46. Mo H, Elson CE. 2004. Studies of the isoprenoid-mediated inhibition of mevalonate syn-
thesis applied to cancer chemotherapy and chemoprevention. Exp Biol Med (Maywood)
229:567–585.
47. Demmig-Adams B, Adams WW, 3rd. 2002. Antioxidants in photosynthesis and human
nutrition. Science 298:2149–2153.
48. Krinsky NI. 2005. The biological activity of carotenoid metabolites. Sight Life Newslett
2:10–15.
49. Wong IYH, Koo SCY, Chan CWN. 2011. Prevention of age-related macular degenera-
tion. Int Ophthalmol 31:73–82.
50. Giovannucci E, Liu Y, Platz EA, Stampfer MJ, Willett WC. 2007. Risk factors for
prostate cancer incidence and progression in the health professionals follow-up study.
Int J Cancer 121:1571–1578.
51. Palozza P, Parrone N, Catalano A, Simone R. 2010. Tomato lycopene and inflammatory
cascade: Basic interactions and clinical implications. Curr Med Chem 17:2547–2563.
52. Chen L, Stacewicz-Sapuntzakis M, Duncan C, Sharifi R, Ghosh L, van Breemen R et al.
2001. Tomato sauce consumption decreases serum PSA and oxidative damage in pros-
tate cancer patients. J Natl Cancer Inst 93:1872–1879.
53. Rischer H, Häkkinen ST, Ritala A, Seppänen-Laakso T, Miralpeix B, Capell T, Christou
P, Oksman-Caldentey KM. 2013. Plant cells as pharmaceutical factories. Curr Pharm
Des 19(31):5640–5660.
54. Giesbrecht T, Rycroft JA, Rowson MJ, De Bruin EA. 2010. The combination of
L-theanine and caffeine improves cognitive performance and increases subjective alert-
ness. Nutr Neurosci 13:283–290.
55. Lee AL, Li Z, Zerlin A, Heber D. 2010. Effects of dihydrocapsiate on adaptive and diet-
induced thermogenesis with a high protein very low calorie diet: A randomized control
trial. Nutr Metab 7:78.
56. Lejeune MPGM, Kovacs EMR, Westerterp-Plantenga MS. 2003. Effect of capsaicin on
substrate oxidation and weight maintenance after modest body-weight loss in human
subjects. Br J Nutr 90:651–659.
57. Lee IO, Lee KH, Pyo JH, Kim JH, Choi YJ, Lee YC. 2007. Anti-inflammatory effect
of capsaicin in Helicobacter pylori-infected gastric epithelial cells. Helicobacter
12:510–517.
58. Wang P, Heber D, Henning SM. 2012. Quercetin increased bioavailability and decreased
methylation of green tea polyphenols in vitro and in vivo. Food Funct 3:635–642.
59. Xie L, Roto AV, Bolling BW. 2012. Characterization of ellagitannins, gallotannins, and
bound proanthocyanidins from California almond (Prunus dulcis) varieties. J Agric
Food Chem 60:12151–12156.
60. Gao K, Xu A, Krul C, Venema K, Liu Y, Niu Y et al. 2006. Of the major phenolic
acids formed during human microbial fermentation of tea, citrus, and soy flavonoid
supplements, only 3,4-dihydroxyphenylacetic acid has antiproliferative activity. J Nutr
136:52–57.
61. El-Seedi HR, El-Said AM, Khalifa SA, Göransson U, Bohlin L, Borg-Karlson AK et al.
2012. Biosynthesis, natural sources, dietary intake, pharmacokinetic properties, and bio-
logical activities of hydroxycinnamic acids. J Agric Food Chem 60:10877–10895.
62. Rodríguez-Arcos RC, Smith AC, Waldron KW. 2004. Ferulic acid crosslinks in aspara-
gus cell walls in relation to texture. J Agric Food Chem 52:4740–4750.
63. Farah A, Monteiro M, Donangelo CM, Lafay S. 2008. Chlorogenic acids from green
coffee extract are highly bioavailable in humans. J Nutr 138:2309–2315.
64. Vinson JA, Burnham BR, Nagendran MV. 2012. Randomized, double-blind, placebo-
controlled, linear dose, crossover study to evaluate the efficacy and safety of a green
coffee bean extract in overweight subjects. Diabetes Metab Syndr Obes 5:21–27.
65. Marlowe FW. 2005. Hunter-gatherers and human evolution. Evol Anthropol 14:54–67.
66. King BM. 2013. The modern obesity epidemic, ancestral hunter-gatherers, and the
sensory/reward control of food intake. Am Psychol 68:88–96.
67. Hamdy O, Porramatikul S, Al-Ozairi E. 2006. Metabolic obesity: The paradox between
visceral and subcutaneous fat. Curr Diabetes Rev 2:367–373.
68. Johnson RJ, Sautin YY, Oliver WJ, Roncal C, Mu W, Sanchez-Lozada LG et al. 2009.
Lessons from comparative physiology: Could uric acid represent a physiologic alarm
signal gone awry in western society? J Comp Physiol B 179:67–76.
69. Ouchi N, Parker JL, Lugus JJ, Walsh K. 2011. Adipokines in inflammation and metabolic
disease. Nat Rev Immunol 11:85–97.
70. Wu CH, Lu FH, Chang CS, Chang TC, Wang RH, Chang CJ. 2003. Relationship
among habitual tea consumption, percent body fat, and body fat distribution. Obes Res
11:1088–1095.
71. Iso H, Date C, Wakai K, Fukui M, Tamakoshi A. 2006. The relationship between green
tea and total caffeine intake and risk for self reported type 2 diabetes among Japanese
adults. Ann Intern Med 144:554–562.
72. Yamaji T, Mizoue T, Tabata S, Ogawa S, Yamaguchi K, Shimizu E et al. 2004. Coffee
consumption and glucose tolerance status in middle-aged Japanese men. Diabetologia
47:2145–2151.
73. Yang YC, Lu FH, Wu JS, Wu CH, Chang CJ. 2004. The protective effect of habitual tea
consumption on hypertension. Arch Intern Med 164:1534–1540.
74. Imai K, Nakachi K. 1995. Cross sectional study of effects of drinking green tea on car-
diovascular and liver diseases. BMJ 310:693–696.
75. Sasazuki S, Kodama H, Yoshimasu K, Liu Y, Washio M, Tanaka K et al. 2000. Relation
between green tea consumption and the severity of coronary atherosclerosis among
Japanese men and women. Ann Epidemiol 10:401–408.
76. Kuriyama S, Shimazu T, Ohmori K, Kikuchi N, Nakaya N, Nishino Y et al. 2006. Green
tea consumption and mortality due to cardiovascular disease, cancer, and all causes in
Japan: The Ohsaki study. JAMA 296:1255–1265.
77. Sesso HD, Gaziano JM, Buring JE, Hennekens CH. 1999. Coffee and tea intake and the
risk of myocardial infarction. Am J Epidemiol 149:162–167.
78. Shimazu T, Kuriyama S, Hozawa A, Ohmori K, Sato Y, Nakaya N et al. Dietary pat-
terns and cardiovascular disease mortality in Japan: A prospective cohort study. Int J
Epidemiol 36:600–609.
79. Nagao T, Komine Y, Soga S, Meguro S, Hase T, Tanaka Y et al. 2005. Ingestion of a tea
rich in catechins leads to a reduction in body fat and malondialdehyde-modified LDL in
men. Am J Clin Nutr 81:122–129.
80. Westerterp-Plantenga MS, Lejeune MP, Kovacs EM. 2005. Body weight loss and weight
maintenance in relation to habitual caffeine intake and green tea supplementation. Obes
Res 13:1195–1204.
81. Auvichayapat P, Prapochanung M, Tunkamnerdthai O, Sripanidkulchai BO,
Auvichayapat N, Thinkhamrop B et al. 2008. Effectiveness of green tea on weight
reduction in obese Thais: A randomized, controlled trial. Physiol Behav 93:486–491.
82. Fukino Y, Shimbo M, Aoki N, Okubo T, Iso H. 2005. Randomized controlled trial for an
effect of green tea consumption on insulin resistance and inflammation markers. J Nutr
Sci Vitaminol (Tokyo) 51:335–342.
83. Fukino Y, Ikeda A, Maruyama K, Aoki N, Okubo T, Iso H. 2008. Randomized controlled
trial for an effect of green tea-extract powder supplementation on glucose abnormalities.
Eur J Clin Nutr 62:953–960.
84. Unno T, Tago M, Suzuki Y, Nozawa A, Sagesaka YM, Kakuda T et al. 2005. Effect
of tea catechins on postprandial plasma lipid responses in human subjects. Br J Nutr
93:543–547.
85. Kim W, Jeong MH, Cho SH, Yun JH, Chae HJ, Ahn YK et al. 2006. Effect of green
tea consumption on endothelial function and circulating endothelial progenitor cells in
chronic smokers. Circ J 70:1052–1057.
86. Widlansky ME, Hamburg NM, Anter E, Holbrook M, Kahn DF, Elliott JG et al. 2007.
Acute EGCG supplementation reverses endothelial dysfunction in patients with coro-
nary artery disease. J Am Coll Nutr 26:95–102.
87. Hsu A, Bruno RS, Löhr CV, Taylor AW, Dashwood RH, Bray TM et al. 2011. Dietary
soy and tea mitigate chronic inflammation and prostate cancer via NFκB pathway in the
Noble rat model. J Nutr Biochem 22:502–510.
88. Youdim KA, McDonald J, Kalt W, Joseph JA. 2002. Potential role of dietary flavonoids
in reducing microvascular endothelium vulnerability to oxidative and inflammatory
insults. J Nutr Biochem 13:282–288.
89. Sartippour MR, Seeram NP, Rao JY, Moro A, Harris DM, Henning SM et al. 2008.
Ellagitannin-rich pomegranate extract inhibits angiogenesis in prostate cancer in vitro
and in vivo. Int J Oncol 32:475–480.
90. Rettig MB, Heber D, An J, Seeram NP, Rao JY, Liu H et al. Pomegranate extract
inhibits androgen-independent prostate cancer growth through a nuclear factor-kappaB-
dependent mechanism. Mol Cancer Ther 7:2662–2671.
91. Bagchi D, Bagchi M, Stohs S, Ray SD, Sen CK, Preuss HG et al. 2002. Cellular
protection with proanthocyanidins derived from grape seeds. Ann NY Acad Sci
957:260–270.
92. Wen W, Lu J, Zhang K, Chen S. 2008. Grape seed extract inhibits angiogenesis via sup-
pression of the vascular endothelial growth factor receptor signaling pathway. Cancer
Prev Res (Phila Pa) 1:554–561.
93. Singletary KW, Meline B. 2001. Effect of grape seed proanthocyanidins on colon aber-
rant crypts and breast tumors in a rat dual-organ tumor model. Nutr Cancer 39:252–258.
94. Zhao J, Wang J, Chen Y, Agarwal R. 1999. Anti-tumor-promoting activity of a polyphe-
nolic fraction isolated from grape seeds in the mouse skin two-stage initiation-promotion
protocol and identification of procyanidin B5-3′-gallate as the most effective antioxidant
constituent. Carcinogenesis 20:1737–1745.
95. Agarwal C, Sharma Y, Agarwal R. 2000. Anticarcinogenic effect of a polyphenolic frac-
tion isolated from grape seeds in human prostate carcinoma DU145 cells: Modulation of
mitogenic signaling and cell-cycle regulators and induction of G1 arrest and apoptosis.
Mol Carcinog 28:129–138.
96. Singh RP, Tyagi AK, Dhanalakshmi S, Agarwal R, Agarwal C. 2004. Grape seed extract
inhibits advanced human prostate tumor growth and angiogenesis and upregulates
insulin-like growth factor binding protein-3. Int J Cancer 108:733–740.
97. Kaur M, Singh RP, Gu M, Agarwal R, Agarwal C. 2006. Grape seed extract inhib-
its in vitro and in vivo growth of human colorectal carcinoma cells. Clin Cancer Res
12:6194–6202.
98. Kaur M, Agarwal C, Agarwal R. 2009. Anti-cancer and cancer chemopreventive poten-
tial of grape seed extract and other grape-based products. J Nutr 139:1806S–1812S.
99. Raina K, Singh RP, Agarwal R, Agarwal C. 2007. Oral grape seed extract inhibits pros-
tate tumor growth and progression in TRAMP mice. Cancer Res 67:5976–5982.
100. Jia Z, Song Z, Zhao Y, Wang X, Liu P. 2011. Grape seed proanthocyanidin extract pro-
tects human lens epithelial cells from oxidative stress via reducing NF-кB and MAPK
protein expression. Mol Vis 17:210–217.
101. Rusconi M, Conti A. 2010. Theobroma cacao L., the food of the Gods: A scientific
approach beyond myths and claims. Pharmacol Res 61:5–13.
102. Andújar I, Recio MC, Giner RM, Cienfuegos-Jovellanos E, Laghi S, Muguerza B et al.
2011. Inhibition of ulcerative colitis in mice after oral administration of a polyphenol-
enriched cocoa extract is mediated by the inhibition of STAT1 and STAT3 phosphoryla-
tion in colon cells. J Agric Food Chem 59:6474–6483.
103. Tomás-Barberán FA, Cienfuegos-Jovellanos E, Marín A, Muguerza B, Gil-Izquierdo A,
Cerda B et al. 2007. A new process to develop a cocoa powder with higher flavonoid
monomer content and enhanced bioavailability in healthy humans. J Agric Food Chem
55:3926–3935.
104. Mellor DD, Sathyapalan T, Kilpatrick ES, Beckett S, Atkin SL. 2010. High-cocoa poly-
phenol-rich chocolate improves HDL cholesterol in type 2 diabetes patients. Diabetic
Med 27:1318–1321.
105. Monagas M, Khan N, Andres-Lacueva C, Casas R, Urpi-Sarda M, Llorach R et al. 2009.

Effect of cocoa powder on the modulation of inflammatory biomarkers in patients at
high risk of cardiovascular disease. Am J Clin Nutr 90:1144–1150.
106. Williamson G. 2009. Bioavailability and health effects of cocoa polyphenols. Inflammo
pharmacology 17:2.
107. Holt RR, Lazarus SA, Sullards MC, Zhu QY, Schramm DD, Hammerstone JF et al.
2002. Procyanidin dimer B2 [epicatechin-(4β-8)-epicatechin] in human plasma after the
consumption of a flavanol-rich cocoa. Am J Clin Nutr 76:798–804.
108. Gómez-Juaristi M, González-Torres L, Bravo L, Vaquero MP, Bastida S, Sánchez-
Muniz FJ. 2011. Beneficial effects of chocolate on cardiovascular health. Nutr Hosp
26:289–292.
109. Khawaja O, Gaziano JM, Djoussé L. 2011. Chocolate and coronary heart disease:
A systematic review. Curr Atheroscler Rep 13:447–452.
110. Schroeter H, Heiss C, Balzer J, Kleinbongard P, Keen CL, Hollenberg NK et al. 2006.
(–)-Epicatechin mediates beneficial effects of flavanol-rich cocoa on vascular function
in humans. Proc Natl Acad Sci USA 103:1024–1029.
111. Fahey JW, Zhang Y, Talalay P. 1997. Broccoli sprouts: An exceptionally rich source of
inducers of enzymes that protect against chemical carcinogens. Proc Natl Acad Sci USA
94:10367–10372.
112. Zhang Y, Kensler TW, Cho CG, Posner GH, Talalay P. 1994. Anticarcinogenic activities
of sulforaphane and structurally related synthetic norbornyl isothiocyanates. Proc Natl
Acad Sci USA 91:3147–3150.
113. Gerhauser C, You M, Liu J, Moriarty RM, Hawthorne M, Mehta RG et al. 1997. Cancer
chemopreventive potential of sulforamate, a novel analogue of sulforaphane that induces
phase 2 drug-metabolizing enzymes. Cancer Res 57:272–278.
114. Zhang Y, Talalay P, Cho CG, Posner GH. 1992. A major inducer of anticarcinogenic
protective enzymes from broccoli: Isolation and elucidation of structure. Proc Natl Acad
Sci USA 89:2399–2403.
115. http://www.brassica.com/emerging-science/sulforaphane-and-cancer (accessed on
November 21, 2013).
116. Fahey JW, Talalay P. 1999. Antioxidant functions of sulforaphane: A potent inducer of
phase II detoxification enzymes. Food Chem Toxicol 37:973–979.
117. Zhang Y, Talalay P. 1994. Anticarcinogenic activities of organic isothiocyanates:
Chemistry and mechanisms. Cancer Res 54:1967s–1981s.
118. Tanito M, Masutani H, Kim YC, Nishikawa M, Ohira A, Yodoi J. 2005. Sulforaphane
induces thioredoxin through the antioxidant responsive element and attenuates retinal
light damage in mice. Invest Ophthalmol Vis Sci 46:979–987.
119. Youn HS, Kim YS, Park ZY, Kim SY, Choi NY, Joung SM et al. 2010. Sulforaphane sup-
presses oligomerization of TLR4 in a thiol-dependent manner. J Immunol 184:411–419.
120. Marcotorchino J, Romier B, Gouranton E, Riollet C, Gleize B, Malezet-Desmoulins
C et al. 2012. Lycopene attenuates LPS-induced TNF-α secretion in macrophages and
inflammatory markers in adipocytes exposed to macrophage-conditioned media. Mol
Nutr Food Res 56:725–732.
6 Genetic and
Environmental Modifiers
of Immune Function
David Heber
CONTENTS
Introduction............................................................................................................... 85
Genetic Variation in Complex Diseases.................................................................... 86
Genetic Variation in the Immune System................................................................. 87
Interaction of the Immune System with the Microbes in the Environment.............. 89
Obese Microbiota: Interactions with the Immune System........................................90
Autoimmune Disorders and the Gut Microflora....................................................... 91
Conclusion................................................................................................................96
References.................................................................................................................96
INTRODUCTION
Most common disorders that have a genetic component such as obesity, diabetes,
heart disease, common forms of cancer, most autoimmune diseases, and allergic
diseases, including asthma, have both a genetic and an environmental component.
It is now recognized that an underlying mechanism of disease common to these
various disorders is inflammation. Both environmental factors and multiple genes,
each with modest contributions to the total variance in association with common
diseases, are found universally in common diseases. Although the number of known
mutations underlying complex traits is still relatively small, advances in genomics
have greatly enhanced both genetic discovery and the analysis of genomic and epi-
genomic influences on disease risk. The methods of linkage analysis and gene asso-
ciation studies have been enhanced with recent technological advances in genome
mapping, sequencing, and analysis of individual variation. Genome-wide associa-
tion studies (GWASs) and the use of single-nucleotide polymorphisms (SNPs) are
being widely applied to research on chronic diseases using commercially available
microarrays.
However, genetics simply sets the stage for a disease while the environmental
influences including the impact of diet, lifestyle, and obesity on inflammation trigger
the ultimate development of that disease or disorder. The understanding of common
environmental influences has also advanced. The World Health Organization has
recognized the impact of malnutrition on immune function as well as the effects
85
of the global epidemic of overweight and obesity on chronic diseases. It is now

clear that not only is the immune system of the overweight individual affected by
visceral fat associated with overweight and obesity but the gut microflora are also
affected by both nutrition and obesity. Research combining influences of nutrition
and genetics on both body fat and the microbiome is progressing toward an improved
understanding of the interaction of genetics and environment in the modulation of
immune function and inflammation.
GENETIC VARIATION IN COMPLEX DISEASES

In order to understand gene–environment interactions modifying the immune
response, it is necessary to have an understanding of the methods used in genetic
studies of common diseases [1]. Analysis of SNPs is by far the most commonly
applied analysis used in the study of genetic variation in association with the risk of
age-related chronic diseases and inflammation. The human genome has tens of mil-
lions of these SNPs as small insertions and deletions (indels) as well as having copy
number variants (CNVs) of various genes. The database called sbSNP, containing
short human genetic variations, lists more than 60 million entries (http://www.ncbi.
nlm.nih.gov/projects/SNP/). Commercially available microarrays that permit the
screening of individuals for hundreds of thousands of genomic variants in a single
assay are widely available. For example, a particular chip for human SNPs contains
markers for more than 900,000 SNPs and 900,000 CNVs.
Identification of genes responsible for monogenic traits has become straightfor-
ward with resources such as GeneCards, a database of human disease genes (http://
www.genecards.org/cgi-bin/listdiseasecards.pl?type = full), which lists 5600 genes
that are associated with human diseases. Most of these diseases are monogenic. The
Online Mendelian Inheritance of Man (http://www.omim.org) contains information
on all known Mendelian disorders and over 12,000 genes. Even in these large data-
bases, only a few genes underlying complex human disease are known.
Another technique for genetic analysis of disease association is the mapping
of quantitative trait loci (QTLs), the chromosomal regions (locations) that contain
genomic elements contributing to variation in a trait such as obesity. While several
thousand genes have been associated with obesity through mapping QTLs, only
a handful of the actual mutations responsible for genetic variation have been
identified.
The most logically intuitive approach to the identification of specific genes and
mutations responsible for disease associations is the candidate gene approach. Prior
knowledge of the function of a gene, coupled with some understanding of the physi-
ological basis of the disease being studied, has been a historical first approach to the
discovery of gene/disease relationships. This logic has been exploited effectively for
a number of monogenic human disease genes. The positional candidate approach
to gene discovery employs linkage mapping to map a trait related to the disease
and then searching for most likely candidates among the genes known to lie in this
region. While this has been effective in monogenic traits, it has had limited success
for QTLs. One reason is that the function of most genes is still unknown. Another is
that mapping resolution is usually severely limited. A typical confidence interval in
Genetic and Environmental Modifiers of Immune Function 87
a QTL mapping study might be 20 or more centimorgans, corresponding to roughly

20 million base pairs and potentially 200 or more genes.
GWASs have been increasingly used in studies of genetic variation [2]. A catalog
of published GWAS can be accessed at www.genome.gov/gwasstudies. This catalog
includes over 1200 publications on over 6000 SNPs.
While large numbers of QTLs underlying complex genetic disorders are known,
the causative mutations remain elusive. Recent advances in genome sequencing
projects, particularly the discovery of vast numbers of SNPs and their availability
in affordable arrays, have been an advance in gene discovery. When used in GWAS,
these techniques may lead ultimately to the discovery of large numbers of genes
involved in complex ways in chronic diseases impacted by inflammation and in host
resistance to infectious diseases.
The understanding of the genetics of disease and of the immune system is still
evolving and is somewhat restricted in its potential. Put simply, genes set the stage
for a disorder, but environmental influences trigger the actual response to disease.
The classic example that has received great attention has been the Native Americans
living in southern Arizona who are from the same genetic background as the
Tarahumara Indians of northern Mexico. The incidence of diabetes, hypertension,
and heart diseases is at epidemic proportions among the Native Americans but is
much less common in the Tarahumara Indians despite their similar genetics. The
influence of the high-fat/high-sugar diet of the Native Americans combined with a
more sedentary lifestyle is typically viewed as being responsible for the observed
differences in disease incidence. Another example is the increase in cancer incidence
among Asian-Americans living in California within a single generation of migrating
from Asia to California. As discussed later, the genetic influence of diet now
extends also to the microbiome as well as the human genome, and the interaction
of the microbiome and the human immune system must be considered as a separate
pathway to inflammation-associated chronic diseases.
GENETIC VARIATION IN THE IMMUNE SYSTEM

The immune system response to pathogens is considered as being divided into
innate and adaptive immunity, recognizing that the two interact in the human body.
The innate immune response provides a very rapid defense mechanism involving
inflammation, complement activation, phagocytosis, and destruction of pathogens.
The innate immune response is critically dependent on pattern-recognition recep-
tors (PRRs), such as Toll-like receptors (TLRs) found on the cell surface or endo-
somes in effector cells (including macrophages, neutrophils, and dendritic cells),
that recognize pathogen-associated molecular patterns typically located on the sur-
face of pathogen cells. The resulting gene activation leads to cytokine and chemo-
kine release and generation of an inflammatory response. On the other hand, the
adaptive immune response initiates an antigen-specific response after first being
exposed via an interaction with the innate immune system. The adaptive response
involves B and T lymphocytes with recognition of antigens leading to the generation
of a specific antibody protein. There is also a cell-mediated immune response initi-
ated involving T helper cells and cytotoxic T cells particularly involved in cancer
surveillance. The antibody response eliminates pathogens and allows for generation
of long-term immune memory. Antibodies may act, for example, to neutralize bac-
terial toxins, opsonize bacteria in order to target them for phagocytosis or destroy
them via complement activation.
GWASs have resulted in the identification of 200 genomic loci in autoimmune
diseases [3–5]. Crohn’s disease has been highlighted as a significant GWAS suc-
cess story in which new pathogenic pathways and potential drug targets have been
identified. Genes encoding components of cytokine signaling, as well as of innate
immunity including autophagy, and of a sensor of bacterial peptidoglycan have been
identified [6]. Genetic variation involving genes at diverse genomic locations that
encode proteins involved in a given immune pathway is associated with several
related phenotypes. For example, strong associations have been recognized between
the IL-23/IL-12 signaling pathways and several autoimmune diseases, including
inflammatory bowel disease, psoriasis, and primary biliary cirrhosis [3,7].
GWAS has uncovered overlap between the pathways and mechanisms involved
in immune-related diseases [8]. In a recent analysis, among 107 independent SNP
markers associated at genome-wide significance across seven common autoimmune
diseases, 44% associated with at least two diseases [9]. Moreover, in 8% of the
observed associations, the effects were shared but in an opposite direction, with
increased risk in some diseases and protection in others. This is seen, for example,
with PTPN22 R620W; it is associated with increased risk of rheumatoid arthri-
tis, thyroid disease, and type 1 diabetes mellitus but with protection for Crohn’s
disease, and no effect on multiple sclerosis [3]. Associations involving cytokine
signaling, pathways involved in B- and T-cell activation, innate immunity, and
response to pathogens also demonstrated this type of overlap [3]. These shared pat-
terns support the notion of underlying central mechanisms across diseases most
likely involving inflammation. The initial response to antigens or microbial pat-
tern can also be affected [10]. Since their discovery, innate immunity microbial
sensors have been shown to play a critical role in innate immune responses to
microbes in several experimental in vitro, ex vivo, and animal models. However,
their role in the human response to infection in natural conditions has just started
to be deciphered by means of clinical studies of primary immunodeficiencies and
epidemiological genetic studies. There have been a number of studies of the genetic
diversity of the various families of microbial sensors in humans and of other mol-
ecules involved in the signaling pathways they trigger. The genetic associations,
revealed by both clinical and epidemiological genetic studies, of microbial sensors
concentrate on five different families: TLRs, C-type lectin receptors, NOD-like
receptors (NLRs), RIG-I-like receptors, and cytosolic DNA sensors. Variations at
the genes encoding these molecules have been related to the susceptibility to and
the severity of infectious diseases and other clinical conditions associated with
immune dysfunction, including autoimmunity, inflammation, allergy, and cancer.
At this time, the genetic links between innate immunity sensors and human disor-
ders remain to be established.
Epigenetics consists of those factors such as methylation and histone deacety-
lation, which can modulate gene expression. Epigenetics has been studied in the
immune system [11,12] where it is involved in the regulation of immune-cell identity
and function during development and differentiation in order to generate multiple

cell lineages, with remodeling of the epigenome acting to restrict or promote gene
expression and determine cell fate. Epigenetic regulation can mediate nutritional and
environmental influences on both the development and the function of the immune
system. Recent work has illustrated that genetic and epigenetic studies can help to
define this variation and improve our understanding of the immune response in
health and disease [13,14].
INTERACTION OF THE IMMUNE SYSTEM WITH

THE MICROBES IN THE ENVIRONMENT
Just as plants interact to protect plants from pathogenic soil bacteria through innate
immune functions, so vertebrates and mammals have evolved over millions of years
with the bacteria in the environment to establish a host gut microbiome and to repel
pathogenic gut bacteria with IgA, a secreted antibody [15]. Both the innate and
adaptive immune systems require microbial interactions during normal development
[16,17]. The secretion of IgA is critical in the development of mucosal immunity, as
it is initiated in response to bacterial colonization by specific bacterial species so as
to protect the integrity of the mucosal barriers to the external world and so establish
the host–microbiome interaction characteristic of an individual [18–20]. Germ-free
mice have abnormalities of gut secretory IgA, reduced gut-associated lymphoid tis-
sues with smaller Peyer’s patches and smaller mesenteric lymph nodes [21].
The innate immune system recognizes microbe-associated molecular patterns
found in a wide variety of bacterial species. These are most often integral compo-
nents of the cell wall, including lipopolysaccharides, peptidoglycans, and flagellin.
The recognition proteins of the innate immune system include TLRs and NLRs.
When TLRs are not present or are functionally mutated due to genetic abnormalities
or purposeful modification, the gut and mucosal immune systems do not develop
a healthy host–microbiome interaction [17]. The commensal bacteria appear to
be important in suppressing inflammatory response and promoting immunologi-
cal tolerance, and this interaction also occurs through TLRs [17,22]. NLRs also
recognize microbial molecules and can form oligomers (inflammasomes) that serve
as sensors of damage-associated patterns. Deficiency of NLPRP6, for example,
results in reduced IL-18 levels, an altered composition of the microbiota, and intes-
tinal hyperplasia [23].
The adaptive immune system is also influenced strongly by the gut microbiota,
and bacteria have been shown to influence the differentiation of T-cell populations,
which can be not only determined by self-/non-self-discrimination mechanisms but
also educated by the gut microbiota [24,25]. Gut bacteria are also capable of modu-
lating the host innate immune system to promote their own survival in the intestinal
microenvironment. For instance, Bacteroides species have been shown to induce
peptides with bactericidal activity targeting other intestinal microbes [26]. Being
able to stimulate the secretion of low amounts of IgA by modulating immune func-
tion is another way that host bacteria can promote their own survival in the gut
environment [20]. An interesting hypothesis in this regard is that modern cleanli-
ness may have increased the incidence of autoimmune disease by eliminating the
necessary exposure of the infant gastrointestinal tract to host bacteria that are ben-
eficial in stimulating normal immune function and tolerance of self. These examples
demonstrate the impact of microbial interactions with the immune systems on host
health.
OBESE MICROBIOTA: INTERACTIONS

WITH THE IMMUNE SYSTEM
Obesity is the result of the gene–environment imbalance resulting from industrial
food production and sedentary lifestyles. Obesity has emerged as a major health
concern in populations that have adopted a Western diet and is closely tied to the
microbiota [27]. In animal models of obesity, the distribution of the dominant gut
phyla, Bacteroidetes and Firmicutes, is shifted with a significant reduction of the
Bacteroidetes and a corresponding increase in Firmicutes [28]. The same trend has
been observed in individuals on weight-reduction diets [27]. One study in human
twins showed that in obese individuals the decrease in Bacteroidetes was accompa-
nied by an increase in Actinobacterium rather than in Firmicutes [29]. The observed
shift in the relative abundances of these bacterial phyla results in an increased effi-
ciency in harvesting energy from food and produces low-level inflammation.
Several changes in host genetics and environmental factors have been used to
induce obesity in animal models and thus provoke a change in microbiota composi-
tion. Remarkably, the energy harvest phenotype is transmissible simply by trans-
planting the obese microbiota into healthy lean donors [30,31]. Bacteria may be
enriched in enzymes such as amylase that break down starches to simple sugars
increasing the energy liberated from complex carbohydrates. On the other hand,
Zhang et al. suggest that increased energy harvest in obese individuals is related to
hydrogen transfer between taxa of gut bacteria, but no clear principle has been estab-
lished to explain the increased energy efficiency in mice with bacteria compared to
germ-free mice [32].
The inflammation characteristic of visceral obesity is a low-level inflamma-
tion very distinct from classical inflammation [33]. The obesity-associated pattern
includes moderate induction of inflammatory cytokines such as TNF-α, IL-1β,
and CCL2, as well as an increase in mast cells, T cells, and macrophages [33]. An
increase in Bifidobacterium species has also been shown to modulate inflamma-
tion in obese mice by increasing the production of glucagon-like peptide-2, which
reduces intestinal permeability and thus reduces the translocation of lipopolysaccha-
rides [34]. Although the elucidation of the exact mechanisms responsible for obesity
is still an open and complex problem, these studies demonstrate the link between an
imbalanced gut microbiota and diseased states and suggest hypotheses to be tested
in future research.
The Toll-like receptor TLR5 used to recognize flagellin from microbes in the
gut, and normally activates the innate immune system on interacting with flagellin
[35]. Mice lacking TLR5 develop characteristics of the metabolic syndrome along
with significant changes in their gut microbiota. It is likely that the alterations in the
gut flora secondary to a lack of TLR5 induced a low-grade inflammatory signaling
that eventually resulted in the development of metabolic syndrome. Interestingly,
this obesity phenotype was transmissible to wild-type mice simply by transferring

the microbiota [35]. A recent study, however, was unable to reproduce some of these
results, suggesting some colony-specific effects to be elucidated [36].
While human GWASs have identified a number of loci contributing to obesity,
a major limitation of these studies is the inability to assess environmental interac-
tions common to obesity. Using a systems genetics approach, Lusis and coworkers
[37] measured obesity traits, global gene expression, and gut microbiota composi-
tion in response to a high-fat/high-sucrose (HF/HS) diet of more than 100 inbred
strains of mice. In this study, HF/HS feeding promoted robust, strain-specific
changes in obesity that were not accounted for by food intake and provided evi-
dence for a genetically determined set point for obesity. GWAS analysis identified
11 genome-wide significant loci associated with obesity traits, several of which
overlapped with loci identified in human studies. These studies also demonstrated
strong relationships between genotype and gut microbiota plasticity during HF/
HS feeding and identified gut microbial phylotypes associated with obesity (see
Figures 6.1 and 6.2).
These studies demonstrated a high heritability of about 80% for body fat per-
centage across the study timeline. Moreover, changes in body fat percentage after
HF/HS feeding were also highly heritable (>70%), suggesting that dietary responses
are strongly controlled by genetics. These results are consistent with the heritabil-
ity estimates for body mass index and obesity in humans [38,39] and emphasize the
importance of genetics in controlling obesity traits, such as gene–nutrient interac-
tions. Overconsumption of high-calorie, energy-rich foods is a key environmental
factor contributing to the global obesity epidemic [40] making these model studies
in mice highly relevant to understanding the gene–environment interaction operative
in humans.
AUTOIMMUNE DISORDERS AND THE GUT MICROFLORA

Further evidence of the influence of the gut microflora on normal immune func-
tion comes from the study of autoimmune diseases. In mouse models of autoim-
mune type 1 diabetes, it has been shown that the interaction of the gut microflora
with the innate immune system modifies predisposition toward developing diabetes
[41]. Children at high genetic risk for type 1 diabetes have different gut microbiota
with decreased diversity over time and higher relative abundances of Bacteroides
ovatus and firmicute strains compared to children without an increased risk of type
1 diabetes [42]. Use of animal models of autoimmune multiple sclerosis and rheu-
matoid arthritis demonstrates a pronounced influence of the gut microflora in that
these autoimmune diseases do not develop in germ-free mice identical to those that
develop these models of disease [25]. When germ-free mice are then colonized by
specific bacteria, multiple sclerosis occurs in the animal model of the disease [25].
In our modern environment, many people are not exposed to the microbiota of our
evolutionary past. In the absence of appropriate microbial signals, the immune sys-
tem does not develop normally [21]. Autoimmune diseases, in general, and allergies,
in particular, have significantly increased in developed countries over the last few
years, which has been attributed to a burgeoning list of potential factors. The hygiene
MRI
Food intake
Weeks: 0 8 10 12 14 16
End study
HF/HS feeding
Weeks: 0 8
(a)
Before HF/HS feeding After HF/HS feeding
AXB19b/PgnJ
AXB19/PgnJ
BxH20/K J
BXA14/Pgn
ccJ
BXD 0/TyJ
BX A12/P J
CX A13 gnJ
Bx 11/Ty
SJL B12 /PgnJ

iAJ
C58/J
2
/H
BXD
BX A1/ /HI J
Bx D /T gnJ
0
BX K Rww yJ
D3 Pg J
J
BXxH2 2/P
BX
/T ww
D 87/ yJ
PL/ J
9/T nJ
Ax /J
B B1
D4 XD
12 R
BX
yJ
K
10
y
4/ 9/T
yJ wJ
B
D
B 2 /T /Rw AJ
Body fat percentage
AX xD 4b/ 1 i
BX B1 19 Ty D 61
D6 0/ /Ty J 20 Bx XD 13/H nJ
BX 0/R Pgn J B XB /Pg wwJ
w J C xA4 5/R J
BX A2/P wJ B D7 hiLt
BX D32/ gnJ 30 BX D/S yJ
BXD D21/ TyJ NO D15/T
T BX BL/6J nJ
BXD 43/Rw yJ
BXD 85/Rww J
w 40 C57 19a/Pg
66/R J AxB 5/RwwJ
ww BXD4 /TyJ
RIIIS J 50 BXH19
BXD16/T /J CxB3/ByJ
yJ
SEA/GnJ BXD8/TyJ
BTBRTtf/J CXB6/ByJ
LG/J BXD34/TyJ
129X1/SvJ BXD49/RwwJ
BUB/BnJ
BxA1 PgnJ
1/ SWR/J
CBA/J
BXD6
AKR/J AxB8 8/RwwJ
2J
DBA/ yJ BXD /PgnJ
x D 31/T wJ BX 13/Ty
B Rw BX D55/R J
62/ iLtJ Cx A24/ wwJ
BXD N/SH gnJ C5 B7/ PgnJ
P
NO xA7/ MyJ J
/ c BX 7L/J ByJ
B A C H6
M 2/Kc cJ B 3H /T
H 2 /La wJ Ax XD /He yJ
Bx ZW Rw /J B6 40/ J
N 51/ A /P Ty
BX D 86/ gnJ
H /T J
Bx 38 Pgn
D gn J
BX XD 6/P wwJ
CX XD3 9/T yJ
D 74/ Rw
BX J
BX B11 6/T yJ
B A1 0/R wJ
D /
84 Rw wJ
Bx xA8
Bx D5 /Rw J
D7 /H yJ
/R w
BX D64 /Rww
ww J
ww J
9/R iA
B
BX D71 wwJ
J
BX D48/R wJ
BxHVB/NJ
CXB /TyJ
BX 56/Rw
J
BXD 8/TyJ
yJ
AxB15 SM/J
BXH /TyJ
BXD6
CE/J
/PgnJ
BXD70/Rw /J
4/B
BxD5/TyJ
BALB/cJ
BXD14/TyJ
AXB2/PgnJ
wJ
BXD73/RwwJ
B
KS
F
C57BL
(b)
FIGURE 6.1 (See color insert.) Natural variation in gene-by-diet interactions. (a) Schematic
of study design with indicated time points for HF/HS feeding, magnetic resonance imaging
(MRI), food intake monitoring, and end of study. (b) Body fat percentage in male mice (108
strains) before and after 8 weeks of HF/HS feeding. Error bars represent SEM.
0%–50%
400 50%–100%
100%–150%
150%–200%
Body fat percentage growth
200%–250%
300 250%–300%
>300%
200
100
0 2 4 6 8
(c) Weeks on diet
5.0 5.0
4.5 4.5
Food intake (g/day)
Food intake (g/day)
4.0 4.0
3.5 3.5
3.0 3.0
2.5 r = 0.45 2.5 r = 0.52
2.0 p = 4.18e–33 2.0 p = 1.49e–45
20 30 40 50 60 15 20 25 30 35 40
(d) Body weight—4 weeks on diet (g) (e) Lean mass—4 weeks on diet (g)
5.0 5.0
4.5 4.5
Food intake (g/day)
Food intake (g/day)
4.0 4.0
3.5 3.5
3.0 3.0
2.5 2.5 r = 0.01
r = 0.18 p = 0.807
2.0 p = 4.33e–06 2.0
10 20 30 40 0 100 200 300 400
(f) Body fat percentage— (g) Body fat percentage
4 weeks on diet growth—0–4 weeks
FIGURE 6.1 (continued) (See color insert.) Natural variation in gene-by-diet interactions.
(c) Biweekly percent body fat percentage increase in male mice with indicated body fat
percentage increase after 8 weeks of HF/HS feeding. (d–g) Correlation of food intake
(g/day/mouse) with body weight (d), lean mass (e), body fat percentage—4 weeks on HF/HS
diet (f), and body fat percentage growth—0–4 weeks (g), regression line. r, bi-weight mid-
correlation; p, p value. (From Parks, B.W. et al., Cell Metab., 17, 141, 2013. With permission.)
hypothesis postulates that lack of exposure to pathogenic and nonpathogenic micro-

bial products early in life might result in an asthmatic phenotype due to an impaired
development of the immune system [41]. The concept is that individuals raised in
developing countries have lower rates of allergies due to larger family sizes, a larger
percentage of the population living in rural environments with poor sanitary condi-
tions, lower antibiotic use, and prevalence of helminths. A potential mechanism that
has been proposed to support this concept is the counterregulatory role of inter-
leukin-10 (IL-10) [42]. IL-10 is an anti-inflammatory cytokine that downregulates
responses from both the innate and adaptive immune systems. Infection by micro-
bial organisms results in the upregulation of IL-10, which subsequently suppresses
Chow diet HF/HS diet
Actinobacteria
Bacteroidetes
Firmicutes
Other
Proteobacteria
Tenericutes
Verrucomicrobia
(a)
PC2 (4%)
HF/HS
Chow
Verrucomicrobia
Actinobacteria
Proteobacteria
Firmicutes
Other Bacteroidetes
Tenericutes
PC3 (3.3%)
PC1 (9.5%)
(b)
FIGURE 6.2 (See color insert.) Robust shifts in gut microbiota composition after HF/HS
feeding. (a) Relative abundances of the different phyla after chow diet and HF/HS feeding
(average among 52 matched strains). (b) Principal coordinates analysis (PCoA) plot of the
unweighted UniFrac distances. Each circle representing a different mice strain is colored
according to the dietary conditions. PC1, PC2, and PC3 values for each mouse sample are
plotted; percent variation explained by each PC is shown in parentheses.
Chow HF/HS
Akkermansia
Lachnospiraceae_unclassified
Ruminococcaceae_unclassified
Clostridium
Bifidobacterium
Turicibacter
Clostridiaceae_unclassified
Dorea
Roseburia
Hydrogenoanaerobacterium
Erysipelotrichaceae_unclassified
Lactococcus
Butyricicoccus
Anaeroplasma
Oscillibacter
Barnesiella
Porphyromonadaceae_unclassified
–6.0 –4.8 –3.6 –2.4 –1.2 0.0 1.2 2.4 3.6 4.8 6.0
(c) LDA score (log 10)
FIGURE 6.2 (continued) (See color insert.) Robust shifts in gut microbiota composi-
tion after HF/HS feeding. (c) Linear discriminant analysis (LDA) coupled with effect size
measurements identifies the most differentially abundant taxons between chow and HF/HS
diets. HF/HS-diet-enriched taxa are indicated with a positive LDA score and taxa enriched in
normal chow diet have a negative score. Only taxa meeting an LDA significant threshold >2
are shown. (From Parks, B.W. et al., Cell Metab., 17, 141, 2013. With permission.)
inflammation and a predisposition to allergies. A reduced exposure early in life to

infectious agents and normal gut microflora could result in a weakened systemic
response to counterregulate inflammatory responses. As a result, there would be an
increase in the prevalence of allergies in these individuals raised in environments
that are too clean.
Although numerous genes could predispose individuals to allergic diseases [43],
it is the interaction of multiple genes and the environmental triggers to allergy that
seems to be most likely involved in atopic individuals. In one study, elevated serum
IgE levels associated with atopy have been correlated statistically with an SNP in the
promoter region of CD14, a coreceptor for lipopolysaccharides [44]. Children carry-
ing this SNP and who have regular contact with pets have higher levels of serum IgE
[45]. There is anecdotal evidence that taking hookworm eggs reduces or eliminates
allergic responses since parasites can increase IgE and distract the immune system
from the offending allergen [46].
An interesting and counterintuitive example of the effect of losing a normal bacte-
rial species in the human gut is Helicobacter pylori. H. pylori coevolved with the gut
microbiome and has adapted to the acidic environment of the stomach by evolving
a urease enzyme that makes ammonia to neutralize the acid around the bacterium
[47]. Gastric colonization by H. pylori usually takes place within the first 10 years
of life persisting throughout life in the absence of antibiotics due to the attachment
of H. pylori to the gastric epithelium with a corkscrew-like action [48,49]. Nearly
all adults in developing countries harbor H. pylori, and this bacterium has probably
colonized mankind for much of human history. There is strong evidence linking the
decreased prevalence of H. pylori in the Western world to increased antibiotic use.
At the same time, its absence is associated with elevated rates of asthma and allergic
disorders in the same Western populations [50,51]. Moreover, increased prevalence
of gastroesophageal reflux disease, adenocarcinoma of the esophagus, and Barrett’s
esophagus has been linked to decreased prevalence of certain strains of H. pylori
[52]. Overall, the strong link between H. pylori and modern diseases illustrates how
the disruption of the human microbiota can influence the overall health. To date,
there is no mechanistic evidence linking the absence of H. pylori to differences in
specific immune functions that could increase the risk of allergies.
An increasing variety of disease states and disorders is being found to corre-
late with the host microbiota [53], including susceptibility to influenza [54], retro-
virus transmission [55], colon cancer [56], autoimmune demyelination [56], and
even behavior [35,57]. Government-funded sequencing projects such as the Human
Microbiome Project [58,59] and the Earth Microbiome Project [60] may ultimately
lead to some more unified and comprehensive understanding of the link between the
microbiome and health.
CONCLUSION
As reviewed in this chapter, genetics and epigenetics affect both innate and adaptive
immune function. However, the largest part of the immune system, the gut-associated
immune system, interacts with the external world at the level of the intestinal micro-
biota and represents a modifiable and responsive organ of immune function. Nutrition
impacts the microflora in the gut and so impacts immune function. The gene–
environment interactions underlying diet and immune function provide an opportu-
nity to impact the incidence of immune-related and inflammation-driven common
diseases through changes in diet and lifestyle that impact multiple pathways.
REFERENCES
1. Knight JC. 2013. Genomic modulators of the immune response. Trends Genet. 29:74–83.
2. Hirschhorn JN, Daly MJ. 2005. Genome-wide association studies for common diseases
and complex traits. Nat. Rev. Genet. 6:95–108.
3. Cho JH, Gregersen PK. 2011. Genomics and the multifactorial nature of human autoim-
mune disease. N. Engl. J. Med. 365:1612–1623.
4. Kuchroo V.K. 2012. Dysregulation of immune homeostasis in autoimmune diseases.
Nat. Med. 18:42–47.
5. Lessard CJ. 2012. The genomics of autoimmune disease in the era of genome-wide
association studies and beyond. Autoimmun. Rev. 11:267–275.
6. Mathew CG. 2008. New links to the pathogenesis of Crohn disease provided by genome-
wide association scans. Nat. Rev. Genet. 9:9–14.
7. Wang K. 2009. Diverse genome-wide association studies associate the IL12/IL23 path-
way with Crohn disease. Am. J. Hum. Genet. 84:399–405.
8. Zhernakova A. 2009. Detecting shared pathogenesis from the shared genetics of

immune-related diseases. Nat. Rev. Genet. 10:43–55.
9. Cotsapas C, Voigt BF, Rossin E, Lage K, Neale BM, Wallace C et al. 2011. Pervasive
sharing of genetic effects in autoimmune disease. PLoS Genet. 7:e1002254.
10. Pothlichet J, Quintana-Murci L. 2013. The genetics of innate immunity sensors and
human disease. Int. Rev. Immunol. 32:157–208.
11. Fernandez-Morera JL. 2010. Epigenetic regulation of the immune system in health and
disease. Tissue Antigens. 76:431–439.
12. Suarez-Alvarez B. 2012. DNA methylation: A promising landscape for immune system-
related diseases. Trends Genet. 28:506–514.
13. Karlson EW. 2010. Gene–environment interaction between HLA-DRB1 shared epitope
and heavy cigarette smoking in predicting incident rheumatoid arthritis. Ann. Rheum.
Dis. 69:54–60.
14. Ramagopalan SV. 2009. Expression of the multiple sclerosis-associated MHC class II
Allele HLA-DRB1*1501 is regulated by vitamin D. PLoS Genet. 5:e1000369.
15. Ley RE, Lozupone CA, Hamady M, Knight R, Gordon JI. 2008. Worlds within worlds:
Evolution of the vertebrate gut microbiota. Nat. Rev. Microbiol. 6:776–788.
16. Chow J, Lee SM, Shen Y, Khosrav A, Mazmanian SK. 2010. Hostbacterial symbiosis in
health and disease. Adv. Immunol. 107:243–274.
17. O’Hara AM, Shanahan F. 2006. The gut flora as a forgotten organ. EMBO Rep.
7:688–693.
18. Bouskra D, Brezillon C, Berard M, Werts C, Varona R, Bonec IG et al. 2008. Lymphoid
tissue genesis induced by commensals through NOD1 regulates intestinal homeostasis.
Nature 456:507–510.
19. Macpherson AJ, Geuking MB, McCoy KD. 2011. Immunoglobulin A: A bridge between
innate and adaptive immunity. Curr. Opin. Gastroenterol. 27:529–533.
20. Peterson DA, McNulty NP, Guruge JL, Gordon JI. 2007. IgA response to symbiotic
bacteria as a mediator of gut homeostasis. Cell Host Microbe 2:328–339.
21. Round JL, Mazmanian SK. 2009. The gut microbiota shapes intestinal immune
responses during health and disease. Nat. Rev. Immunol. 9:313–323.
22. Round JL, Lee SM, Li J, Tran G, Jabri B, Chatila TA et al. 2011. The Toll-like receptor
2 pathway establishes colonization by a commensal of the human microbiota. Science
332:974–977.
23. Elinav E, Strowig T, Kau AL, Henao-Mejia J, Thaiss CA, Booth CJ et al. 2011.
NLRP6 inflammasome regulates colonic microbial ecology and risk for colitis. Cell
145:745–757.
24. Lathrop SK, Bloom SM, Rao SM, Nutsch K, Lio CW, Santacruz N et al. 2011.
Peripheral education of the immune system by colonic commensal microbiota. Nature
478:250–254.
25. Lee YK, Mazmanian SK. 2010. Has the microbiota played a critical role in the evolution
of the adaptive immune system? Science 330:1768–1773.
26. Hooper LV, Stappenbeck TS, Hong CV, Gordon, JI. 2003. Angiogenins: A new class of
microbicidal proteins involved in innate immunity. Nat. Immunol. 4:269–273.
27. Ley RE. 2010. Obesity and the human microbiome. Curr. Opin. Gastroenterol.
26:5–11.
28. Ley RE, Backhed F, Turnbaugh P, Lozupone CA, Knight RD, Gordon JI. 2005. Obesity
alters gut microbial ecology. Proc. Natl. Acad. Sci. USA 102:11070–11075.
29. Turnbaugh PJ, Hamady M, Yatsunenko T, Cantarel BL, Duncan A, Ley RE et al. 2009.
A core gut microbiome in obese and lean twins. Nature 457:480–484.
30. Turnbaugh PJ, Ley RE, Mahowald MA, Magrini V, Mardis ER, Gordon JI. 2006. An
obesity-associated gut microbiome with increased capacity for energy harvest. Nature
444:1027–1031.
31. Turnbaugh PJ, Backhed F, Fulton L, Gordon JI. 2008. Diet-induced obesity is linked to
marked but reversible alterations in the mouse distal gut microbiome. Cell Host Microbe
3:213–223.
32. Zhang H, DiBaise JK, Zuccolo A, Kudrna D, Braidotti M, Yu Y et al. 2009. Human
gut microbiota in obesity and after gastric bypass. Proc. Natl. Acad. Sci. USA
106:2365–2370.
33. Gregor MF, Hotamisligil GS. 2011. Inflammatory mechanisms in obesity. Annu. Rev.
Immunol. 29:415–445.
34. Cani PD, Possemiers S, Van de Wiele T, Guiot Y, Everard A, Rottier O et al. 2009.
Changes in gut microbiota control inflammation in obese mice through a mechanism
involving GLP-2-driven improvement of gut permeability. Gut 58:1091–1103.
35. Vijay-Kumar M, Aitken JD, Carvalho FA, Cullender TC, Mwangi S, Srinivasan S et al.
2010. Metabolic syndrome and altered gut microbiota in mice lacking Toll-like receptor
5. Science 328:228–231.
36. Letran SE, Lee SJ, Atif SM, Flores-Langarica A, Uematsu S, Akira S et al. 2011. TLR5-
deficient mice lack basal inflammatory and metabolic defects but exhibit impaired CD4
T cell responses to a flagellated pathogen. J. Immunol. 186:5406–5412.
37. Parks BW, Nam E, Org E, Kostem E, Norheim F, Hui ST et al. 2013. Genetic control
of obesity and gut microbiota composition in response to high-fat, high-sucrose diet in
mice. Cell Metab. 17:141–152.
38. Barsh GS, Farooqi IS, O’Rahilly S. 2000. Genetics of body-weight regulation. Nature
404:644–651.
39. Stunkard AJ, Foch TT, Hrubec Z. 1986. A twin study of human obesity. JAMA 256:51–54.
40. McCaffery JM, Papandonatos GD, Peter I, Huggins GS, Raynor HA, Delahanty LM
et al. 2012. Obesity susceptibility loci and dietary intake in the Look AHEAD Trial. Am.
J. Clin. Nutr. 95:1477–1486.
41. Strachan DP. 1989. Hay fever, hygiene, and household size. BMJ 299:1259–1260.
42. Wills-Karp M, Santeliz J, Karp CL. 2001. The germless theory of allergic disease:
Revisiting the hygiene hypothesis. Nat. Rev. Immunol. 1:69–75.
43. Moffatt MF, Kabesch M, Liang L, Dixon AL, Strachan D, Heath S et al. 2007. Genetic
variants regulating ORMDL3 expression contribute to the risk of childhood asthma.
Nature 448:470–473.
44. Vercelli D. 2003. Learning from discrepancies: CD14 polymorphisms, atopy and the
endotoxin switch. Clin. Exp. Allergy 33:153–155.
45. Eder W, Klimecki W, Yu L, von Mutius E, Riedler J, Braun-Fahrlander C et al. 2005.
Opposite effects of CD 14/-260 on serum IgE levels in children raised in different envi-
ronments. J. Allergy Clin. Immunol. 116:601–607.
46. Feary J, Britton J, Leonardi-Bee J. 2011. Atopy and current intestinal parasite infection:
A systematic review and meta-analysis. Allergy 66:569–578.
47. Blaser M. 2011. Antibiotic overuse: Stop the killing of beneficial bacteria. Nature
476:393–394.
48. Brown LM. 2000. Helicobacter pylori: Epidemiology and routes of transmission.
Epidemiol. Rev. 22:283–297.
49. Perry S, de la Luz Sanchez M, Yang S, Haggerty TD, Hurst P, Perez-Perez G et al. 2006.
Gastroenteritis and transmission of Helicobacter pylori infection in households. Emerg.
Infect. Dis. 12:1701–1708.
50. Anderson HR. 2005. Prevalence of asthma. BMJ 330:1037–1038.
51. Banatvala N, Mayo K, Megraud F, Jennings R, Deeks JJ, Feldman RA. 1993. The cohort
effect and Helicobacter pylori. J. Infect. Dis. 168:219–221.
52. Atherton JC, Blaser MJ. 2009. Coadaptation of Helicobacter pylori and humans:
Ancient history, modern implications. J. Clin. Invest. 119:2475–2487.
53. Virgin HW, Todd JA. 2011. Metagenomics and personalized medicine. Cell 147:44–56.
54. Ichinohe T, Pang IK, Kumamoto Y, Peaper DR, Ho JH, Murray TS et al. 2011. Microbiota
regulates immune defense against respiratory tract influenza A virus infection. Proc.
Natl. Acad. Sci. USA 108:5354–5359.
55. Kane M, Case LK, Kopaskie K, Kozlova A, MacDearmid C, Chervonsky AV et al. 2011.
Successful transmission of a retrovirus depends on the commensal microbiota. Science
334:245–249.
56. Kostic AD, Gevers D, Pedamallu CS, Michaud M, Duke F, Earl AM et al. 2011.
Genomic analysis identifies association of Fusobacterium with colorectal carcinoma.
Genome Res. 22:292–298.
57. Heijtz RD, Wang S, Anuar F, Qian Y, Bjorkholm B, Samuelsson A et al. 2011. Normal
gut microbiota modulates brain development and behavior. Proc. Natl. Acad. Sci. USA
108:3047–3052.
58. Peterson J, Garges S, Giovanni M, McInnes P, Wang L, Schloss JA et al. 2009. The NIH
Human Microbiome Project. Genome Res. 19:2317–2323.
59. Turnbaugh PJ, Ley RE, Hamady M, Fraser-Liggett CM, Knight R, Gordon JI. 2007. The
Human Microbiome Project. Nature 449:804–810.
60. Gilbert JA, Meyer F, Antonopoulos D, Balaji P, Brown CT, Brown CT et al. 2010.
Meeting report: The terabase metagenomics workshop and the vision of an Earth micro-
biome project. Stand. Genomic Sci. 3:243–248.
7 Cancer and Inflammation
David Heber
CONTENTS
Introduction............................................................................................................. 101
Acute Inflammation and Cancer............................................................................. 102
Chronic Inflammation and Cancer.......................................................................... 102
Colon Cancer and Gut Microflora...................................................................... 102
Helicobacter and Stomach Cancer..................................................................... 103
Hepatitis Viruses and Liver Cancer.................................................................... 103
Prostate Cancer and Inflammation..................................................................... 103
Inflammation and Lymphomas........................................................................... 104
Inflammation in Tumor Promotion, Angiogenesis, and Metastasis........................ 104
Targeted Immunotherapy: Inflammation and Tumor Cell Killing.......................... 105
Inflammation as a Side Effect of Tumor Treatment................................................ 107
Inflammation as a Causative Factor in Cancer Cachexia........................................ 108
Conclusion.............................................................................................................. 109
References............................................................................................................... 109
INTRODUCTION
Inflammation and cancer are closely associated, and invasive cancers have been
conceptualized as being linked to persistent pathogens with metabolic and immune
adaptations that make them resistant to killing, starvation, or clearance by immune
surveillance while growing and consuming host nutrients. While cancer can result in
a chronic inflammation, the opposite is also true as inflammation can lead to cancer.
Many of the known risk factors for common forms of cancer are associated with
some form of chronic inflammation. Up to 20% of cancers are linked to chronic
infections, 30% can be attributed to tobacco smoking and inhaled pollutants (such
as silica and asbestos), and 35% to dietary factors (with 20% of the overall cancer
burden attributed to obesity) [1]. Inherited forms of cancer account for only about
10% of all cancers with the vast majority due to somatic mutations secondary to
some of the environmental factors provided earlier. Many of the common forms of
cancer occur in epithelial cells impacted by the microenvironment through chronic
inflammation from stromal cells or invading immune cells.
On the other hand, inflammation is an integral component of all tumors as
first discovered by the pathologist Virchow in the nineteenth century. Induced
inflammation was also used historically and, to a limited extent, currently as a means
of killing tumor cells by upregulating the immune system using various bacterial
mixtures. Targeted immunotherapy uses antibodies or immune cells to kill tumor
101
cells specifically using antigens or other identifying substances on or in tumor cells

that identify them separately from host cells. More recently, the side effects of antitu-
mor therapies that activate the immune system such as radiation therapy have become
the target of nutritional interventions designed to limit the impact of immune activa-
tion on the host following tumor treatments. Finally, it is the systemic inflammation
induced by the tumor, which is a key factor in the development and progression of
cancer cachexia. This chapter will review the entire spectrum of inflammation and
cancer from causation to its role in tumor therapy.
ACUTE INFLAMMATION AND CANCER

The acute inflammation triggered by an infection is a part of normal host defense
and leads to cancer formation only when it persists over a long term as is characteris-
tic of some chronic infections or chemical irritations. Acute inflammation triggered
by mixtures or single species of bacteria was used historically with some success to
treat cancer in the 1890s, and one such preparation is currently used in the treatment
of bladder cancer [2]. Therefore, acute inflammation and a brisk immune response
can be used to try to kill tumor cells. A more sophisticated and recent version of this
is the field of immunotherapy using targeted antibodies.
CHRONIC INFLAMMATION AND CANCER

Colon Cancer and Gut Microflora
Colorectal cancer (CRC) is one of the leading causes of cancer death in the world
[3] with the majority of cases having no previous family history. Sporadic CRC has
been linked to several risk factors including accumulation of genetic mutations,
inflammatory states such as inflammatory bowel disease, and environmental factors
such as smoking and obesity. More recently, CRC has been linked to changes in the
gut microbiota [4–7]. While bacterial dysbiosis has been associated with CRC, the
mechanism by which it promotes colon carcinogenesis has not been established.
Increased populations of Proteobacteria together with decreased Bacteroides,
and a disproportionate colonization of the gut with predominant Escherichia coli
have been associated with precancerous adenomas of the colon [8,9]. Fusobacteria
have been found to be increased in patients with CRC supporting a role of gram-
negative bacteria in colorectal carcinogenesis [4]. One of the proposed mechanisms
by which bacteria promote the development of colorectal tumors is through the pro-
duction of inflammatory cytokines by the release of their cell wall antigens [10].
Thus, changes in bacterial flora in the gut that favor a higher abundance of gram-
negative bacteria could contribute to the formation of adenomas via increased
endotoxin release from bacterial membranes and inflammation. Endotoxin, or lipo-
polysaccharide (LPS), is a component of the cell wall of gram-negative bacteria and
is released into the host environment by the destruction of the cell wall. Studies
have shown that certain bacterial types are correlated with elevated concentrations
of plasma endotoxin [11,12], which can have detrimental effects.
Cancer and Inflammation 103
Endotoxin can lead to the activation of toll-like receptor 4 (TLR-4), initiation

of innate inflammatory response, activation of macrophages and monocytes, and
production of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α),
interleukin (IL)-6, and IL-23 [11–16]. An overabundance of LPS-rich bacteria in
the gut may provide an environment that is conducive for chronic inflammation and
increased production of proinflammatory cytokines and reactive oxygen species.
These cytokines can also activate NF-κB leading to carcinogenesis [13–16].
Helicobacter and Stomach Cancer

In many countries with poor hygiene and lack of refrigeration, the incidence of
gastric and liver cancers is higher than that in developed countries. Helicobacter
pylori is a bacterium that lodges in the gastric mucosa and survives the acid environ-
ment by virtue of a urease enzyme, which creates ammonia and a resulting halo of
neutral pH in the acid environment of the stomach. Infections with Helicobacter are
lifelong unless they are treated and are endemic in many developing countries. This
infection leads to atrophic gastritis, and this chronic inflammation is associated with
gastric cancer and lymphoma of the mucosa-associated lymphoid tissue [17].
Hepatitis Viruses and Liver Cancer

Hepatomas are very common in developing countries where both environmental
toxins such as aflatoxin and viral infections may contribute to the incidence of these
cancers. Infections with hepatitis B or C viruses increase the risk of hepatocellular
carcinoma [18]. Chronic inflammation secondary to autoimmunity can also increase
the risk of cancer as is common with inflammatory bowel disease, especially ulcer-
ative colitis, which is linked to CRC risk [19].
Prostate Cancer and Inflammation

Asymptomatic prostatic inflammation is found in virtually all prostate glands of
adult males in the developed world. In countries where prostate-specific antigen
(PSA) screening is carried out, prostate cancer is the second most common can-
cer diagnosed in men affecting about 300,000 men per year in the United States.
Studies examining men who undergo biopsy for prostate cancer due to elevated
PSA levels and test negative for cancer [20–23], autopsy studies [24], and findings
from transurethral resections for benign prostatic hyperplasia (BPH) [25] have
all demonstrated inflammation in the prostate. In fact, evidence of inflammation
is found in over 97% of prostate glands at the time of prostatectomy for prostate
cancer. The preneoplastic lesion proliferative inflammatory atrophy is thought to
lead to prostatic intraepithelial neoplasia and prostate cancer. Prostate cancers occur
more commonly in a region of the prostate that is poorly perfused and more suscep-
tible to oxidative stress and inflammation. Furthermore, prostatic inflammation has
been linked to arguably all major diseases of the human prostate including BPH,
prostatitis syndromes, and prostate cancer [26–29].
Pathogens that are known to infect and induce inflammation in the human prostate
include E. coli and sexually transmitted organisms [28]. Recently, Propionibacterium
acnes was reported as the predominant bacterium detected in tissues from patients
with BPH [29] and prostate cancer [30]. P. acnes is a highly proinflammatory bacte-
rium that is implicated as the causative agent in the etiology of common and invasive
acne and in many other inflammatory conditions [31]. P. acnes was previously called
Corynebacterium parvum and was studied in the 1970s and 1980s as an immunos-
timulatory agent in the treatment of cancer [32,33].
Anti-inflammatory phytochemicals including lycopene, green tea polyphenols,
and pomegranate polyphenols among others have been shown to affect prostate
tumor growth at least in part by inhibiting NF-κB and inflammation [34–36].
Inflammation and Lymphomas

Chronic inflammation and some infections have been related to the etiology of lym-
phomas and other soft-tissue tumors. Repeated immune system stimulation, auto-
immunity, and inflammation are all potential risk factors for chronic lymphocytic
leukemia (CLL), which accounts for 30% of all leukemias [37]. Cytokines including
IL-4 and some chemokines promote the proliferation of CLL cells and suppress
apoptosis [38,39]. Leukemic cells also interact with inflammatory cells in the micro-
environment surrounding lymphoid tissues.
In multiple myeloma, cytokines including vascular endothelial growth factor
(VEGF), IL-6, IGF-1, TNF-α, and SDF-1 promote the survival and migration of
abnormal cells [40]. Interestingly, transgenic IL-6-deficient mice are resistant to
induction of multiple myeloma [41].
INFLAMMATION IN TUMOR PROMOTION,

ANGIOGENESIS, AND METASTASIS
Tumor promotion is the phase of carcinogenesis in which a single initiated cell
grows into a clinically detectable primary tumor while tumor progression is the
clinical process of tumor metastasis. During both tumor promotion and progression,
inflammation drives cell proliferation and inhibits apoptosis through multiple paral-
lel mechanisms. Angiogenesis is critical to this process, enabling a small dormant
tumor to receive adequate blood flow required to increase in size beyond 800 μm
[42,43] and to establish metastases.
Autocrine and paracrine secretion of cytokines, including TNF-α, stimulate car-
cinogenesis [44] by activating transcription factors such as AP-1 and NF-κB, which
promote cell proliferation and growth, as well as angiogenesis, invasiveness, and
motility [45,46]. Moreover, this cytokine stimulation leads to further secretion of
chemokines and cytokines leading to an amplification of the initial stimulation of the
multistep process of carcinogenesis.
Pathogens such as bacteria and viruses, through pattern recognition receptors
on innate immune cells in the tumor microenvironment, can stimulate these same
pathways underlying inflammation and tumor promotion, angiogenesis, and metas-
tasis [2]. The interior environments of most solid tumors are oxygen poor due to
inadequate perfusion by the angiogenesis-derived microvasculature. The activation

of angiogenic factors as a result of the hypoxia via increases in hypoxia-inducible
factor 1-alpha (HIF-1α) and increases in proinflammatory cytokines occurs together,
so that the impact of these parallel pathways is difficult to disentangle. Tumor hypoxia
as a result of the poor oxygenation of tumor tissue by the blood vessels formed via
angiogenesis increases the levels of HIF-1α and in turn the levels of VEGF, which
promotes angiogenesis and increases the probability of metastasis.
Most cancer mortality is ultimately related to tumor metastasis rather than the
primary tumor. Exceptions include brain tumors in sensitive areas where a primary
tumor can be fatal, leukemias that begin as systemic diseases, and those indolent
cancers such as localized prostate cancer where death occurs secondary to cardio-
vascular disease rather than cancer.
As sometimes stated as a soil and seed phenomenon, metastatic cells migrate to
an environment where they can seed and grow. This process involves not only the
tumor cells but also ongoing inflammation and angiogenesis in the microenviron-
ment stimulated by tumor-associated macrophages (TAMs). Not only hypoxia, but
also the recruitment of TAMs contributes to tumor angiogenesis. Macrophages also
produce chemokines and proangiogenic factors in response to hypoxia [47,48]. As
with autocrine cytokine production, these factors amplify the process of angiogen-
esis, which contributes to metastasis (see Figure 7.1).
Inflammation promotes vascular permeability, enabling tumor cells to more easily
enter the circulation via blood vessels and lymphatics once they have gone through
an epithelial–mesenchymal transition and lost E-cadherin. It is estimated that only
0.01% of circulating cancer cells survive long enough to give rise to metastases [49].
Immune, inflammatory, and stromal cells interact with those tumor cells that prolif-
erate locally at the metastatic site [50]. Some tumor cells generate inflammatory sig-
nals prior to seeding the metastatic sites [51], which is consistent with the conception
of the tumor cell as a seed seeking a niche in friendly soil where it can proliferate.
Within the circulation, inflammatory mediators released by immune cells in
response to cancer-derived or pathogen-derived stimuli from tumor cells can pro-
mote cell survival [52,53]. Cytokines present in the tumor microenvironment,
including TNF-α and IL-6, have been shown to aid circulating metastatic cells in
their attempts to survive [54]. Chemokines attract the tumor cells, influencing their
movement toward metastatic sites [55]. Finally, cancer cells in the circulation may
interact with platelets or macrophages shielding them from destruction by circulat-
ing immune cells [56].
TARGETED IMMUNOTHERAPY: INFLAMMATION

AND TUMOR CELL KILLING
There are several forms of cancer therapy that can be considered broadly as
targeted immunotherapy. Targeted immunotherapy attempts to have an advan-
tage over systemic therapies by delivering growth inhibitory or cytotoxic agents to
tumor cells while sparing surrounding normal cells. Generally, immunotherapy is
free of many common side effects of chemotherapy including immune suppression,
nausea, and vomiting.
3
Immunosuppression
PGE2, IL-10, TGF-B1
Basement Hypoxic areas

membrane MMPs, uPA,
1
Invasion cathepsins
? VEGF, bFGF, PDGF, MMPs,

?
Stroma IL-8, Ang1
2
Angiogenesis
EGF EGF
4
Metastasis
FIGURE 7.1 (See color insert.) The roles of different subpopulations of TAMs in tumor pro-
gression. (1) Invasion: TAMs secrete a variety of proteases to break down the basement mem-
brane around areas of proliferating tumor cells (e.g., ductal carcinoma in situ in the breast),
thereby prompting their escape into the surrounding stroma where they show deregulated
growth. (2) Angiogenesis: In areas of transient (avascular) and chronic (perinecrotic) tumor
hypoxia, macrophages cooperate with tumor cells to induce a vascular supply for the area by
upregulating a number of angiogenic growth factors and enzymes. These diffuse away from
the hypoxic area and, together with other proangiogenic stimuli in the tumor microenviron-
ment, stimulate endothelial cells in neighboring, vascularized areas to migrate, proliferate,
and differentiate into new vessels. (3) Immunosuppression: Macrophages in hypoxic areas
secrete factors that suppress the antitumor functions of immune effectors within the tumor. (4)
Metastasis: A subpopulation of TAMs associated with tumor vessels secretes factors like EGF
to guide tumor cells in the stroma toward blood vessels where they then escape into the circu-
lation. In the stromal compartment (both acellular regions and others where they are in close
contact with tumor cells), TAMs secrete growth factors to stimulate tumor cell division and/
or undefined factors that promote tumor cell motility. Note: ? refers to undefined factors that
promote tumor cell motility. (From Lewis, C.E. and Pollard, J.W., Cancer Res., 66, 605, 2006.)
The ability of vaccinations to prevent and treat many infectious diseases encour-
aged research on the use of vaccines against cancer. While cancer cells express sur-
face antigens that differ from those of normal cells, tumor cells are only generally
tolerated by the host. As the cancer evolves within the body due to the inherent
genetic instability of tumor cells, the cells are capable of no longer expressing tumor
antigens or launching defenses against immune attack. As the tumor grows, it favors
the activation and the expansion of adaptive regulatory T (Treg) cells and, therefore,
the generation of a microenvironment that tolerates the cancer cells. The evasion of
immune surveillance can thus increase as a tumor grows.
One form of immunotherapy seeks to block the immune tolerance of the host for
cancer cells. This strategy seeks to restore a vigorous antitumor immune response
by blocking those aspects of immune function leading to tolerance of the tumor
cells. Immune checkpoint proteins can be blocked by human antibodies with pro-
found effects in vitro, in animal tumor systems, and in patients. Promising clinical
data have already been generated in melanoma and other tumor types with human
antibodies directed against cytotoxic T lymphocyte antigen-4 (CTLA-4) and the
programmed death-1 (PD-1) protein [57,58]. CTLA-4 is an immunoglobulin pro-
tein expressed on the surface of T cells that transmits an inhibitory signal. An
antibody against CTLA-4 is the basis of a drug called ipilimumab, which is FDA
approved for melanoma. It acts by inhibiting immune system tolerance to tumors
and thereby provides a potentially useful immunotherapy strategy for patients with
cancer. In humans, the exact mechanism by which CTLA-4 inhibition induces an
antitumor effect is still unclear. PD-1 is related to the CTLA-4 family of T cell reg-
ulators. Inhibition of PD-1 functions via immune signaling pathways is different
from CTLA-4 and is likely to have a different spectrum of effects from blocking
CTLA-4. The clinical development of anti-PD-1 antibody so far has shown that it
has a potent effect when administered alone, and trials of vaccines with anti-PD-1
are being done currently.
The use of antibodies to deliver drugs as targeted chemotherapies has success-
fully entered clinical practice and holds promise [59]. These drugs consist of an anti-
body and toxin–drug combined together via a chemical linker. These antibody–drug
combinations are being further developed with less toxic drugs, and other reviews
go into detail on the design and testing of such antibody–drug combinations. For our
purposes in this chapter, it is important to understand the potential and limitations
of these approaches as the killing of tumor cells also results in the stimulation of the
innate immune system and inflammation as discussed in the following.
INFLAMMATION AS A SIDE EFFECT OF TUMOR TREATMENT

When cancer cells are killed by either chemotherapy or radiation, the dead cells
are cleared by immune cells, leading to a systemic inflammatory response. This
response is responsible for much of the fatigue and the flu-like symptoms, which
occur after cancer therapy.
In research directed at reducing these side effects, Longo and colleagues have
shown that a limited exposure to a severely restricted diet (short-term starvation or
fasting) can protect yeast, mammalian cells, mice, and some patients from the toxic
effects of oxidative and chemotherapeutic agents without causing chronic weight loss
[60–64]. For example, fasting for 48–60 h protected mice of three different genetic
backgrounds from the side effects of the chemotherapy drug etoposide [62]. Fasting
works to protect normal cells from inflammation by reallocating energy toward
maintenance pathways from reproduction and growth processes when nutrients are
scarce or absent [60,63,64]. This switch to a fasting mode occurs only in normal cells,
but not in cancer cells. Oncogenes prevent the activation of this response to nutrient
deprivation. One strategy that is being explored to kill cancer cells is to take advan-
tage of this difference in metabolic response [60–62]. In mouse and mammalian
cells, the adaptive response to nutrient deprivation in normal cells is mediated in part
by the reduction of extracellular glucose and IGF-1 concentration and intracellular
signaling [60–62,65]. Fasting for two to three days before and for 24 h after chemo-
therapy is well tolerated by cancer patients receiving a variety of toxic treatments
[62]. In mice, fasting protects against ischemia–reperfusion injury [66]. The depriva-
tion of a single essential amino acid results in both lower IGF-1 levels and protection
against renal and hepatic ischemic injury [67] in response to ischemia–reperfusion.
Therefore, short-term fasting and special nutrition regimens are both being investi-
gated to determine whether side effects of chemotherapy are lessened and whether
there is a potential to enhance cancer cell killing at the same time.
INFLAMMATION AS A CAUSATIVE FACTOR IN CANCER CACHEXIA

As many forms of cancer progress, they lead to protein–energy malnutrition with a
loss of lean body mass and adipose tissue, known as cancer cachexia. Approximately
half of all cancer patients develop cachexia [68]. At the end of life, up to 86% of
patients have cachexia in the last one to two weeks of life [69]. About 20% of cancer
deaths are attributed to cancer cachexia [70]. Malnutrition is extremely common
among cancer patients with almost half of all patients losing more than 10% of their
preillness weight as their disease progresses [71].
Cancer patients often develop loss of appetite and taste changes leading to sig-
nificant reduction in food intake characterized as anorexia [72]. Loss of appetite and
weight loss are presenting clinical features of many forms of cancer. While reduced
food intake can contribute to cachexia, weight losses observed in advanced can-
cer cannot be attributed to decreased food intake alone. Cancer cells are known to
develop abnormalities of protein, carbohydrate, and lipid metabolism, which benefit
tumor cells but contribute to the negative energy balance of the cancer patient [73].
For example, muscle wasting is accelerated as the result of increased proteolysis and
reduced anabolism. The development of the anorexia–cachexia syndrome increases
morbidity and mortality, and reduces overall quality of life.
There are complex metabolic, molecular, and cellular alterations associated with
cancer cachexia. Increased plasma levels of C-reactive protein have been used as a
clinical marker of cancer cachexia, when combined with reduced food intake and
weight loss [74]. Inflammation appears to play a significant role in cancer anorexia as
well. In animal models of cancer anorexia, increased brain levels of cytokines includ-
ing IL-1 and TNF-α have been demonstrated [75,76]. Blocking circulating TNF-α or
intrahypothalamic IL-1 receptors in anorexic tumor-bearing mice improved food
intake [77,78]. Finally, inflammatory cytokines derange brain chemistry, leading to
the release of neurotransmitters such as serotonin [79], which can influence food
intake [80]. In humans, evidence directly linking cancer anorexia to inflammation is
still lacking, but in patients with renal failure who suffer from anorexia, the degree
of appetite lost is correlated to the intensity of systemic inflammation [81]. Both
patients with cancer and kidney failure have anorexia so this observation of the effect
of inflammation in renal failure may also have implications for cancer cachexia.
However, clinical trials in this area are difficult due to the heterogeneity of the cancer
patient population in most centers.
CONCLUSION
Inflammation plays a critical role in the causation and progression of many forms of
cancer. Since inflammation can be affected by diet, the relationship of inflammation
to cancer is one of the key connections between diet and cancer as well [82]. For
many of the known or suspected dietary risk factors for common forms of cancer, the
mediator may be chronic low-grade inflammation engendered by the Western diet
and a sedentary lifestyle. The immune response is also affected by many different
nutrients including omega-3 fatty acids, vitamin D, and other vitamins and minerals.
Therefore, optimizing nutrient intake by including known antioxidants and natural
products with anti-inflammatory activities represents a unique opportunity to apply
immunonutrition to cancer prevention and treatment.
REFERENCES
1. Aggarwal BB, Vijayalekshmi RV, Sung B. 2009. Targeting inflammatory pathways for
prevention and therapy of cancer: Short-term friend, long-term foe. Clin Cancer Res
15:425–430.
2. Rakoff-Nahoum S, Medzhitov R. 2009. Toll-like receptors and cancer. Nat Rev Cancer
9:57–63.
3. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. 2011. Global cancer statistics.
CA Cancer J Clin 61:69–90.
4. Kostic AD, Gevers D, Pedamallu CS et al. 2012. Genomic analysis identifies association
of Fusobacterium with colorectal carcinoma. Genome Res 22:292–298.
5. Scanlan PD, Shanahan F, Clune Y et al. 2008. Culture-independent analysis of the gut
microbiota in colorectal cancer and polyposis. Environ Microbiol 10:789–798.
6. Sobhani I, Tap J, Roudot-Thoraval F et al. 2011. Microbial dysbiosis in colorectal can-
cer (CRC) patients. PLoS One 6(1):e16393.
7. Wang T, Cai G, Qiu Y et al. 2012. Structural segregation of gut microbiota between
colorectal cancer patients and healthy volunteers. ISME J 6:320–329.
8. Sanapareddy N, Legge RM, Jovov B et al. 2012. Increased rectal microbial richness is
associated with the presence of colorectal adenomas in humans. ISME J 6:1858–1868.
9. Shen XJ, Rawls JF, Randall T et al. 2010. Molecular characterization of mucosal adher-
ent bacteria and associations with colorectal adenomas. Gut Microbes 1:138–147.
10. Abdulamir AS, Hafidh RR, Abu Bakar F. 2011. The association of Streptococcus bovis/
gallolyticus with colorectal tumors: The nature and the underlying mechanisms of its
etiological role. J Exp Clin Cancer Res 30:11.
11. Cani PD, Bibiloni R, Knauf C et al. 2008. Changes in gut microbiota control metabolic
endotoxemia-induced inflammation in high-fat diet-induced obesity and diabetes in
mice. Diabetes 57:1470–1481.
12. Cani PD, Neyrinck AM, Fava F et al. 2007. Selective increases of bifidobacteria in
gut microflora improve high-fat-diet-induced diabetes in mice through a mechanism
associated with endotoxaemia. Diabetologia 50:2374–2383.
13. Atreya R, Neurath MF. 2008. Signaling molecules: The pathogenic role of the IL-6/
STAT-3 trans signaling pathway in intestinal inflammation and in colonic cancer. Curr
Drug Targets 9:369–374.
14. Langowski JL, Zhang X, Wu L et al. 2006. IL-23 promotes tumour incidence and
growth. Nature 442:461–465.
15. Greten FR, Eckmann L, Greten TF et al. 2004. IKKbeta links inflammation and
tumorigenesis in a mouse model of colitis associated cancer. Cell 118:285–296.
16. Puppa MJ, White JP, Sato S, Cairns M, Baynes JW, Carson JA. 2011. Gut barrier
dysfunction in the Apc(Min/+) mouse model of colon cancer cachexia. Biochim Biophys
Acta 1812:1601–1606.
17. Wroblewski LE, Peek RM Jr. 2013. Helicobacter pylori in gastric carcinogenesis:
Mechanisms. Gastroenterol Clin North Am 42:285–298.
18. Karin M. 2006. Nuclear factor-kappaB in cancer development and progression. Nature
441:431–436.
19. Waldner MJ, Neurath MF. 2009. Colitis-associated cancer: The role of T cells in tumor
development. Semin Immunopathol 31:249–226.
20. Gui-zhong LI, Libo M, Guanglin H, Jianwei W. 2011. The correlation of extent and
grade of inflammation with serum PSA levels in patients with IV prostatitis. Int Urol
Nephrol 43:295–301.
21. Stimac G, Reljic A, Spajic B et al. 2009. Aggressiveness of inflammation in histological
prostatitis—Correlation with total and free prostate specific antigen levels in men with
biochemical criteria for prostate biopsy. Scott Med J 54:8–12.
22. Ugurlu O, Yaris M, Oztekin CV, Kosan TM, Adsan O, Cetinkaya M. 2010. Impacts of
antibiotic and anti-inflammatory therapies on serum prostate-specific antigen levels in
the presence of prostatic inflammation: A prospective randomized controlled trial. Urol
Int 84:185–190.
23. Fujita K, Hosomi M, Tanigawa G, Okumi M, Fushimi H, Yamaguchi S. 2011. Prostatic
inflammation detected in initial biopsy specimens and urinary pyuria are predictors of
negative repeat prostate biopsy. J Urol 185:1722–1727.
24. Delongchamps NB, de la Roza G, Chandan V et al. 2008. Evaluation of prostatitis in
autopsied prostates—Is chronic inflammation more associated with benign prostatic
hyperplasia or cancer? J Urol 179:1736–1740.
25. Nickel JC, Downey J, Young I, Boag S. 1999. Asymptomatic inflammation and/or
infection in benign prostatic hyperplasia. BJU Int 84:976–981.
26. Chughtai B, Lee R, Te A, Kaplan S. 2011. Role of inflammation in benign prostatic
hyperplasia. Rev Urol 13:147–150.
27. De Marzo AM, Platz EA, Sutcliffe S et al. 2007. Inflammation in prostate carcinogenesis.
Nat Rev Cancer 7:256–269.
28. Sfanos KS, De Marzo AM. 2012. Prostate cancer and inflammation: The evidence.
Histopathology 60:199–215.
29. Alexeyev O, Bergh J, Marklund I et al. 2006. Association between the presence of
bacterial 16S RNA in prostate specimens taken during transurethral resection of prostate
and subsequent risk of prostate cancer (Sweden). Cancer Causes Control 17:1127–1133.
30. Cohen RJ, Shannon BA, McNeal JE, Shannon T, Garrett KL. 2005. Propionibacterium
acnes associated with inflammation in radical prostatectomy specimens: A possible link
to cancer evolution? J Urol 173:1969–1974.
31. Jakab E, Zbinden R, Gubler J, Ruef C, von Graevenitz A, Krause M. 1996. Severe
infections caused by Propionibacterium acnes: An underestimated pathogen in late
postoperative infections. Yale J Biol Med 69:477–482.
32. Lichtenstein AK, Kahle J, Berek J, Zighelboim J. 1984. Successful immunotherapy with
intraperitoneal Corynebacterium parvum in a murine ovarian cancer model is associated
with the recruitment of tumor-lytic neutrophils into the peritoneal cavity. J Immunol
133:519–526.
33. Currie GA, Bagshawe KD. 1970. Active immunotherapy with Corynebacterium parvum
and chemotherapy in murine fibrosarcomas. BMJ 1:541–544.
34. Wertz K. 2009. Lycopene effects contributing to prostate health. Nutr Cancer
61:775–783.
35. Henning SM, Wang P, Heber D. 2011. Chemopreventive effects of tea in prostate cancer:
Green tea versus black tea. Mol Nutr Food Res 55:905–920.
36. Rettig MB, Heber D, An J et al. 2008. Pomegranate extract inhibits androgen-independent
prostate cancer growth through a nuclear factor-kappaB-dependent mechanism. Mol
Cancer Ther 7:2662–2671.
37. Chiorazzi N, Rai KR, Ferrarini M. 2005. Chronic lymphocytic leukemia. N Engl J Med
352:804–815.
38. Granziero L, Ghia P, Circosta P 2001. Survivin is expressed on CD40 stimulation and
interfaces proliferation and apoptosis in B-cell chronic lymphocytic leukemia. Blood
97:2777–2783.
39. Pedersen IM, Kitada S, Leoni LM et al. Protection of CLL B cells by a follicular
dendritic cell line is dependent on induction of Mcl-1. Blood 100:1795–1801.
40. Kastritis E, Palumbo A, Dimopoulos MA. 2009. Treatment of relapsed/refractory
multiple myeloma. Semin Hematol 46:143–157.
41. Hodge DR, Hurt EM, Farrar WL. 2005. The role of IL-6 and STAT3 in inflammation and
cancer. Eur J Cancer 41:2502–2512.
42. Folkman J, Merler E, Abernathy C, Williams G. 1971. Isolation of a tumor factor
responsible for angiogenesis. J Exp Med 133:275–288.
43. Chen CT, Hung MC. 2013. Beyond anti-VEGF: Dual-targeting antiangiogenic and anti-
proliferative therapy. Am J Transl Res 5:393–403.
44. Moore RJ, Owens DM, Stamp G et al. 1999. Mice deficient in tumor necrosis factor-
alpha are resistant to skin carcinogenesis. Nat Med 5:828–831.
45. Grivennikov S, Karin E, Terzic J et al. 2009. IL-6 and Stat3 are required for survival
of intestinal epithelial cells and development of colitis-associated cancer. Cancer Cell
15:103–113.
46. Yu H, Pardoll D, Jove R. 2009. STATs in cancer inflammation and immunity: A leading
role for STAT3. Nat Rev Cancer 9:798–809.
47. Kujawski M, Kortylewski M, Lee H, Herrmann A, Kay H, Yu H. 2008. Stat3 mediates
myeloid cell-dependent tumor angiogenesis in mice. J Clin Invest 118:3367–3377.
48. Rius J, Guma M, Schachtrup C et al. 2008. NF-kappaB links innate immunity to
the hypoxic response through transcriptional regulation of HIF-1alpha. Nature
453:807–811.
49. Joyce JA, Pollard JW. 2009. Microenvironmental regulation of metastasis. Nat Rev
Cancer 9:239–252.
50. Polyak K, Weinberg RA. 2009. Transitions between epithelial and mesenchymal states:
Acquisition of malignant and stem cell traits. Nat Rev Cancer 9:265–273.
51. Kaplan RN, Riba RD, Zacharoulis S et al. 2005. VEGFR1-positive haematopoietic bone
marrow progenitors initiate the premetastatic niche. Nature 438:820–827.
52. Kim S, Takahashi H, Lin WW et al. 2009. Carcinoma produced factors activate myeloid
cells through TLR2 to stimulate metastasis. Nature 457:102–106.
53. Luo JL, Maeda S, Hsu LC, Yagita H, Karin M. 2004. Inhibition of NF-kappaB in cancer
cells converts inflammation-induced tumor growth mediated by TNFalpha to TRAIL-
mediated tumor regression. Cancer Cell 6:297–305.
54. Nguyen DX, Bos PD, Massague J. 2009. Metastasis: From dissemination to organ-
specific colonization. Nat Rev Cancer 9:274–284.
55. Bonecchi R, Galliera E, Borroni EM, Corsi MM, Locati M, Mantovani A. 2009.
Chemokines and chemokine receptors: An overview. Front Biosci 14:540–551.
56. Palumbo JS, Talmage KE, Massari JV et al. 2007. Tumor cell-associated tissue factor
and circulating hemostatic factors cooperate to increase metastatic potential through
natural killer cell-dependent and-independent mechanisms. Blood 110:133–141.
57. Weber J. 2010. Immune checkpoint proteins: A new therapeutic paradigm for cancer—
Preclinical background: CTLA-4 and PD-1 blockade. Semin Oncol 37:430–439.
58. Callahan MK, Wolchok JD. 2013. At the bedside: CTLA-4- and PD-1-blocking
antibodies in cancer immunotherapy. J Leukoc Biol 94:41–53.
59. Kovtun YV, Audette CA, Ye Y et al. 2006. Antibody-drug conjugates designed to erad-
icate tumors with homogeneous and heterogeneous expression of the target antigen.
Cancer Res 66:3214–3221.
60. Raffaghello L, Lee C, Safdie FM et al. 2008. Starvation-dependent differential stress
resistance protects normal but not cancer cells against high-dose chemotherapy. Proc
Natl Acad Sci USA 105:8215–8220.
61. Lee C, Safdie FM, Raffaghello L et al. 2010. Reduced levels of IGF-I mediate
differential protection of normal and cancer cells in response to fasting and improve
chemotherapeutic index. Cancer Res 70:1564–1572.
62. Safdie FM, Dorff T, Quinn D et al. 2009. Fasting and cancer treatment in humans:
A case series report. Aging 1:988–1007.
63. Longo VD, Ellerby LM, Bredesen DE, Valentine JS, Gralla EB. 1997. Human Bcl-2
reverses survival defects in yeast lacking superoxide dismutase and delays death of
wild-type yeast. J Cell Biol 137:1581–1588.
64. Wei M, Fabrizio P, Hu J, Ge H, Cheng C, Li L, Longo VD. 2008. Life span extension by
calorie restriction depends on Rim15 and transcription factors downstream of Ras/PKA,
Tor, and Sch9. PLoS Genet 4:e13.
65. Li Y, Xu W, McBurney MW, Longo VD. 2008. SirT1 inhibition reduces IGF-I/IRS-2/
Ras/ERK1/2 signaling and protects neurons. Cell Metab 8:38–48.
66. Mitchell JR, Verweij M, Brand K et al. 2010. Short-term dietary restriction and fasting
precondition against ischemia reperfusion injury in mice. Aging Cell 9:40–53.
67. Peng W, Robertson L, Gallinetti J et al. 2012. Surgical stress resistance induced by
single amino acid deprivation requires Gcn2 in mice. Sci Transl Med 4:118ra11.
68. Tijerina AJ. 2004. The biochemical basis of metabolism in cancer cachexia. Dimens Crit
Care Nurs 23:237–243.
69. Teunissen SCCM, Wesker W, Kruitwagen C et al. 2007. Symptom prevalence in patients
with incurable cancer: A systematic review. J Pain Symptom Manag 34:94–104.
70. Skipworth RJE, Stewart GD, Dejong CHC, Preston T, Fearon KCH. 2007
Pathophysiology of cancer cachexia: Much more than host-tumour interaction? Clin
Nutr 26:667–676.
71. Argiles JM. 2005. Cancer-associated malnutrition. Eur J Oncol Nurs 9(Suppl 2):
S39–S50.
72. Laviano A, Meguid MM, Inui A et al. 2005. Therapy insight: Cancer anorexia—
Cachexia syndrome: When all you can eat is yourself. Nat Clin Pract Oncol 2:158–165.
73. Muscaritoli M, Bossola M, Aversa Z et al. 2006. Prevention and treatment of cancer
cachexia: New insights into an old problem. Eur J Cancer 42:31–41.
74. Fearon KC, Voss AC, Hustead DS and Cancer Cachexia Study Group. 2006. Definition
of cancer cachexia: Effect of weight loss, reduced food intake, and systemic inflamma-
tion on functional status and prognosis. Am J Clin Nutr 83:1345–1350.
75. Guijarro A, Laviano A, Meguid MM. 2006. Hypothalamic integration of immune
function and metabolism. In: Hypothalamic Integration of Energy Metabolism, eds.
A. Kalsbeek, E. Fliers, M.A. Hofman, D.F. Swaab, E.J.W. van Someren, and R.M. Buijs,
pp. 367–405. Amsterdam, the Netherlands: Elsevier.
76. Plata-Salaman CR, Ilyin SE, Gayle D. 1998. Brain cytokine mRNAs in anorectic rats
bearing prostate adenocarcinoma tumor cells. Am J Physiol 275:R566–R573.
77. Torelli GF, Meguid MM, Moldawer LL et al. 1999. Use of recombinant human soluble
TNF receptor in anorectic tumor-bearing rats. Am J Physiol 277:R850–R855.
78. Laviano A, Gleason JR, Meguid MM et al. 2000. Effects of intra-VMN mianserin and
IL-1ra on meal number in anorectic tumor-bearing rats. J Invest Med 48:40–48.
79. Yang ZJ, Blaha V, Meguid MM et al. 1999. Interleukin-1alpha injection into ventrome-
dial hypothalamic nucleus of normal rats depresses food intake and increases release of
dopamine and serotonin. Pharmacol Biochem Behav 62:61–65.
80. Heisler LK, Cowley MA, Tecott LH et al. 2002. Activation of central melanocortin path-
ways by fenfluramine. Science 297:609–611.
81. Kalantar-Zadeh K, Block G, MacAllister CJ et al. 2004. Appetite and inflammation,
nutrition, anemia, and clinical outcome in hemodialysis patients. Am J Clin Nutr
80:299–307.
82. Lewis CE, Pollard JW. 2006. Distinct role of macrophages in different tumor environ-
ments. Cancer Res 66:605–612.
8 Pathophysiology and Related
Abdominal Obesity
Metabolic Complications
Ana F.T.A. Junqueria and Caroline M. Apovian
CONTENTS
Introduction............................................................................................................. 115
Abdominal Obesity and Disease Risk..................................................................... 117
Metabolic Abnormalities Related to Visceral Obesity....................................... 117
Adipose Organ........................................................................................................ 119
Adipose Tissue Dysfunction in Obesity.................................................................. 120
Adipose Tissue Distribution.................................................................................... 121
Heterogeneity among Adipose Tissue Depots........................................................ 124
Differences in Developmental Roots of Adipose Tissue.................................... 124
Differences in Cellularity, Growth, and Remodeling......................................... 124
Differences in Adipocyte Metabolism................................................................ 125
Theory of the Portal Circulation.................................................................... 126
Theory of Ectopic Fat Deposition................................................................. 126
Adipokine and Cytokine Secretion.................................................................... 127
Lessons from Fat-Tissue Removal and Transplantation.................................... 130
Measuring Abdominal Fat in Clinical Practice....................................................... 130
Conclusion.............................................................................................................. 134
References............................................................................................................... 134
INTRODUCTION
A significant worldwide increase in the prevalence of obesity has been noticed
in the last decades, which may be associated with an excess of more the 100,000
deaths per year in the United States [1,2]. With the development of obesity, the
adipose tissue becomes increasingly dysfunctional. Excess fat mass is often associ-
ated with elevated systemic free fatty acids (FFAs), altered adipokine and cytokine
secretion, and local and systemic inflammation. Those changes are linked to the
development of abnormalities such as insulin resistance, hyperglycemia, dyslip-
idemia, hypertension, metabolic syndrome, and a chronic proinflammatory and
prothrombotic state. Eventually, the metabolic derangements observed in obese
individuals increase the risk of the development of type 2 diabetes, nonalcoholic
115
(a) (b)
FIGURE 8.1 Two types of body fat distributions: (a) android or apple-shaped and
(b) gynecoid or pear-shaped. Weight gain in the area around the waist (android type) is asso-
ciated with obesity-related metabolic diseases. Weight gained around the hips and flank area
(gynecoid type) may be protective. There is evidence that adipose tissues in the upper body
have different phenotypic characteristics than those found in hips and thighs. (Reprinted
from Ophthalmology, 117(1), Kesler, A., Kliper, E., Shenkerman, G., and Stern, N., Idiopathic
intracranial hypertension is associated with lower body adiposity, 169–174, Copyright 2010,
with permission from Elsevier.)
hepatic steatosis (NASH), cardiovascular disease, various types of cancers, and

overall mortality [3].
Although obesity may be defined as an excess of body adiposity, it is currently
well recognized that the distribution of body fat matters and influences disease risk.
Epidemiological studies clearly show that a central android pattern of fat accumula-
tion confers an independent risk of obesity-related diseases, while the quantity of
gluteal–femoral adipose tissue may be protective (Figure 8.1). Thus, abdominal fat
accumulation, particularly visceral fat, is strongly associated with the development
of multiple metabolic diseases, including type 2 diabetes. This phenomenon is the
result of differences in anatomical location and intrinsic developmental properties of
different white adipose depots. There is mounting evidence to support that visceral
adipocytes are phenotypically different from subcutaneous adipocytes, as a result of
genetic and developmental events [4]. In this chapter, we discuss the heterogeneity
of white adipose tissue distribution and function and the mechanisms that link the
depot-specific properties to obesity-related metabolic diseases.
Abdominal Obesity 117
ABDOMINAL OBESITY AND DISEASE RISK

In 1947, Professor Jean Vague first proposed that, more importantly than total body
fat mass, a central android rather than a peripheral gynecoid pattern of fat deposi-
tion is linked to increased disease risk [5]. The subsequent work focused on the
detrimental role of visceral adipose tissue (VAT) and the heterogeneity between the
different fat depots. Clinical and epidemiological data have established that the glu-
teal–femoral adipose tissue acts as a protective energy store while excessive abdomi-
nal fat (particularly visceral) is associated with deleterious metabolic consequences
[6]. People with accumulation of excess fat in the abdomen, called abdominal obe-
sity, central adiposity, android obesity, male-type obesity, or apple-shaped obesity
(Figure 8.1), are at increased risk of developing type 2 diabetes, hypertension, dys-
lipidemia, various cancers, and cardiovascular disease.
Anthropometric measures of abdominal adiposity, waist circumference (WC)
and waist-to-hip ratio (WHR), are strongly and positively associated with morbidity
and mortality independently of body mass index (BMI). The absolute WC >102 cm
(40 in.) in men and >88 cm (35 in.) in women and the WHR >0.9 for men and >0.85
for women are generally used as measures of central obesity [7]. Various studies
have found that markers of abdominal adiposity are more strongly related to the risk
of obesity-associated complications compared to BMI alone.
The relationship between abdominal obesity and mortality risk was evaluated in
a cohort of 44,636 women in the Nurses’ Health Study. The relative risk of cardio-
vascular, cancer, and overall mortality increased significantly from the lowest to the
highest WC quintiles after adjustment for BMI and other confounders, during 16
years of follow-up. Elevated WC was associated with significantly increased cardio-
vascular mortality even among normal-weight women (BMI 18.5 to <25 kg/m2) [8].
A case-control study using data from 27,098 participants in 52 countries assessed
the relation between BMI, waist and hip circumferences, and WHR to the risk of
myocardial infarction. WHR and waist and hip circumferences were closely asso-
ciated with risk of myocardial infarction even after adjustment for other risk fac-
tors. The population-attributable risk of myocardial infarction for increased WHR
in the top two quintiles was 24.3% (95% CI 22.5–26.2) compared with only 7.7%
(6.0–10.0) for the top two quintiles of BMI [9] (Figure 8.2). Another large trial, the
Cancer Prevention Study II Nutrition Cohort, examined the association between
WC and mortality among 48,500 men and 56,343 women, 50 years or older. WC
was positively associated with mortality within all categories of BMI [10].
Metabolic Abnormalities Related to Visceral Obesity

Intraperitoneal, or VAT, are associated with digestive organs and include the omental
(hangs off the stomach), the mesenteric (associated with the intestine), and epiploic
(along the colon) [4]. In parallel to the epidemiological studies using anthropometric
measures to predict risk in patients with central obesity, high intra-abdominal adipos-
ity was also found to be closely related with the metabolic complications seen in obe-
sity. Increased visceral fat has been associated with a reduction in the concentration
of high-density lipoprotein (HDL), elevation in triglycerides, glucose intolerance,
4.0
3.5
3.0
2.5
OR (95% Cl)
2.0
1.5
1.25
1.0
0.9
0.8
<20 20–23 23.1–25 25.1–27 27.1–30 >30
BMI
FIGURE 8.2 Association of WHR within BMI categories with myocardial infarction
risk. (Reprinted from The Lancet, Vol. 366, Yusuf, S., Hawken, S., Ôunpuu, S.,
Bautista, L., Franzosi, M.G., Commerford, P., Lang, C.C., Rumboldt, Z., Onen, C.L.,
Lisheng, L., Tanomsup, S., Wangai, Jr P., Razak, F., Sharma A.M., and Anand, S.S., On behalf
of the INTERHEART Study Investigators, Obesity and the risk of myocardial infarction
in 27,000 participants from 52 countries: A case-control study, 1640–1649, Copyright 2005,
with permission from Elsevier.)
hypertension, and a proinflammatory and prothrombotic profile [11–13]. Findings

from case-control and small longitudinal studies suggest that excess visceral adipos-
ity may, in fact, be predictive of increased cardiovascular risk. Prospective studies
are underway to test this hypothesis [14].
It has been proposed that the dyslipidemic state frequently observed in patients
with visceral obesity is a major cardiovascular risk factor [13,14]. It includes high
levels of triglycerides, low levels of HDL cholesterol, relatively normal total and
low-density lipoprotein (LDL) cholesterol levels, but more LDL particles (as quanti-
fied by high apolipoprotein B levels) that are smaller and denser than normal [14].
The combination of high triglyceride, low HDL cholesterol levels, and small, dense
LDL particles has been termed the atherogenic lipid triad and has been recognized
as a major cardiovascular risk factor [15,16]. Another triad of metabolic abnormali-
ties often found among individuals with visceral obesity, the atherogenic metabolic
triad of hyperinsulinemia, elevated apolipoprotein B, and small LDL particles, has
been shown to increase coronary heart disease (CHD) risk by 20-fold in middle-aged
men, such risk being largely independent of traditional risk factors and blood lipid
variables [14,17]. Visceral obesity in nondiabetic obese men is a predictor of ectopic
fat accumulation within and around the heart, which has been related to increased
risk of heart disease [18].
ADIPOSE ORGAN
In mammals, adipose tissue exists in two forms, white adipose tissue (WAT) and
brown adipose tissue (BAT). The primary role of BAT is to store small amounts of
fat that can be used, when needed, to produce heat and maintain body temperature
[19]. WAT, on the other hand, is designed to store large amounts of excess energy in
the form of triglycerides for use during periods of food deprivation. This requires
the process of fatty acid uptake as well as lipogenesis for accumulation of fat and the
mobilization of this energy for use by other cells of the organism through the process
of lipolysis [20]. This was thought to be the only function of the adipose organ, but
for the past two decades, the traditional view of adipose tissue as a passive energy
reservoir is no longer valid. Besides a depot of highly energetic molecules, the adi-
pose tissue is a complex, essential, and active metabolic and endocrine organ [21].
As early as 1987, adipose tissue was identified as a major site for metabolism of
sex steroids, but it was mainly in 1994 that adipose tissue was firmly established as an
endocrine organ, with the identification of leptin, an adipocyte-derived hormone that
regulates energy intake and expenditure. Since that time, a substantial number of other
factors secreted from the adipose organ have been recognized (Table 8.1). Many of
these factors act locally within the adipose tissue through autocrine/paracrine mecha-
nisms, but others act systemically to influence the function of distant tissues like the
brain, skeletal muscle, liver, pancreas, and heart. In addition to these efferent signals,
TABLE 8.1
Examples of Adipocyte-Derived Proteins with
Endocrine Functions
Cytokines and Leptin
cytokine-related proteins TNF-α
IL-6
Other immune-related proteins MCP-1
Proteins involved in the PAI-1
fibrinolytic system Tissue factor
Complement and Adipsin (complement factor D)
complement-related proteins Complement factor B
ASP
Adiponectin
Lipids and proteins for lipid Lipoprotein lipase (LPL)
metabolism or transport Cholesterol ester transfer protein (CETP)
Apolipoprotein E
NEFAs
Enzymes involved in steroid Cytochrome P450-dependent aromatase
metabolism 17βHSD
11βHSD1
Proteins of the RAS AGT
Other proteins Resistin
TABLE 8.2
Examples of Receptors Expressed in Adipose Tissue
Receptors for traditional Insulin receptor
endocrine hormones Glucagon receptor
GH receptor
TSH receptor
Gastrin/CCK-B receptor
Glucagon like peptide-1 receptor
Angiotensin II receptors type 1 and 2
Nuclear hormone receptors Glucocorticoid receptor
Vitamin D receptor
Thyroid hormone receptor
Androgen receptor
Estrogen receptor
Progesterone receptor
Cytokine receptors Leptin receptor
IL-6 receptor
TNF-α receptor
Catecholamine receptors β1, β2, β3 receptors
α1, α2 receptors
adipose tissue expresses numerous receptors that allow it to respond to afferent signals
from traditional hormone systems and the central nervous system (Table 8.2) [21].
It must be outlined that adipocytes are not the only cell type present in the adipose
organ. Preadipocytes, immune cells, mesenchymal cells, and connective, vascular
and nervous elements are found in the adipose organ and outnumber adipocytes in
the tissue. Although adipocytes secrete several endocrine hormones, many secreted
proteins are derived from the nonadipocyte fraction of adipose tissue. Those compo-
nents function together as an integrated unit, and through an interactive network, the
adipose tissue is integrally involved in coordinating a variety of biological processes
including energy metabolism, neuroendocrine function, and immune function [4,21].
ADIPOSE TISSUE DYSFUNCTION IN OBESITY

With the development of obesity, the adipose tissue becomes increasingly dysfunctional.
Both elevated FFAs and altered adipokine and cytokine production are found in obe-
sity and are thought to play critical roles in the etiology of obesity-related metabolic
complications. Elevated circulating FFAs increase pancreatic insulin secretion, decrease
insulin sensitivity in muscle and liver, increase hepatic VLDL secretion, and induce
endothelial dysfunction. Heightened lipolysis and immune cell infiltration from dysfunc-
tional visceral adipose tissue is thought to contribute to visceral obesity–related meta-
bolic complications, by increasing FFA and cytokine delivery directly to the liver [4].
Obesity is also frequently associated with chronic low-grade inflammation, and
a causal relationship between such inflammation and the development of insulin
resistance has been described [22]. Likewise, increased inflammation has been found
in adipose tissue of insulin-resistant compared with insulin-sensitive obese patients,
suggesting that it could be a key factor that distinguishes the two populations [23]. As
adipocytes become hypertrophic, macrophages and other immune cells infiltrate the
tissue, become activated, and secrete proinflammatory molecules, while adipocyte
production of anti-inflammatory adipokines such as adiponectin is suppressed.
Unless the vasculature expands in proportion to the expansion of adipocyte vol-
ume, microhypoxia results and contributes to the inflammation within the tissue
[24]. Yet another abnormality observed in obese humans is an increase in oxidative
stress, which may represent a mechanistic link between several components of meta-
bolic syndrome and cardiovascular disease. The balance of these factors, acting on
target tissues, especially liver, hypothalamus, muscle, and pancreas, influences insu-
lin action, substrate utilization, and inflammation, playing a major role in increasing
metabolic risk of cardiovascular diseases (Figure 8.3).
ADIPOSE TISSUE DISTRIBUTION

The size of the white adipose organ is highly variable, representing from 5% to 60%
of total body weight. It is distributed throughout the body in various depots, gener-
ally divided into two main components: subcutaneous and internal adipose tissues
(Figure 8.4) [4,26]. Subcutaneous adipose depots generally store more than 80% of total
body fat in the body. The most commonly studied subcutaneous depots are the abdomi-
nal, gluteal, and femoral. In the anterior abdominal wall, the superficial and deep sub-
cutaneous fat layers are separated by a fascial plane, the Scarpa’s fascia [4]. Internal
adipose tissues are associated with internal organs and include intrathoracic and intra-
abdominal fat depots [26]. Intra-abdominal fat depots represent 10%–20% of total body
fat in men and 5%–10% in women [4] and include intraperitoneal and extraperitoneal
depots [4,26]. Intraperitoneal, or VAT are associated with digestive organs and are com-
posed of three main compartments: the omental (hangs off the stomach), the mesenteric
(associated with the intestine), and the epiploic (along the colon) [4]. Retroperitoneal adi-
pose tissues include preperitoneal and retroperitoneal. There are also numerous smaller
internal adipose depots such as epicardial (intrathoracic) and intermuscular that may
serve specialized functions related to their neighboring tissues [4].
Morphological differences have been observed between the adipose tissue com-
partments. Superficial subcutaneous adipose tissue layers are organized in a regular
fashion, whereas deep subcutaneous layers and internal compartments, especially
the greater omentum, are large, irregular, and less organized. Vascularization, blood
flow, and innervation may also be different among the various compartments [14,27].
Gender, ethnicity, age, genetics, and, likely, environmental factors affect the
WAT distribution. However, the mechanisms involved are not very well understood.
Women generally have higher adiposity than men and accumulate more the lower-
body subcutaneous area (gluteal–femoral), while men accumulate more fat the cen-
tral abdominal region (both VAT and abdominal subcutaneous) [4]. Sex steroids
(estrogen and testosterone) are thought to affect adipose tissue mass and distribution,
although the mechanisms remain poorly understood. Women with polycystic ovary
syndrome, characterized by a hyperandrogenic state, are prone to central obesity
122
Adipose tissue Insulin sensitivity

Insulin clearance
FFA, SAA adn Liver
leptin, IL-6
Hepatic glucose output
VLDL-TG
CRP
FFA,
adn
leptin, IL-6
SAA, IL-8,
Leptin, IL-6 Muscle
IL-6, TSP-1
FFA Insulin sensitivity
Brain Blood vessels

Pancreas
Insulin secretion
Food intake
Energy Inflammation
expenditure Atherogenesis
FIGURE 8.3 (See color insert.) Adipose signals influence systemic metabolism and appetite. Dysfunctional adipose tissue in obesity produces more
proinflammatory factors (e.g., FFA, SAA, IL-6) and less anti-inflammatory factors (e.g., adiponectin). These exacerbate inflammation and hence risk
for metabolic diseases by affecting liver, skeletal muscle, beta cells, as well as blood vessels. Insulin–glucose homeostasis becomes impaired as a result
of increased hepatic glucose output and muscle insulin resistance, and basal insulin secretion from pancreas is increased, most likely by FAs. Leptin
normally regulate food intake and energy expenditure through its effects on the central nervous system. Besides leptin levels are commonly elevated in
the obese state, most obese persons are resistant to the weight-reducing effects of leptin. (Reprinted from Mol Aspects Med, 34(1), Lee, M.J., Wu, Y.,
and Fried, S.K., Adipose tissue heterogeneity: Implication of depot differences in adipose tissue for obesity complications, 1–11, Copyright 2013, with
Immunonutrition: Interactions of Diet, Genetics, and Inflammation
permission from Elsevier.)

Fatty liver
Retroperitoneal Preperitoneal
Pancreas
Stomach
Retroperitoneal
perinephric Abdominal sc
(superficial)
Intestine
sc deep
Mesenteric
Omental
Gluteal sc
Thigh
(femoral) sc
FIGURE 8.4 (See color insert.) Major adipose depots in humans. Subcutaneous adipose
tissues include abdominal, femoral, and gluteal. Intraperitoneal (visceral) adipose tissues are
associated with digestive organs. Omental is attached to the stomach and mesenteric and epi-
ploic are associated with the intestine and colon, respectively. Retroperitoneal fat is located in
the retroperitoneal compartment. (Reprinted from Mol Aspects Med, 34(1), Lee, M.J., Wu, Y.,
and Fried, S.K., Adipose tissue heterogeneity: Implication of depot differences in adipose
tissue for obesity complications, 1–11, Copyright 2013, with permission from Elsevier.)
whereas testosterone-treated men have less fat mass with selective loss of central
fat [28,29]. In addition, while premenopausal women often have increased amounts
of subcutaneous adipose tissue, postmenopausal women are prone to increases in
intra-abdominal fat, and this is attenuated by hormone replacement therapy [30].
Factors that govern this sexual dimorphism in humans better clarification, but may
contribute to the etiology of differences observed in cardiovascular disease risks
among men and women. In addition to gender differences, age- and ethnicity-related
variations in fat distribution are observed. Fat tends to accumulate in central areas
with aging (both subcutaneous and visceral depots) [31]. Compared to Caucasians,
African Americans and Hispanics have relatively less visceral fat, while South
Asians seem to have more central adiposity [32,33].
Genetics as well as environmental factors play a role in determining differences

in fat distribution. Twin and population studies have revealed that both BMI and
WHR are heritable traits, with genetics accounting for 30%–70% of the variability
[34]. The occurrence of diverse phenotypes in partial lipodystrophies, which are the
result of mutations in different genes, indicates the developmental heterogeneity of
the different adipose depots [20].
HETEROGENEITY AMONG ADIPOSE TISSUE DEPOTS

When excessive fat accumulation occurs in obesity, whether it is deposited in the
subcutaneous or visceral depots has a different impact on the development of insulin
resistance, hepatic fat infiltration, proinflammatory state, and, ultimately, obesity-
related diseases. Regional differences exist in adipose tissue depots with regards
to cellular composition, microvasculature, innervation, metabolic characteristics,
extracellular matrix composition, and secretory products. The differences collectively
comprise the microenvironment that will contribute to heterogeneity in metabolism
and endocrine function within each depot [4].
Differences in Developmental Roots of Adipose Tissue

There is substantial evidence supporting the theory that different white adipose
depots may be derived from distinct precursors [20]. Cloned human preadipocytes
from subcutaneous adipose tissue exhibit a greater ability to differentiate and accu-
mulate lipids in culture than those from mesenteric or omental adipose depots. These
differences are associated with differences in expression of C/EBP-α, PPAR-γ, and
many other adipocyte-related genes [35]. Similarly, intrinsic variations in gene
expression have been observed in adipocyte and preadipocyte fractions taken from
different intra-abdominal and subcutaneous adipose tissue depots in mice [36]. In
humans, HoxA5, Gpc4, and Tbx15 expression has been shown to be highly correlated
with both obesity (measured by BMI) and fat distribution (measured by WHR). The
differences in gene expression pattern persist even after in vitro differentiation of
preadipocytes, suggesting that the differences are independent of extrinsic factors,
and different adipocyte progenitors are programmed through epigenetic modula-
tion during early stages in the development [36]. These observations indicate that
the adipogenic lineage for the development of white adipose tissue differs from one
depot to another and may participate in determining functional differences observed
between visceral and subcutaneous adipose tissue. In addition, a recent study also
showed differences in developmental gene expression patterns between abdominal
and gluteal subcutaneous depots, suggesting that programmed differences may con-
tribute to the distinct phenotypic characteristics of peripheral fat as well [37].
Differences in Cellularity, Growth, and Remodeling

In terms of cellularity, VAT have a greater number of stromal cells (nonadipocytes)
and immune cells, per gram of tissue, compared to subcutaneous adipose tissues
(SATs) [38]. SAT contain higher numbers of preadipocytes [39]. These differences in
cellularity contribute to the heterogeneity in cytokine production observed and the

association of the VAT with a proinflammatory state.
In response to a high caloric diet, the growth of adipose tissues occurs by a combi-
nation of hypertrophy and hyperplasia. In rodents, the preadipocyte capacity of pro-
liferation and recruitment is lower in visceral than subcutaneous depots. The limited
capacity for hyperplasia in intraperitoneal depots may contribute to the excessive
hypertrophy and thus stress on the existing adipocytes. Accordingly, in obese mouse
models, the epididymal fat exhibits much greater rates of adipocyte death/tissue
remodeling as reflected in an increased number of macrophages forming crown-like
structures (CLS) around dead adipocytes [40,41]. CLSs are also more numerous in
VAT than SAT in humans, although the number of CLS in human adipose tissues
is considerably lower than that observed in epididymal fat of rodents [42–44]. The
numbers of CLS in the omentum correlate with the severity of hepatic fibroinflam-
matory lesions and liver fat content as well as insulin sensitivity [43,45,46]. Several
cytokines implicated in macrophage chemotaxis and activation are expressed at
higher levels in the omentum, potentially contributing to the preferential macro-
phage infiltration into the depot [47,48]. In vitro, femoral compared to abdominal
subcutaneous preadipocytes exhibit a lower differentiation capacity, which may
affect the capacity of adipose depots to form new fat cells [49,50]. After 8 weeks of
overfeeding, upper-body SAT expands through hypertrophy while lower-body SAT
expands through hyperplasia, suggesting intrinsic differences in expansion capacity
between the two subcutaneous depots in humans [51]. In addition, human preadipo-
cytes from VAT are more susceptible to apoptotic stimuli, suggesting that there are
also differences in apoptosis between preadipocytes from the two depots [52,53].
Differences in Adipocyte Metabolism

Fat accumulation in the adipocyte is achieved by fatty acid uptake as well as syn-
thesis of fatty acids (de novo lipogenesis), while fat mobilization is achieved by
lipolysis. Both processes are regulated by various hormones including insulin and
catecholamines [20]. Studies of post-meal fatty acid uptake provide evidence for
heterogeneity in the metabolism of visceral and subcutaneous adipose tissue. In the
post-absorptive state, FFA uptake is greater in intraperitoneal than subcutaneous
abdominal adipose tissue in both sexes (comparing the same mass of tissue) [54,55].
The direct uptake of plasma FFA delivered intravenously is also greater in omental
compared to abdominal subcutaneous fat of women, so this mechanism may contrib-
ute to the preferential accumulation of VAT [4,56].
During lipolysis, the hydrolysis of triglycerides results in the efflux of nonesteri-
fied fatty acids (NEFAs) and glycerol in the bloodstream, which can be used as sub-
strates by other tissues. Insulin regulation is critical in the postprandial state where
it favors substrate uptake and storage and limits hydrolysis of triglyceride in adipo-
cytes. Omental adipocytes are reported to be less sensitive to the antilipolytic effects
of insulin in vivo [57]. Catecholamines (epinephrine and norepinephrine) exert a
bimodal regulation in lipolysis dependence throughout its interaction with different
adrenergic receptors. Binding of catecholamines to β-adrenergic receptors leads to
the activation of lipolysis, while binding to the α2-adrenergic receptor leads to its
inhibition. Adipose tissue depots are heterogeneous with regard to their response to
catecholamine-stimulated lipolysis due to expression differences of these two recep-
tors [20]. Intra-abdominal adipose tissues are more responsive to catecholamine-
stimulated lipolysis than SAT, due to a greater presence of β-adrenergic receptors
than α2-adrenergic receptors on the cell membrane [58,59]. By contrast, catechol-
amines have a very small lipolytic effect in gluteal subcutaneous adipose tissue of
normal and obese women and subcutaneous abdominal adipose tissue of obese men,
due to a concomitant increase in α2-adrenergic and decrease in β-adrenergic respon-
siveness [60]. With regard to heterogeneity in lipolysis between subcutaneous depots
(femoral, gluteal, and abdominal), most studies indicate that upper body adipocytes
are more responsive to β-adrenergic agonists, and lower-body adipocytes are more
responsive to the antilipolytic effects of α2-adrenergic agonists with lower lipolytic
responses to mixed agonists [61].
Theory of the Portal Circulation

There is ample evidence that an impaired NEFA metabolism could contribute to
the insulin-resistant state observed among individuals with abdominal obesity.
Hypertrophied intraperitoneal adipocytes are characterized by a hyperlipolytic state
that is resistant to the antilipolytic effect of insulin [62]. The theory of the portal
circulation hypothesizes that an increase in NEFAs originating from excess visceral
adipose tissue would be drained directly into the portal system and could have an
important role in the etiology of insulin resistance, particularly as it relates to hepatic
carbohydrate and lipid metabolism [63,64].
Fatty acids released from visceral fat to the portal vein increase with increasing
mass of visceral fat, although its relative contribution is highly variable at a given
visceral adiposity (5%–50%) [65]. The resulting NEFA flux to the liver would impair
liver metabolism, leading to reduced hepatic insulin sensitivity and increased hepatic
glucose production (gluconeogenesis). Hepatic insulin resistance is associated with
decreased apolipoprotein B degradation and increased production of triacylglycerol-
rich lipoproteins. Although there is a correlation between visceral fat accumulation
and portal delivery of NEFAs to the liver, the fact that most portal NEFAs originate
from the systemic circulation suggests that there are other factors that might explain
the altered metabolic profile of viscerally obese patients [66]. In addition, visceral
fat–derived FFA is not a significant contributor of FFA delivery to the periphery
(i.e., skeletal muscle). Considering upper (abdominal) and lower-body fat accumula-
tion, lipolytic activity is greater in subcutaneous adipose tissue on upper body than
lower body, so that upper body subcutaneous fat mass accounts for ∼70% of systemic
FFA rate of appearance [57,67,68]. Although depot differences in adipocyte metabo-
lism and endocrine function are clearly important in the etiology of obesity-related
diseases, the relative contribution of visceral compared to abdominal subcutaneous
fat is still controversial.
Theory of Ectopic Fat Deposition

The altered NEFA metabolism and portal circulation hypothesis imply that visceral
adipose tissue is causally involved in the pathophysiology of the metabolic syndrome
that is often found in patients with visceral obesity. However, another possibility,
which does not exclude a contribution from the mechanism described earlier, is that
subcutaneous adipose tissue could reach its limit of expansion (maximum hypertro-
phy of existing adipocytes and failure to recruit new adipocytes) with excess energy
intake, and so energy would be stored in visceral fat [68,69] (Figure 8.5). Such a rela-
tive deficit in the capacity of subcutaneous fat to store excess energy would result in
increased accumulation of fat at undesired sites such as the liver, the skeletal muscle,
the heart, and even pancreatic β-cells, a phenomenon that has been described as
ectopic fat deposition [70]. Consistent with this theory is the fact that transgenic
mice that are essentially fatless owing to the expression of A-ZIP/F-1 protein, which
blocks the activity of several transcription factors, also show liver and muscle insulin
resistance and eventually develop diabetes. Surgical implantation of adipose tissue in
these mice improves the insulin sensitivity of their liver and muscles, consistent with
the idea that subcutaneous fat is a metabolic sink to buffer an energy surplus [71,72].
In humans, lipodystrophy, the loss of the ability to store excess lipids in adipose tis-
sue, can lead to the overdevelopment of ectopic fat stores and hence metabolic per-
turbations. In accordance with this hypothesis, treatment with glitazones increases
subcutaneous fat deposition, which might help to explain the beneficial effects of
this class of drug on muscle and liver insulin sensitivity [68,73]. Given evidence for
heterogeneity in the metabolic consequences of obesity for liver and muscle metabo-
lism, it seems likely that both direct effects of an expanded VAT and limitations of
SAT storage capacity that result in ectopic fat contribute to tissue-specific metabolic
impairments within an individual [4].
Adipokine and Cytokine Secretion

The adipose organ is not only specialized in the storage and mobilization of lipids,
but it is also a remarkable endocrine organ releasing numerous peptide hormones
and bioactive molecules that regulate systemic metabolism. Adipocytes produce sev-
eral hormones, most notably leptin and adiponectin, which modulate appetite, fuel
metabolism, innate immune function, and reproduction [74]. Adipocytes also secrete
complement factors and acute-phase response proteins including serum amyloid A
(SAA) and retinol-binding protein 4 (RBP4), which can influence systemic inflam-
mation and insulin resistance [75,76]. Other cells produce additional factors, includ-
ing omentin, visfatin, resistin, TNF-α, IL-6, and IL-8 (Tables 8.1 and 8.2) [21]. The
macrophage and immune cell infiltration in adipose tissue contributes to the inflam-
matory profile reported in abdominally obese patients and for the development of
obesity-related diseases [4,68].
There are differences in cellularity composition between the adipose depots.
VAT includes an abundance of milky spots and lymph nodes where lymphocytes
accumulate [77,78]. Pond hypothesized that VAT plays a special role in immunity,
potentially explaining the growth of VAT in response to infections such as HIV [79].
Undoubtedly, depot differences in cell populations contribute to depot differences
in adipokine production and can contribute to variations in adipocyte function via
paracrine interactions [4].
Proteomic screening approaches have identified over 250 proteins secreted
by human visceral adipose tissue [80]. A quantitative analysis of the secretomes
Normal adiposity
Energy-dense food Lack of physical

( fat+sugar content) activity/exercise
Positive
energy balance
Smoking
Unfavorable genotype
Maladaptive response
to stress
Subcutaneous obesity Visceral obesity

Healthy adipose tissue Dysfunctional adipose tissue
Altered FFA Altered release

metabolism of adipokines
No ectopic fat Lipid overflow–ectopic fat
Muscle fat
Low muscle fat ( intracellular lipid)
Low epicardial fat Epicardial fat
Low liver fat and

normal function Liver fat and
altered function
Normal metabolic profile Altered metabolic profile
Absence of clinical criteria Presence of clinical criteria

for metabolic syndrome for metabolic syndrome
(including hypertrigly-
ceridemic waist)
comparing visceral and subcutaneous fat showed that visceral adipose tissue has
a higher secretory capacity than subcutaneous adipose tissue [81]. Generally, the
expression of proinflammatory cytokines (IL-6, IL-8, MCP-1, RANTES, MIP-1α,
PAI-1) is higher in visceral fat. In addition, molecules involved in innate immu-
nity and the acute-phase response and complement factors are overexpressed in
visceral adipose tissue. The depot differences in TNF-α expression levels are
inconsistent [4]. Plasma levels of C-reactive protein (CRP), an inflammatory marker
that is predictive of a risk of myocardial infarction, are increased in patients with
visceral obesity [11].
Leptin is a hormone secreted by adipose tissue in direct proportion to the amount
of body fat. The circulating leptin levels serve as a gauge of energy stores, thereby
directing the regulation of energy homeostasis, neuroendocrine function, and metab-
olism. Persons with congenital deficiency are obese, and treatment with leptin results
in dramatic weight loss through decreased food intake and possible increased energy
expenditure. However, most obese persons are resistant to the weight-reducing
effects of leptin [82]. Fat distribution contributes to the variability in serum leptin in
obese patients; in particular, subcutaneous abdominal fat is a stronger determinant
of leptin concentration [83].
The protein adiponectin is specifically derived from adipose tissue. As opposed
to proinflammatory adipokines, adiponectin levels are reduced in obese individu-
als, particularly among patients with excess visceral adiposity [84]. Adiponectin has
been found to have many effects in vitro that are compatible with improved insulin
signaling and potential protection against atherosclerosis [85]. The reduced adipo-
nectin levels observed in viscerally obese patients could therefore be a contribut-
ing factor responsible for their atherogenic and diabetogenic metabolic risk-factor
profile [68].
Omentin is a protein expressed and secreted from visceral but not subcutane-
ous adipose tissue that increases insulin sensitivity in human adipocytes. Decreased
omentin levels are associated with increasing obesity and insulin resistance [86]. In
vitro, omentin increases insulin sensitivity of glucose uptake in human adipocytes,
suggesting that omentin might protect this depot from insulin resistance associated
FIGURE 8.5 (See color insert.) The lipid overflow–ectopic fat model. Excess visceral
fat accumulation might be causally related to the features of insulin resistance, but might
also be a marker of a dysfunctional adipose tissue being unable to appropriately store the
energy excess. According to this model, the body’s ability to cope with the surplus of calo-
ries (resulting from excess caloric consumption, a sedentary lifestyle, or a combination
of both factors) might, ultimately, determine the individual’s susceptibility to developing
metabolic syndrome. There is evidence suggesting that if the extra energy is channeled into
insulin-sensitive subcutaneous adipose tissue, the individual, although in positive energy
balance, will be protected against the development of the metabolic syndrome. However, in
cases in which adipose tissue is absent, deficient, or insulin resistant with a limited ability
to store the energy excess, the triacylglycerol surplus will be deposited at undesirable sites
such as the liver, the heart, the skeletal muscle and in VAT—a phenomenon described as
ectopic fat deposition. (Reprinted by permission from Macmillan Publishers Ltd. Nature,
Després, J.P. and Lemieux, I., Abdominal obesity and metabolic syndrome, 444(14),
881–887, Copyright 2006.)
with high levels of inflammatory cytokines that are detected in visceral fat [87].
Visfatin was identified as a visceral specific adipokine, but in humans its expression
levels are similar between VAT and SAT [88]. Adipose tissue has also been identified
as a site of expression of resistin (expressed in monocytes in humans) and thrombo-
spondin-1, but their functional importance and depot differences in their expression
levels are as yet unclear [4,74].
Lessons from Fat-Tissue Removal and Transplantation

The removal of visceral fat improves while removal of subcutaneous fat does not
affect insulin sensitivity in rodents [89,90]. Transplantation of intra-abdominal fat
into the mesentery (conferring a portal venous drainage) leads to the development of
glucose intolerance and hepatic insulin resistance, whereas transplantation of intra-
abdominal fat into the parietal peritoneum (conferring a caval/systemic venous drain-
age) has no effect [91]. These deleterious effects of portally drained intra-abdominal
transplantation appeared to be mediated by the production of IL-6, as these effects
are abolished when transplants are derived from IL-6 knockout mice. Several stud-
ies involving the transplantation of subcutaneous adipose tissue into the visceral
cavity have provided some perspectives. Transplantation of inguinal subcutaneous
to the visceral cavity leads to lower body-weight gain with improvement in glucose
metabolism in a mouse [92]. While these studies suggest that subcutaneous adipose
tissue is intrinsically different from visceral, location also seems to be important as
subcutaneous to visceral transplants are more beneficial than subcutaneous to sub-
cutaneous transplantation. Whether these interesting findings can be extrapolated to
humans remains to be determined [4].
The beneficial effects of adipose tissue removal on metabolic risks in humans are
controversial [4]. Several studies using omentectomy in addition to Roux-en-Y gas-
tric bypass resulted in controversial findings, commonly confounded by weight loss
due to gastric bypass (93–95). In addition, removal of a large quantity of abdominal
subcutaneous fat through liposuction has been shown to be ineffective [96]. In con-
trast, weight loss through diet, exercise, or in combination has been proven to be
effective in improving metabolic diseases. Diet and exercise cause preferential fat
loss from intra-abdominal fat than the subcutaneous tissue [97].
MEASURING ABDOMINAL FAT IN CLINICAL PRACTICE

Numerous techniques are available to estimate body composition and fat distribu-
tion, and the preferred methodology will depend on the medical indication, whether
it is to be used in clinical practice or a research study, the economic resources, and
availability (Table 8.3) [98].
BMI is a practical surrogate for body fat mass, generally providing a fair correla-
tion with total body fat in a nonathlete population [99]. It is calculated by taking a
person’s weight (in kilograms) and dividing by their height squared (in meters). BMI
is widely used for guidance of therapy, with specific medical and surgical interven-
tions being recommended based on which category a given individual is included.
Major national and international health institutes adopt BMI cutoffs to categorize
TABLE 8.3
Capability of Different Body Fat Measurements to Estimate
Total Body Fat and Fat Distribution
Capability Capability Applicability in
Measuring Measuring Fat Large Population
Method Total Body Fat Distribution Studies
CT Moderate Very high Low
MRI High Very high Low
DXA Very high High Moderate
Densitometry Very high Very low Low
Dilution techniques High Very low Moderate
BIA Moderate Very low High
Anthropometry
BMI Moderate Very low Very high
WC, HC, WHR, SAD Low High Very high
Skinfolds Moderate Moderate High
Notes: CT, computed tomography; MRI, magnetic resonance imaging; DXA, dual-
energy X-ray absorptiometry; BIA, bioelectrical impedance analysis; BMI,
body mass index; WC, waist circumference; HC, hip circumference; WHR,
waist-to-hip ratio; SAD, sagittal abdominal diameter.
adult men and women as underweight (BMI < 18.5), normal weight (BMI 18.5—
24.9), overweight (BMI 25—29.9), or obese (class 1 BMI 30—34.9, class 2 BMI
35—39.9, class 3 BMI ≥ 40). For children and adolescents, the calculated BMI num-
ber can be plotted on BMI-for-age growth charts for either girls or boys to obtain
a percentile ranking [100]. Large epidemiological studies in adult populations have
shown a positive correlation between cardiovascular mortality risk with BMI values
higher than 25 kg/m2 [101]. However, the use of BMI alone has its limitations in risk
prediction and in generalizations for populations from different ethnic backgrounds
(Table 8.4). It does not take into consideration the body-fat distribution, while it is
clearly established that more fat in the abdomen is a risk factor for the development
of metabolic syndrome and cardiovascular disease. In addition, BMI cannot distin-
guish between fat mass and lean mass or the presence of edema. For example, well-
trained bodybuilders have a low percentage of body fat, but their BMI may be on the
overweight range because of their large muscle (lean) mass [98].
As exposed anteriorly, numerous studies have indicated that abdominal obesity is
a better predictor of risk of metabolic and cardiovascular disease than weight or BMI
alone [8–10,102]. Abdominal fat can be easily assessed in clinical practice by per-
forming anthropometric measurements of the WC and the WHR. Dual-energy x-ray
absorptiometry (DXA), computed tomography (CT), and magnetic resonance imag-
ing (MRI) are more accurate but are impractical for routine clinical use [103]. In the
clinical setting, the WC is generally the preferred method to assess abdominal fat
content given its ease of use, when performed properly. Whether WHR imparts any
TABLE 8.4
Combined Recommendations of Body Mass Index and Waist Circumference
Cut-Off Points Made for Overweight or Obesity, and Association
with Disease Risk
Disease Riska Relative to Normal Weight
and Waist Circumference
BMI Obesity Men 102 cm (40 in.) or less Men > 102 cm (40 in.)
(kg/m2) Class Women 88 cm (35 in.) or less Women > 88 cm (35 in.)
Underweight < 18.5 – –
Normal 18.5–24.9 – –
Overweight 25.0–29.9 Increased High
Obesity 30.0–34.9 I High Very High
35.0–39.9 II Very High Very High
Extreme 40.0 + III Extremely High Extremely High
Obesity
Note: +, Increased waist circumference also can be a marker for increased risk, even in persons of
normal weight.
a Disease risk for type 2 diabetes, hypertension, and CVD.
independent information about disease risk beyond WC is uncertain, but between

the two, the WC appears to be simplest to measure and interpret. Despite recom-
mendations by major national and international health organizations, anthropomet-
ric measurements of abdominal obesity are still underused by clinicians. Greater use
is recommended in addition to BMI to help identify appropriate high-risk patients for
further screening and more intensive goals of therapy to treat coexistent factors, as
blood pressure and hyperlipidemia [104]. Table 8.3 incorporates both BMI and WC
in the classification of overweight and obesity and provides an indication of relative
disease risk.
Waist circumference can be measured in the clinical setting with a flexible tape
placed on a horizontal plane at the level of the iliac crest as recommended by the
(US) National Institutes of Health (NIH). According to the National Health and
Nutrition Examination Survey (NHANES) III protocol, to define the level at which
WC is measured, a bony landmark is first located and marked (Figure 8.6) [105]. The
subject stands, and the examiner, positioned at the right of the subject, locates the
upper hip bone and the top of the right iliac crest. Just above the uppermost lateral
border of the right iliac crest, a horizontal mark is drawn and then crossed with a
vertical mark on the midaxillary line. The measuring tape is placed in a horizontal
plane around the abdomen at the level of this marked point on the right side of the
trunk. The plane of the tape is parallel to the floor, and the tape is snug but does
not compress the skin. The measurement is made at a normal minimal respiration
[104,106]. The World Health Organization (WHO) STEPwise approach to surveil-
lance protocol for measuring WC instructs that the measurement should be made at
a higher level in the abdomen, compared with the Centers for Disease Control and
Waist circumference measurement
To measure waist
circumference, locate
the upper hip bone
and the top of the right
iliac crest. Place a
measuring tape in a
horizontal plane around
the abdomen at the level
of the iliac crest. Before
reading the tape measure,
ensure that the tape is
snug, but does not
compress the skin, and
is parallel to the floor.
The measurement is
made at the end of a
normal expiration.
Measuring-tape position for waist

(abdominal) circumference in adults
FIGURE 8.6 Measuring WC according to the National Health and Nutrition Examination
Survey III protocol. (Reprinted from the National Heart, Lung, and Blood Institute,
The Practical Guide to the Identification, Evaluation, and Treatment of Overweight
and Obesity in Adults, National Heart, Lung, and Blood Institute, Bethesda, MD, 2000;
National Institutes of Health, National Heart, Lung, and Blood Institute, Obes Res., 6, 51S,
1998; U.S. Department of Health and Human Services, Public Health Service, NHANES
III Anthropometric Procedures Video, U.S. Government Printing Office, Washington, DC,
1996. With permission.)
Prevention (CDC) protocol. The WHO recommends measuring at the approximate

midpoint between the lower margin of the last palpable rib and the top of the iliac
crest [7]. The WHR is the ratio of the WC to the hip circumference. The hip circum-
ference should be measured around the widest portion of the buttocks, with the tape
parallel to the floor [7].
The absolute WC >102 cm (40 in.) in men and >88 cm (35 in.) in women are used
as measures of central obesity [104]. The prevalence of abdominal obesity accord-
ing to these cut-points in the United States has tripled in men from 13% in 1960–
62 to 38% in 1999–2000. In women, the prevalence increased from 19% to 60%
over the same period [107]. Abdominal obesity is further defined as WHR above
0.90 for males and above 0.85 for females, accordingly with the WHO [7]. There
are ethnic and age-related differences in body-fat composition that need consider-
ation [104]. For older individuals, WC assumes greater value for estimating risk of
obesity-related diabetes. Asian ethnic groups generally have a smaller WC compared

with Caucasians but, despite the smaller WC, the visceral fat mass is higher for
Asians. Moreover, for specific Asian groups, disease risks may already be increased
at a lower level of WC, suggesting that lower WC cut-points should be used for these
ethnic groups. In Asian females, a WC >80 cm and in Asian males, a value >90 cm
are considered abnormal [104].
CONCLUSION
Although an increase in total body adiposity is associated with an increase in disease
risk, the amount of central upper-body fat, particularly intraperitoneal, has been asso-
ciated with an increase in risk of type 2 diabetes, hypertension, dyslipidemia, insulin
resistance, inflammation, cardiovascular disease, and various types of cancers. This
phenomenon is usually verified at any level of total body fat. In clinical practice,
measuring WC in addition to the BMI may be helpful for the identification and man-
agement of a subgroup of overweight or obese patients at high cardiometabolic risk.
Evidence supports that adipocytes distributed in the various fat depots in the
body are phenotypically different, as a result of genetic and developmental events.
Moreover, important regional distinctions exist between the depots regarding cel-
lular composition, microvasculature, metabolic characteristics, and secretory prod-
ucts. These differences collectively comprise the microenvironment that contributes
to heterogeneity in metabolism and endocrine function within each depot and may
help in understanding the pathophysiological association of visceral fat and cardio-
metabolic risk.
REFERENCES
1. Caballero B. 2007. The global epidemic of obesity: An overview. Epidemiol Rev. 29: 1–5.
2. Flegal KM, Graubard BI, Williamson DF, Gail MH. 2005. Excess deaths associated with
underweight, overweight, and obesity. JAMA. 293: 1861–1867.
3. Apovian CM, Gokce N. 2012. Obesity and cardiovascular disease. Circulation. 6;125:
1178–1182.
4. Lee MJ, Wu Y, Fried SK. 2013. Adipose tissue heterogeneity: Implication of depot
differences in adipose tissue for obesity complications. Mol Aspects Med. 34: 1–11.
5. Vague J. 1947. La diffférenciacion sexuelle, facteur déterminant des formes de l’obésité.
Presse Med. 30: 339–340.
6. Manolopoulos KN, Karpe F, Frayn KN. 2010. Gluteofemoral body fat as a determinant
of metabolic health. Int J Obes (Lond). 34: 949–959.
7. WHO STEPwise approach to surveillance (STEPS). 2008. Waist circumference and
Waist–Hip ratio: Report of a WHO expert consultation. World Health Organization
(WHO), Geneva, Switzerland.
8. Zhang C, Rexrode KM, van Dam RM, Li TY, Hu FB. 2008. Abdominal obesity and the
risk of all-cause, cardiovascular, and cancer mortality: Sixteen years of follow-up in US
women. Circulation. April 1;117: 1658–1667.
9. Yusuf S, Hawken S, Ôunpuu S, Bautista L, Franzosi MG, Commerford P et al., On
behalf of the INTERHEART Study Investigators. 2005. Obesity and the risk of myocar-
dial infarction in 27,000 participants from 52 countries: A case-control study. Lancet.
366: 1640–1649.
10. Jacobs EJ, Newton CC, Wang Y, Patel AV, McCullough ML, Campbell PT et al. 2010.
Waist circumference and all-cause mortality in a large US cohort. Arch Intern Med. 170:
1293–1301.
11. Lemieux I, Pascot A, Prud’homme D, Alméras N, Bogaty P, Nadeau A et al. 2001.
Elevated C-reactive protein: Another component of the atherothrombotic profile of
abdominal obesity. Arterioscler Thromb Vasc Biol. 21: 961–967.
12. Janand-Delenne B, Chagnaud C, Raccah D, Alessi MC, Juhan-Vague I, Vague P. 1998.
Visceral fat as a main determinant of plasminogen activator inhibitor 1 level in women.
Int J Obes Relat Metab Disord. 22: 312–317.
13. Després JP, Moorjani S, Lupien PJ, Tremblay A, Nadeau A, Bouchard C. 1990.
Regional distribution of body fat, plasma lipoproteins, and cardiovascular disease.
Arteriosclerosis. 10: 497–511.
14. Tchernof A, Després JP. 2013. Pathophysiology of human visceral obesity: An update.
Physiol Rev. 93: 359–404.
15. Austin MA, King MC, Vranizan KM, Krauss RM. 1990. Atherogenic lipoprotein pheno-
type. A proposed genetic marker for coronary heart disease risk. Circulation. 82: 495–506.
16. Grundy SM. 1998. Hypertriglyceridemia, atherogenic dyslipidemia, and the metabolic
syndrome. Am J Cardiol. 1998 Feb 26;81(4A):18B–25B. under review.
17. Lamarche B, Tchernof A, Mauriège P, Cantin B, Dagenais GR, Lupien PJ et al. 1998.
Fasting insulin and apolipoprotein B levels and low-density lipoprotein particle size as
risk factors for ischemic heart disease. JAMA. 279: 1955–1961.
18. Granér M, Siren R, Nyman K, Lundbom J, Hakkarainen A, Pentikäinen MO et al. 2013.
Cardiac steatosis associates with visceral obesity in nondiabetic obese men. J Clin
Endocrinol Metab. 98: 1189–1197.
19. Nicholls DG, Locke RM. 1994. Thermogenic mechanisms in brown fat. Physiol Rev.
64: 1–64.
20. Gesta S, Kahn CR. 2012. White adipose tissue. In: Adipose Tissue Biology, Symonds
M.E. (ed.). New York: Springer Science+Business Media, LLC.
21. Kershaw EE, Flier JS. 2004. Adipose tissue as an endocrine organ. J Clin Endocrinol
Metab. 89: 2548–2556.
22. Wellen KE, Hotamisligil GS. 2005. Inflammation, stress, and diabetes. J Clin Invest.
115: 1111–1119.
23. Barbarroja N, Lopez-Pedrera R, Mayas MD, Garcia-Fuentes E, Garrido-Sánchez L,
Macías-González M et al. 2010. The obese healthy paradox: Is inflammation the answer?
Biochem J. 430: 141–149.
24. Pasarica M, Sereda OR, Redman LM, Albarado DC, Hymel DT, Roan LE et al. 2009.
Reduced adipose tissue oxygenation in human obesity: Evidence for rarefaction, mac-
rophage chemotaxis, and inflammation without an angiogenic response. Diabetes. 58:
718–725.
25. Kesler A, Kliper E, Shenkerman G, Stern N. 2010. Idiopathic intracranial hypertension
is associated with lower body adiposity. Ophthalmology. 117: 169–174.
26. Shen W, Wang Z, Punyanita M, Lei J, Sinav A, Kral JG et al. 2003. Adipose tissue quan-
tification by imaging methods: A proposed classification. Obes Res. 11: 5–16.
27. Markman B, Barton FE Jr. 1987. Anatomy of the subcutaneous tissue of the trunk and
lower extremity. Plast Reconstr Surg. 80: 248–254.
28. Escobar-Morreale HF, San Millan JL. 2007. Abdominal adiposity and the polycystic
ovary syndrome. Trends Endocrinol Metab. 18: 266–272.
29. Allan CA, McLachlan RI. 2010. Androgens and obesity. Curr Opin Endocrinol Diabetes
Obes. 17: 224–232.
30. Mayes JS, Watson GH. 2004. Direct effects of sex steroid hormones on adipose tissues
and obesity. Obes Rev. 5: 197–216.
31. Kuk JL, Saunders TJ, Davidson LE, Ross R. 2009. Age-related changes in total and
regional fat distribution. Ageing Res Rev. 8: 339–348.
32. Carroll JF, Chiapa AL, Rodriquez M, Phelps DR, Cardarelli KM, Vishwanatha JK et al.
2008. Visceral fat, waist circumference, and BMI: Impact of race/ethnicity. Obesity
(Silver Spring). 16: 600–607.
33. Anand SS, Tarnopolsky MA, Rashid S, Schulze KM, Desai D, Mente A et al. 2011.
Adipocyte hypertrophy, fatty liver and metabolic risk factors in South Asians: The molec-
ular study of health and risk in ethnic groups (mol-SHARE). PLoS ONE. 6: e22112.
34. Nelson TL, Vogler GP, Pedersen NL, Hong Y, Miles TP. 2000. Genetic and environmen-
tal influences on body fat distribution, fasting insulin levels and CVD: Are the influences
shared? Twin Res. 3: 43–50.
35. Tchkonia T, Giorgadze N, Pirtskhalava T, Tchoukalova Y, Karagiannides I, Forse RA
et al. 2002. Fat depot origin affects adipogenesis in primary cultured and cloned human
preadipocytes. Am J Physiol Regul Integr Comp Physiol. 282: R1286–R1296.
36. Gesta S, Bluher M, Yamamoto Y, Norris AW, Berndt J, Kralisch S et al. 2006. Evidence
for a role of developmental genes in the origin of obesity and body fat distribution. Proc
Natl Acad Sci USA. 103: 6676–6681.
37. Karastergiou K, Fried SK, Xie H, Lee MJ, Divoux A, Rosencrantz MA et al. 2013.
Distinct developmental signatures of human abdominal and gluteal subcutaneous adi-
pose tissue depots. J Clin Endocrinol Metab. 98: 362–371.
38. Van HV, Rohrig K, Hauner H. 2004. Comparison of proliferation and differentiation
capacity of human adipocyte precursor cells from the omental and subcutaneous adipose
tissue depot of obese subjects. Metabolism. 53: 632–637.
39. Tchkonia T, Tchoukalova YD, Giorgadze N, Pirtskhalava T, Karagiannides I, Forse
RA et al. 2005. Abundance of two human preadipocyte subtypes with distinct capaci-
ties for replication, adipogenesis, and apoptosis varies among fat depots. Am J Physiol
Endocrinol Metab. 288: E267–E277.
40. Strissel KJ, Stancheva Z, Miyoshi H, Perfield JW, DeFuria J, Jick Z et al. 2007.
Adipocyte death, adipose tissue remodeling, and obesity complications. Diabetes. 56:
2910–2918.
41. Nishimura S, Manabe I, Nagasaki M, Seo K, Yamashita H, Hosoya Y et al. 2008. In vivo
imaging in mice reveals local cell dynamics and inflammation in obese adipose tissue.
J Clin Invest. 118: 710–721.
42. Cinti S, Mitchell G, Barbatelli G, Murano I, Ceresi E, Faloia E et al. 2005. Adipocyte
death defines macrophage localization and function in adipose tissue of obese mice and
humans. J Lipid Res. 46: 2347–2355.
43. Cancello R, Tordjman J, Poitou C, Guilhem G, Bouillot JL, Hugol D et al. 2006.
Increased infiltration of macrophages in omental adipose tissue is associated with
marked hepatic lesions in morbid human obesity. Diabetes. 55: 1554–1561.
44. Harman-Boehm I, Bluher M, Redel H, Sion-Vardy N, Ovadia S, Avinoach E et al. 2007.
Macrophage infiltration into omental versus subcutaneous fat across different popula-
tions: Effect of regional adiposity and the comorbidities of obesity. J Clin Endocrinol
Metab. 92: 2240–2247.
45. Kolak M, Westerbacka J, Velagapudi VR, Wagsater D, Yetukuri L, Makkonen J et al.
2007. Adipose tissue inflammation and increased ceramide content characterize subjects
with high liver fat content independent of obesity. Diabetes. 56: 1960–1968.
46. Apovian CM, Bigornia S, Mott M, Meyers MR, Ulloor J, Gagua M et al. 2008. Adipose
macrophage infiltration is associated with insulin resistance and vascular endothelial
dysfunction in obese subjects. Arterioscler Thromb Vasc Biol. 28: 1654–1659.
47. Bruun JM, Lihn AS, Pedersen SB, Richelsen B. 2005. Monocyte chemoattractant
protein-1 release is higher in visceral than subcutaneous human adipose tissue (AT):
Implication of macrophages resident in the AT. J Clin Endocrinol Metab. 90: 2282–2289.
48. Sjoholm K, Palming J, Olofsson LE, Gummesson A, Svensson PA, Lystig TC et al.
2005. A microarray search for genes predominantly expressed in human omental adipo-
cytes: Adipose tissue as a major production site of serum amyloid A. J Clin Endocrinol
Metab. 90: 2233–2239.
49. Hauner H, Entenmann G. 1991. Regional variation of adipose differentiation in cultured
stromal-vascular cells from the abdominal and femoral adipose tissue of obese women.
Int J Obes. 15: 121–126.
50. Tchoukalova YD, Koutsari C, Votruba SB, Tchkonia T, Giorgadze N, Thomou T et al.
2010. Sex- and depot-dependent differences in adipogenesis in normal-weight humans.
Obesity (Silver Spring). 18: 1875–1880.
51. Tchoukalova YD, Votruba SB, Tchkonia T, Giorgadze N, Kirkland JL, Jensen MD.
2010. Regional differences in cellular mechanisms of adipose tissue gain with overfeed-
ing. Proc Natl Acad Sci USA. 107: 18226–18231.
52. Niesler CU, Siddle K, Prins JB. 1998. Human preadipocytes display a depot-specific
susceptibility to apoptosis. Diabetes. 47: 1365–1368.
53. Tchkonia T, Giorgadze N, Pirtskhalava T, Thomou T, DePonte M, Koo A et al. 2006. Fat
depot-specific characteristics are retained in strains derived from single human preadi-
pocytes. Diabetes. 55: 2571–2578.
54. Jensen MD, Sarr MG, Dumesic DA, Southorn PA, Levine JA. 2003. Regional uptake of
meal fatty acids in humans. Am J Physiol Endocrinol Metab. 285: E1282–E1288.
55. Marin P, Lonn L, Andersson B, Oden B, Olbe L, Bengtsson BA et al. 1996. Assimilation
of triglycerides in subcutaneous and intraabdominal adipose tissues in vivo in men:
Effects of testosterone. J Clin Endocrinol Metab. 81: 1018–1022.
56. Koutsari C, Ali AH, Mundi MS, Jensen MD. 2011. Storage of circulating free fatty acid
in adipose tissue of postabsorptive humans: Quantitative measures and implications for
body fat distribution. Diabetes. 60: 2032–2040.
57. Meek SE, Nair KS, Jensen MD. 1999. Insulin regulation of regional free fatty acid
metabolism. Diabetes. 48: 10–14.
58. Hellmer J, Marcus C, Sonnenfeld T, Arner P. 1992. Mechanisms for differences in lipol-
ysis between human subcutaneous and omental fat cells. J Clin Endocrinol Metab. 75:
15–20.
59. Mauriege P, Galitzky J, Berlan M, Lafontan M. 1987. Heterogeneous distribution of
beta and alpha-2 adrenoceptor binding sites in human fat cells from various fat deposits:
Functional consequences. Eur J Clin Invest. 17: 156–165.
60. Mauriege P, Despres JP, Prud’Homme D, Pouliot MC, Marcotte M, Tremblay A et al.
1991. Regional variation in adipose tissue lipolysis in lean and obese men. J Lipid Res.
32: 1625–1633.
61. Leibel RL, Edens NK, Fried SK. 1989. Physiologic basis for the control of body fat
distribution in humans. Annu Rev Nutr. 9: 417–443.
62. Mittelman SD, Van Citters GW, Kirkman EL, Bergman RN. 2002. Extreme insulin
resistance of the central adipose depot in vivo. Diabetes. 51: 755–761.
63. Bergman RN, Kim SP, Catalano KJ, Hsu IR, Chiu JD, Kabir M et al. 2006. Why visceral
fat is bad: Mechanisms of the metabolic syndrome. Obesity (Silver Spring). 14 (S2):
16S–19S.
64. Bjorntorp P. 1990. “Portal” adipose tissue as a generator of risk factors for cardiovascu-
lar disease and diabetes. Arteriosclerosis. 10: 493–496.
65. Nielsen S, Guo Z, Johnson CM, Hensrud DD, Jensen MD. 2004. Splanchnic lipolysis in
human obesity. J Clin Invest. 113: 1582–1588.
66. Jensen MD. 2006. Is visceral fat involved in the pathogenesis of the metabolic syn-
drome? Human model. Obesity (Silver Spring). 14 (S1): 20S–24S.
67. Jensen MD. 1995. Gender differences in regional fatty acid metabolism before and after
meal ingestion. J Clin Invest. 96: 2297–2303.
68. Després J.P. and Lemieux I. 2006. Abdominal obesity and metabolic syndrome. Nature.
444: 881–887.
69. Danforth Jr E. 2000. Failure of adipocyte differentiation causes type II diabetes mel-
litus? Nat Genet. 26: 13.
70. Miranda PJ, DeFronzo RA, Califf RM, Guyton JR. 2005. Metabolic syndrome:
Definition, pathophysiology, and mechanisms. Am Heart J. 149: 33–45.
71. Gavrilova O, Marcus-Samuels B, Graham D, Kim JK, Shulman GI, Castle AL, Vinson C
et al. 2000. Surgical implantation of adipose tissue reverses diabetes in lipoatrophic
mice. J Clin Invest. 105: 271–278.
72. Kim JK, Gavrilova O, Chen Y, Reitman ML, Shulman GI. 2000. Mechanism of insulin
resistance in A-ZIP/F-1 fatless mice. J Biol Chem. 275: 8456–8460.
73. Miyazaki Y, Mahankali A, Matsuda M, Mahankali S, Hardies J, Cusi K et al. 2002.
Effect of pioglitazone on abdominal fat distribution and insulin sensitivity in type 2
diabetic patients. J Clin Endocrinol Metab. 87: 2784–2791.
74. Trujillo ME, Scherer PE. 2006. Adipose tissue-derived factors: Impact on health and
disease. Endocr Rev. 27: 762–778.
75. Yang Q, Graham TE, Mody N, Preitner F, Peroni OD et al. 2005. Serum retinol binding
protein 4 contributes to insulin resistance in obesity and type 2 diabetes. Nature. 436:
356–362.
76. Yang RZ, Lee MJ, Hu H, Pollin TI, Ryan AS, Nicklas BJ et al. 2006. Acute-phase serum
amyloid A: An inflammatory adipokine and potential link between obesity and its meta-
bolic complications. PLoS Med. 3: e287.
77. Gabrielsson BG, Johansson JM, Lonn M, Jernas M, Olbers T, Peltonen M et al. 2003.
High expression of complement components in omental adipose tissue in obese men.
Obes Res. 11: 699–708.
78. Litbarg NO, Gudehithlu KP, Sethupathi P, Arruda JA, Dunea G, Singh AK. 2007.
Activated omentum becomes rich in factors that promote healing and tissue regenera-
tion. Cell Tissue Res. 328: 487–497.
79. Pond CM. 2005. Adipose tissue and the immune system. Prostaglandins Leukot Essent
Fatty Acids. 73: 17–30.
80. Varez-Llamas G, Szalowska E, de Vries MP, Weening D, Landman K, Hoek A et al. 2007.
Characterization of the human visceral adipose tissue secretome. Mol Cell Proteomics.
6: 589–600.
81. Hocking SL, Wu LE, Guilhaus M, Chisholm DJ, James DE. 2010. Intrinsic depot-
specific differences in the secretome of adipose tissue, preadipocytes, and adipose
tissue-derived microvascular endothelial cells. Diabetes. 59: 3008–3016.
82. Kelesidis T, Kelesidis I, Chou S, Mantzoros CS. 2010. Narrative review: The role of
leptin in human physiology: Emerging clinical applications. Ann Intern Med. 152(2):
93–100.
83. Minocci A, Savia G, Lucantoni R, Berselli ME, Tagliaferri M, Calò G et al. 2000. Leptin
plasma concentrations are dependent on body fat distribution in obese patients. Int J
Obes Relat Metab Disord. 24: 1139–1144.
84. Côté M, Mauriège P, Bergeron J, Alméras N, Tremblay A, Lemieux I. 2005.
Adiponectinemia in visceral obesity: Impact on glucose tolerance and plasma lipopro-
tein and lipid levels in men. J Clin Endocrinol Metab. 90: 1434–1439.
85. Matsuzawa Y. 2006. Therapy insight: Adipocytokines in metabolic syndrome and related
cardiovascular disease. Nat Clin Pract Cardiovasc Med. 3: 35–42.
86. De Souza Batista CM, Yang RZ, Lee MJ, Glynn NM, Yu DZ, Pray J et al. 2007. Omentin
plasma levels and gene expression are decreased in obesity. Diabetes. 56: 1655–1661.
87. Yang RZ, Lee MJ, Hu H, Pray J, Wu HB, Hansen BC et al. 2006. Identification of
omentin as a novel depot-specific adipokine in human adipose tissue: Possible role in
modulating insulin action. Am J Physiol Endocrinol Metab. 290: E1253–E1261.
88. Varma V, Yao-Borengasser A, Rasouli N, Bodles AM, Phanavanh B, Lee MJ et al. 2007.
Human visfatin expression: Relationship to insulin sensitivity, intramyocellular lipids,
and inflammation. J Clin Endocrinol Metab. 92: 666–672.
89. Barzilai N, She L, Liu BQ, Vuguin P, Cohen P, Wang J et al. 1999. Surgical removal of
visceral fat reverses hepatic insulin resistance. Diabetes. 48: 94–98.
90. Shi H, Strader A, Woods SC, Seeley RJ. 2007. The effect of fat removal on glucose toler-
ance is depot specific in male and female mice. Am J Physiol Endocrinol Metab. 293:
E1012–E1020.
91. Rytka JM, Wueest S, Schoenle EJ, Konrad D. 2011. The portal theory supported by
venous drainage-selective fat transplantation. Diabetes. 60: 56–63.
92. Tran TT, Yamamoto Y, Gesta S, Kahn CR. 2007. Transplantation of subcutaneous fat to
the visceral cavity induced protective metabolic effects: Evidence for intrinsic proper-
ties of subcutaneous fat. Diabetes. 56: A5–A6.
93. Thorne A, Lonnqvist F, Apelman J, Hellers G, Arner P. 2002. A pilot study of long-term
effects of a novel obesity treatment: Omentectomy in connection with adjustable gastric
banding. Int J Obes Relat Metab Disord. 26: 193–199.
94. Herrera MF, Pantoja JP, Velazquez-Fernandez D, Cabiedes J, Aguilar-Salinas C, Garcia-
Garcia E et al. 2010. Potential additional effect of omentectomy on metabolic syndrome,
acute-phase reactants, and inflammatory mediators in grade III obese patients under-
going laparoscopic Roux-en-Y gastric bypass: A randomized trial. Diabetes Care. 33:
1413–1418.
95. Csendes A, Maluenda F, Burgos AM. 2009. A prospective randomized study comparing
patients with morbid obesity submitted to laparotomic gastric bypass with or without
omentectomy. Obes Surg. 19: 490–494.
96. Klein S, Fontana L, Young VL, Coggan AR, Kilo C, Patterson BW et al. 2004. Absence
of an effect of liposuction on insulin action and risk factors for coronary heart disease.
N Engl J Med. 350: 2549–2557.
97. Chaston TB, Dixon JB. 2008. Factors associated with percent change in visceral versus
subcutaneous abdominal fat during weight loss: Findings from a systematic review. Int
J Obes (Lond). 32: 619–628.
98. Snijder MB, Van Dam RM, Visser M, Seidell JC. 2006. What aspects of body fat are
particularly hazardous and how do we measure them? Int J Epidemiol. 35: 83–92.
99. Gray DS, Fujioka K. 1991. Use of relative weight and body mass index for the determi-
nation of adiposity. J Clin Epidemiol. 44: 545–550.
100. National Institutes of Health, National Heart, Lung, and Blood Institute. 1998. Clinical
guidelines on the identification, evaluation, and treatment of overweight and obesity in
adults—The evidence report. Obes Res. 6(Suppl. 2): 51S–209S.
101. Calle EE, Thun MJ, Petrelli JM, Rodriguez C, Heath Jr CW. 1999. Body-mass index and
mortality in a prospective cohort of U.S. adults. N Engl J Med. 341: 1097–1105.
102. Lapidus L, Bengtsson C, Larsson B et al. 1984. Distribution of adipose tissue and risk of
cardiovascular disease and death: A 12 year follow up of participants in the population
study of women in Gothenburg, Sweden. Br Med J (Clin Res Ed). 289: 1257–1261.
103. Jensen MD, Kanaley JA, Reed JE, Sheedy PF. 1995. Measurement of abdominal and
visceral fat with computed tomography and dual-energy x-ray absorptiometry. Am J
Clin Nutr. 61: 274–278.
104. Rosenzweig JL, Ferrannini E, Grundy SM, Haffner SM, Heine RJ, Horton ES et al.
2008. Primary prevention of cardiovascular disease and type 2 diabetes in patients at
metabolic risk: An endocrine society clinical practice guideline. J Clin Endocrinol
Metab. 93: 3671–3689.
105. National Heart, Lung, and Blood Institute. 2000. The Practical Guide to the Identification,
Evaluation, and Treatment of Overweight and Obesity in Adults. Bethesda, MD: National
Heart, Lung, and Blood Institute.
106. U.S. Department of Health and Human Services, Public Health Service. 1996. NHANES
III Anthropometric Procedures Video. Washington, DC: U.S. Government Printing
Office.
107. Okosun IS, Chandra KM, Boev A, Boltri JM, Choi ST, Parish DC et al. 2004. Abdominal
adiposity in U.S. adults: Prevalence and trends, 1960–2000. Prev Med. 39: 197–206.
9 Type 2 Diabetes and
Inflammation
CONTENTS
Introduction............................................................................................................. 141
Cytokines and Inflammation in Diabetes................................................................ 142
Dietary Factors in Inflammation............................................................................. 143
Cellular and Subcellular Mechanisms of Inflammation in Diabetes...................... 144
References............................................................................................................... 146
INTRODUCTION
Over the past 50 years, the understanding of type 1 and type 2 diabetes has changed
drastically. In the 1970s, medical schools were teaching that type 2 diabetes was due
to the aging of the beta cell somehow and that it was otherwise analogous to type 1
diabetes where the most important element would be the control of blood sugar. With
the discovery of the immune actions of adipocytes in abdominal obesity, the role of
inflammation in type 2 diabetes mellitus (T2DM) became evident as discussed in
this chapter.
Type 1 diabetes mellitus (T1DM) is understood as being due to immune
destruction of the beta cells in early life. However, there is some overlap between
these two types of diabetes [1]. Antibodies suggesting autoimmune reaction to islet
cells are found in up to 15% of subjects in the UK Prospective Diabetes Study
(UKPDS) of subjects with T2DM. Autoantibodies to the enzyme glutamic acid
decarboxylase (GAD) and cytoplasmic islet cell antibodies (ICA) were associated
with the amount of insulin required as compared with patients not carrying these
autoantibodies. While the idea that autoimmunity may explain a subset of T2DM
patients as large as the entire population of T1DM in the United States (about two
million individuals) holds open the possibility that immunomodulatory therapeu-
tic strategies could be instituted early in a subset of patients diagnosed as having
T2DM, which could conceivably delay the progression to insulin-requiring status
over time during which systemic inflammation is relentlessly destroying beta cells
in the pancreas.
The worldwide epidemics of obesity and diabetes follow similar demographic
patterns with the largest increases in the next 30 years predicted in China and
India [2].
141
The increased prevalence of T2DM has been attributed to economic and environ-
mental changes that promote a Western diet and sedentary lifestyle leading to excess
adiposity. As discussed elsewhere in this text, inflammation has been proposed as
an underlying pathophysiological mechanism in metabolic syndrome, type 2 dia-
betes mellitus, and many obesity-associated chronic diseases including cardiovas-
cular diseases [3].
CYTOKINES AND INFLAMMATION IN DIABETES

Hotamisiligil et al. showed that tumor necrosis factor α (TNF-α) was produced by
adipose cells and could induce insulin resistance in animal models [4]. Blocking
TNF-α action improved insulin resistance. There have been numerous studies asso-
ciating increased markers of inflammation in obese patients with type 2 diabetes
compared to healthy individuals [5]. The various tissues in the body are affected dif-
ferently in diabetes by inflammation. First, adipose cells are both a source of inflam-
matory markers and a target of inflammation. Abdominal adipocytes are the site for
production of bioactive substances including TNF-α, interleukin IL-1, IL-6, IL-10,
leptin, adiponectin, monocyte chemoattractant protein-1, resistin, angiotensinogen,
visfatin, retinol-binding protein-4, serum amyloid protein, and many others [5]. As
discussed elsewhere in great detail in Chapter 8, adipose tissue expansion in obesity
is associated with angiogenesis that results in poor oxygen delivery, adipose cell
death, ingress of macrophages, and the establishment of systemic chronic low-grade
inflammation. Hypoxia, adipocyte cell death, and increased secretion of chemo-
kines and adipokines mediate the ingress of immune cells to the abdominal adipose
depot. Both adaptive and innate immunity are active in adipose tissue inflammation.
Among macrophages, there is a shift from anti-inflammatory M2-type macrophages
to proinflammatory M1-type macrophages in the process of innate immune activa-
tion by signals coming from dying adipocytes [6]. The cytokines and chemokines
produced are a pathophysiological link between obesity and insulin resistance lead-
ing to exhaustion of beta cells and the development of T2DM (Figure 9.1) [6,7].
Interleukin 1 (IL-1) has been implicated in the destruction of beta cells in the pan-
creas [8,9]. It remains to be established whether the observed inflammatory response
is due to an auto-immune process [9], glucotoxicity [8], circulating adipokines, or
accumulation of insulin-associated polypeptide leading to beta-cell inflammatory
destruction as proposed by Butler and coworkers [10].
In T2DM, pancreatic islets are characterized by a deficit in β-cells, increased
β-cell apoptosis, and extracellular amyloid deposits derived from islet amyloid poly-
peptide (IAPP). Although insulin resistance is a risk factor for T2DM, most indi-
viduals who are insulin-resistant do not develop diabetes. By inference, an increased
β-cell workload results in T2DM in some, but not all, individuals. Butler has sug-
gested that the amount of beta-cell mass that develops during childhood may under-
lie subsequent successful or failed adaptation to insulin resistance in later life [10].
Individuals with a low islet beta-cell mass would be less able to accommodate to
insulin resistance, which requires insulin and IAPP biosynthesis that exceeds the
cellular capacity for protein folding and trafficking. When this occurs, intracellular
Type 2 Diabetes and Inflammation 143
Genetically modified environmental factors

Decreased physical activity, inadequate nutrition, obesity, and infection
Signal (ROS, fatty acids, AGES, etc.)
Cells—macrophages,
PRRs
endothelium, adipocytes
NF-κB
Chronic complications of Nucleus

type 2 diabetes
(atherosclerosis and Pathogenesis of
dyslipidemia) type 2 diabetes
Skeletal muscle—
Blood—clotting insulin resistance
CRP, fibrinogen IL-1β, TNF-α
Endothelium—permeability Cytokines Apoptosis of pancreatic β-cells—
VCAM-1, ICAM-1 impaired insulin secretion
IL-1β, TNF-α
Liver—APPs, glucose
output, free fatty acids Adipose tissue—
IL-6, IL-1β insulin resistance
IL-6, TNF-α
FIGURE 9.1 (See color insert.) Innate immunity and type T2DM. Cell components of
the innate immune system, such as macrophages, endothelial cells, and adipocytes detect,
through pattern-recognition receptors (PRRs), potential environmental threats to the host,
which are represented by signals such as reactive oxygen species (ROS), fatty acids, and
advanced glycation end products (AGES). This process activates nuclear transcription fac-
tors, such as nuclear factor-kappa B (NF-κB), which induce immune inflammatory genes,
which in turn cause the release of cytokines. These cytokines act in many cells in the body
to produce the clinical and biochemical features of type 2 diabetes and its chronic complica-
tions. APPs, acute-phase proteins; CRP, C-reactive protein; IL, interleukin; TNF-α, tissue
necrosis factor alpha; VCAM-1, vascular cell adhesion molecule 1; ICAM-1, vascular endo-
thelial growth factor expression of intercellular adhesion molecule 1. (From Santos-Tunes, R.
et al., J. Can. Dent. Assoc., 76, a35, 2010. With permission.)
toxic IAPP membrane-permeant oligomers (cylindrins) form, compromising β-cell

function and inducing β-cell apoptosis.
Inflammation has also been demonstrated in other tissues and organs, including
the liver [11], hypothalamus [12], and skeletal muscle [13]. The role of inflammation
in the kidney is discussed in Chapter 11 and in muscle in Chapter 15.
DIETARY FACTORS IN INFLAMMATION

High-fat/high-sugar diets characteristic of a Western dietary pattern can cause
changes in the gut microbiota favoring the development of gram-negative bacteria,
which can trigger systemic inflammation via increased production of lipopolysac-
charide (LPS) and/or induction of periodontitis [14]. These diets could also result in
the translocation of live gram-negative bacteria from the gut to adipose tissue [15].
Both saturated fatty acids and n-6 fatty acids can have proinflammatory effects [16],
while n-3 fatty acids can oppose these effects and balance immune function [17].
Periodontitis is a very common low-grade infection related both to Western diets
and to T2DM as well as cardiovascular disease [18,19]. The characteristics of the
mouth flora, especially Streptococcus mutans, which produces acid from glucose,
have been implicated in the etiology of dental caries and periodontal disease. The
gut microflora impacted by a Western diet also can affect host metabolism and
immune function [20,21]. LPS, a highly inflammatory component of the cell wall
of the gram-negative bacteria, has been suggested as a causal link between gut
microflora and systemic low-grade inflammation leading to obesity and diabetes
mellitus (Figure 9.2) [22].
CELLULAR AND SUBCELLULAR MECHANISMS

OF INFLAMMATION IN DIABETES
Insulin, via binding to its membrane receptor, triggers the phosphorylation of several
intracellular docking proteins including insulin receptor substrates 1 and 2 (IRS-1
and IRS-2), Src homology collagen, and associated protein substrate. IRS proteins
are the major and most investigated proteins involved in subcellular signaling. IRS
activates several pathways including the phosphatidylinositol-3 kinase (PI3-K). This
pathway is involved in the regulation of glucose uptake and metabolism, protein syn-
thesis, gene expression, cell survival, growth, development, and differentiation [23].
The PI3-K pathway activates serine/threonine kinases, such as protein kinase C,
glycogen synthase kinase-3, and protein kinase B (PKB, also known as Akt). In insu-
lin resistance, the target tissues of insulin action such as the liver, muscle, and adi-
pose tissue are unable to respond appropriately to insulin. The precise mechanisms
involved in insulin resistance include, among other factors, the increasing levels of
free fatty acid, oxidative stress, altered gene expression, mitochondrial dysfunction,
and subclinical chronic inflammation. It is not likely, as it was once believed, that all
aspects could be explained by alterations in receptor binding.
Clearly, low-grade inflammation affects the action of insulin in cells of target
tissues. Several cell receptors have been identified as the sensors of inflammation
including Toll-like receptors (TLRs), receptors for advanced glycation end prod-
ucts (RAGE), and the nucleotide oligomerization domain (NOD), which links to an
inflammasome. Among the TLRs, TLR4 and TLR2 have been extensively studied
[24]. They activate the JNK/IKK NF-κB pathways and the inflammatory pathways
downstream. Both JNK and IKK can inhibit insulin action by phosphorylating the
serine residues on IRS proteins, therefore blocking the phosphorylation of IRS on
tyrosine residues subsequent to activation by insulin receptors [2,25]. Furthermore,
phosphorylation on serine/threonine residues also increases IRS degradation, fur-
ther increasing insulin resistance [25,26]. In addition, the suppressors of cytokine
signaling (SOCS) 1 and 3, which are induced by cytokines and IL-6 in particular,
lead to ubiquitinylation and degradation of IRS protein [27]. SOCS-3 expression is
increased markedly in insulin-sensitive tissues from patients with type 2 diabetes
Periodontitis Diabetes mellitus/obesity

IL-1β, TNF-α, IL-6, IL-8, PGE2, LPS Fatty acids, lipids, AGES
PRRs
Cell
PKCs JNK IKKβ ROS

PS302
IRS-1 IκB
PS307 NF-κB
Nucleus NF-κB
Inflammatory markers
and mediators
Insulin resistance
Endothelium cells Immune cells
Adipocytes
Hepatocytes Skeletal muscle cells
FIGURE 9.2 (See color insert.) Proposed mechanism by which periodontal inflammatory
mediators may contribute to the development of insulin resistance in individuals with both
type 2 diabetes and periodontitis. The inflammatory mediators originating from periodon-
tal sources can interact systemically with lipids, free fatty acids, and advanced glycation
end products (AGES), all of which are characteristic of diabetes. This interaction induces or
perpetuates activation of the intracellular pathways, such as the I-kappa-B (IκB), I-kappa-B
kinase-β (IKKβ), nuclear factor-kappa B (NF-κβ), and the protein c-Jun N-terminal kinase
(JNK) axes, all of which are associated with insulin resistance. The activation of these
inflammatory pathways in immune cells (monocytes or macrophages), endothelium cells,
adipocytes, hepatocytes, and muscle cells promotes and contributes to an increase in the over-
all insulin resistance, which makes it difficult to achieve metabolic control in patients with
both type 2 diabetes and periodontitis. IL, interleukin; IRS-1, insulin receptor substrate-1;
LPS, lipopolysaccharide; PGE2, prostaglandin E2; PKCs, protein kinases C; PRRs, pattern-
recognition receptors; pS302 (serine-302) and pS307 (serine-307), examples of serine sites;
ROS, reactive oxygen species; TNF-α, tumor necrosis factor alpha. (From Santos-Tunes, R.
et al., J. Can. Dent. Assoc., 76, a35, 2010. With permission.)
and insulin resistance [27]. Animal studies support the role of NOD1 and NOD2 in
glucose intolerance and diabetes induced by high-fat diets [15].
Activation of several pathways including the activation of the c-Jun NH(2)-
terminal kinase (JNK) and the inhibitor of kappa-B kinase (IKK) regulate
downstream transcriptional processes through nuclear factor κB (NF-κB), therefore
amplifying the expression of proinflammatory mediators. Indeed, at the cellular and
molecular level, NF-κB has a central role in promoting the synthesis of mediators of
inflammation that act in a paracrine or endocrine fashion.
Diesel exhaust particles (DEP) less than 2.5 μm in diameter have been associ-
ated with an increased risk of diabetes occurrence and increased mortality in people
with diabetes compared with nondiabetic subjects [28,29]. Inflammation secondary
to oxidative stress has been suggested as an underlying mechanism [28,30]. We have
recently shown that broccoli sprout extract standardized for sulforaphane content
can inhibit DEP-induced nasal inflammation in atopic individuals (Heber et al.,
unpublished observations). These observations would implicate the nrf2/keap path-
way which interacts with the NF-κB pathway of inflammation.
The cellular and molecular mechanisms linking inflammation and T2DM and
related complications has stimulated interest in targeting these pathways as part of
the strategy to prevent or control diabetes mellitus and its complications [31,32].
Lifestyle interventions such as those implemented in diabetes prevention trials lower
levels of inflammatory markers most likely through reduction of abdominal fat and
improved metabolic homeostasis [33]. Salicylates (aspirin), which are nonsteroidal
anti-inflammatory drugs, have been known for over 100 years to improve metabolic
control in diabetes, but the dose necessary would have an unacceptable incidence of
serious adverse effects such as bleeding [31]. The nonacetylated form of salicylates,
which are safer, have been shown in small clinical trials to improve metabolic con-
trol in people with type 2 diabetes [34,35], suggesting possible utility for diabetes
prevention and control, a possibility currently under investigation in much larger
trials [36]. Improving gut microbiota through dietary intervention or probiotics has
been considered as a possible emerging strategy for preventing the development and
progression of diabetes mellitus [37]. In subsequent chapters, the impact of spices
and other phytochemicals with anti-inflammatory effects will be reviewed. In par-
ticular, there is good evidence that cinnamon, which is derived from tree bark and
contains cinnamic acid, has effects on insulin action in humans and may play a role
in controlling blood sugar. However, there are numerous botanicals that lower blood
sugar as do specialized fibers such as alginates.
In summary, all of these approaches targeting inflammation must be put into
proper perspective with reductions in abdominal or visceral adiposity as the primary
goal in prevention and management of the inflammation associated with diabetes
mellitus. Early intervention in patients with metabolic syndrome or hyperglycemia
may delay or prevent progression of type 2 diabetes mellitus in part by decreasing
inflammation.
REFERENCES
1. Syed MA, Barinas-Mitchell E, Pietropaolo SL et al. 2002. Is type 2 diabetes a chronic
inflammatory/autoimmune disease? Diabetes Nutr Metab. 15:68–83.
2. International Diabetes Federation. 2011. In: Unwin N, Whiting D, Guariguata L,
Ghyoot G, Gan D, eds. Updated Diabetes Atlas 2011, 5th edn. Brussels, Belgium:
International Diabetes Federation.
3. Shoelson SE, Lee J, Goldfine AB. 2006. Inflammation and insulin resistance. J Clin
Invest. 116:1793–1801.
4. Hotamisligil GS, Shargill NS, Spiegelman BM. 1993. Adipose expression of tumor
necrosis factor-alpha: Direct role in obesity-linked insulin resistance. Science.
259:87–91.
5. Marques-Vidal P, Schmid R, Bochud M et al. 2012. Adipocytokines, hepatic and inflam-
matory biomarkers and incidence of type 2 diabetes. The CoLaus Study. PLoS One.
7:e51768.
6. Sell H, Habich C, Eckel J. 2012. Adaptive immunity in obesity and insulin resistance.
Nat Rev Endocrinol. 8:709–716.
7. Nikolajczyk BS, Jagannathan-Bogdan M, Shin H, Gyurko R. 2011. State of the
union between metabolism and the immune system in type 2 diabetes. Genes Immun.
12:239–250.
8. Donath MY, Schumann DM, Faulenbach M et al. 2008. Islet inflammation in type 2
diabetes: From metabolic stress to therapy. Diabetes Care. 31(Suppl. 2):S161–S164.
9. Brooks-Worrell B, Palmer JP. 2012. Immunology in the Clinic Review Series; focus
on metabolic diseases: Development of islet autoimmune disease in type 2 diabetes
patients: Potential sequelae of chronic inflammation. Clin Exp Immunol. 167:40–46.
10. Costes S, Langen R, Gurlo T, Matveyenko AV, Butler PC. 2013. β-cell failure in type 2
diabetes: A case of asking too much of too few? Diabetes. 62:327–335.
11. Kiechl S, Wittmann J, Giaccari A et al. 2013. Blockade of receptor activator of nuclear
factor-kappaB (RANKL) signaling improves hepatic insulin resistance and prevents
development of diabetes mellitus. Nat Med. 19:358–363.
12. Cai D. 2013. Neuroinflammation in overnutrition-induced diseases. Vitam Horm.
91:195–218.
13. Varma V, Yao-Borengasser A, Rasouli N et al. 2009. Muscle inflammatory response and
insulin resistance: Synergistic interaction between macrophages and fatty acids leads to
impaired insulin action. Am J Physiol Endocrinol Metab. 296:E1300–E1310.
14. Blasco-Baque V, Serino M, Vergnes JN et al. 2012. High-fat diet induces periodontitis in
mice through lipopolysaccharides (LPS) receptor signaling: Protective action of estro-
gens. PLoS One. 7:e48220.
15. Amar J, Chabo C,Waget A et al. 2011. Intestinal mucosal adherence and translocation
of commensal bacteria at the early onset of type 2 diabetes: Molecular mechanisms and
probiotic treatment. EMBO Mol Med. 3:559–572.
16. Ebbesson SO, Tejero ME, Lopez-Alvarenga JC et al. 2010. Individual saturated fatty
acids are associated with different components of insulin resistance and glucose metab-
olism: The GOCADAN study. Int J Circumpolar Health. 69:344–351.
17. Oh DY, Talukdar S, Bae EJ et al. 2010. GPR120 is an omega-3 fatty acid receptor medi-
ating potent anti-inflammatory and insulin-sensitizing effects. Cell. 142:687–698.
18. Gurav AN. 2012. Periodontitis and insulin resistance: Casual or causal relationship?
Diabetes Metab J. 36:404–411.
19. Pradhan S, Goel K. 2011. Interrelationship between diabetes and periodontitis: A review.
J Nepal Med Assoc. 51:144–153.
20. Nicholson JK, Holmes E, Kinross J et al. 2012. Host-gut microbiota metabolic interac-
tions. Science. 336:1262–1267.
21. Hooper LV, Littman DR, Macpherson AJ. 2012. Interactions between the microbiota
and the immune system. Science. 336:1268–1273.
22. Burcelin R, Garidou L, Pomie C. 2012. Immuno-microbiota cross and talk: The new
paradigm of metabolic diseases. Semin Immunol. 24:67–74.
23. Zeyda M, Stulnig TM. 2009. Obesity, inflammation, and insulin resistance—A mini-
review. Gerontology. 55:379–386.
24. Tanti JF, Ceppo F, Jager J, Berthou F. 2012. Implication of inflammatory signaling path-
ways in obesity-induced insulin resistance. Front Endocrinol. 3:181.
25. Haruta T, Uno T, Kawahara J et al. 2000. A rapamycin-sensitive pathway down-regulates

insulin signaling via phosphorylation and proteasomal degradation of insulin receptor
substrate-1. Mol Endocrinol. 14:783–794.
26. Hiratani K, Haruta T, Tani A et al. 2005. Roles of mTOR and JNK in serine phos-
phorylation, translocation, and degradation of IRS-1. Biochem Biophys Res Commun.
335:836–842.
27. Lebrun P, Van Obberghen E. 2008. SOCS proteins causing trouble in insulin action. Acta
Physiol (Oxf). 192:29–36.
28. Rajagopalan S, Brook RD. 2012. Air pollution and type 2 diabetes: Mechanistic insights.
Diabetes. 61:3037–3045.
29. Andersen ZJ, Raaschou-Nielsen O, Ketzel M et al. 2012. Diabetes incidence and long-
term exposure to air pollution: A cohort study. Diabetes Care. 35:92–98.
30. Liu C, Ying Z, Harkema J et al. 2012. Epidemiological and experimental links between
air pollution and type 2 diabetes. Toxicol Pathol. 41:361–373.
31. Ebstein W. 2002. Invited comment on W. Ebstein: On the therapy of diabetes mellitus,
in particular on the application of sodium salicylate. J Mol Med. 80:618.
32. Hirabara SM, Gorjao R, Vinolo MA et al. 2012. Molecular targets related to inflammation
and insulin resistance and potential interventions. J Biomed Biotechnol. 2012:379024.
33. Haffner S, Temprosa M, Crandall J et al. 2005. Intensive lifestyle intervention or metfor-
min on inflammation and coagulation in participants with impaired glucose tolerance.
Diabetes. 54:1566–1572.
34. Goldfine AB, Fonseca V, Jablonski KA et al. 2010. The effects of salsalate on gly-
cemic control in patients with type 2 diabetes: A randomized trial. Ann Intern Med.
152:346–357.
35. Rumore MM, Kim KS. 2010. Potential role of salicylates in type 2 diabetes. Ann
Pharmacother. 44:1207–1221.
36. Goldfine AB, Fonseca V, Shoelson SE. 2011. Therapeutic approaches to target inflam-
mation in type 2 diabetes. Clin Chem. 57:162–167.
37. Panwar H, Rashmi HM, Batish VK, Grover S. 2013. Probiotics as the potential biothera-
peutics in the management of type 2 diabetes—Prospects and perspectives. Diabetes
Metab Res Rev. 29:103–112.
38. Santos-Tunes R, Foss-Freitas MC, Nogueira-Filho, DaR G. 2010. Impact of periodonti-
tis on the diabetes-related inflammatory status. J Can Dent Assoc. 76:a35.
10 Heart Disease and
Inflammation
Kaveh Daniel Navab
CONTENTS
Cardiovascular Health, Systemic Inflammation, Intestine, and Oxidized Lipids....... 149
LDL Oxidized Phospholipids................................................................................. 150
HDL and Prevention of Lipid Oxidation................................................................ 150
HDL Mimetic Peptides........................................................................................... 151
Inflammatory Reaction in Vascular and Nonvascular Cells
Initiated by Oxidized Lipids................................................................................... 154
Prevention of Inflammatory Reaction by HDL....................................................... 154
Dysfunctional HDL................................................................................................. 155
Small Intestine Is Important in Modulating Systemic Inflammation...................... 157
Conclusion.............................................................................................................. 158
References............................................................................................................... 159
CARDIOVASCULAR HEALTH, SYSTEMIC INFLAMMATION,

INTESTINE, AND OXIDIZED LIPIDS
During the past three decades, there has been continuing evidence indicating that
lipid oxidation plays a key role in atherosclerosis and cardiovascular diseases.
Oxidized phospholipids rise from oxidation of low-density lipoprotein (LDL) phos-
pholipids that contain arachidonic acid. These molecules are recognized by the
innate immune system in humans and in animal models. Lipoxygenase and myelo-
peroxidase pathways generate these oxylipids, and antioxidants including vitamin E
are not able to prevent their formation, partially explaining the failure of antioxidant
vitamins to influence clinical outcomes. High-density lipoprotein (HDL) is capable
of preventing lipid oxidation in many settings. The main function of HDL has been
suggested to be reverse cholesterol transport (RCT), and now the oxidation hypoth-
esis of atherogenesis and RCT seem to have common biological relations and basis.
While normal HDL is capable of preventing oxidative modification of lipids and thus
serving as an anti-inflammatory molecule, HDL from patients with atherosclerosis,
heart failure, diabetes, obesity, lupus, Crohn’s disease, HIV infection, renal failure,
and other metabolic disorders is dysfunctional and not capable of preventing lipid
oxidation.
149
LDL OXIDIZED PHOSPHOLIPIDS

For decades, a role for cell-generated reactive oxygen species in mediating LDL oxi-
dation was postulated [1–5]. The first direct proof in vivo of a role for the lipoxy-
genase pathway was provided in apoE null mice [6–8] that were made genetically
deficient in 12/15-lipoxygenase and that were found to have, as a result, significantly
less atherosclerosis. Decreased atherogenesis was observed following the deletion of
the 12/15-lipoxygenase gene in LDL receptor null mice [9] and in macrophages of
mice that were both LDL receptor null and also deficient in the apoB-editing catalytic
polypeptide-1 enzyme [10]. Conversely, overexpression of the 12/15-lipoxygenase
gene in the endothelium of LDL receptor null mice accelerated atherosclerosis [11].
A transgenic mouse was then generated [12] in a C57BL/6J background that
modestly overexpressed the murine 12/15-lipoxygenase gene. These mice had 2.5-
fold increases in the levels of 12(S)-hydroxyeicosatetraenoic acid (HETE) and a
2-fold increase in the expression of 12/15-lipoxygenase protein in vivo. These mice
developed spontaneous aortic fatty streak lesions on a chow diet, further indicating
the importance of the lipoxygenase pathway in atherogenesis. Our group found that
human artery wall cells required the 12/15-lipoxygenase gene to generate the oxi-
dized phospholipids found in mildly modified LDL (MM-LDL). Myeloperoxidase
null mice had increased, not decreased, atherosclerosis [13], but mouse macro-
phages, unlike human macrophages, have little myeloperoxidase. Products of the
myeloperoxidase reaction have been found in human atherosclerotic lesions [14], and
there is increasing evidence that the myeloperoxidase pathway can generate proin-
flammatory oxidized lipids [15–21]. Another pathway that may be a potential source
of reactive oxygen species for the generation of proinflammatory oxidized lipids is
the NADPH oxidase pathway [22,23].
HDL AND PREVENTION OF LIPID OXIDATION

Unlike vitamin E, HDL, apolipoprotein (apo) A-I, and apoA-I mimetic peptides
have been shown to prevent LDL oxidation in cell-free systems [2,3,24] and in the
artery wall coculture studies [25,26]. HDL, apoA-I, and apoA-I mimetics have addi-
tionally been shown to decrease lesions and improve vascular reactivity in animal
models of atherosclerosis [27–34] and in humans [35–37]. As stated, the mechanism
by which HDL and apoA-I and apoA-I mimetic peptides exert their beneficial effect
has been presumed to primarily be the enhancement of RCT [29,38–40]. ApoA-I,
however, has also been shown to be capable of removing seeding molecules from
LDL, thus preventing the oxidation of LDL-derived phospholipids to those that are
thought to be responsible for the inflammatory response characteristic of atheroscle-
rosis [25,26]. A subpopulation of freshly isolated LDL was reported [41] to contain
lipid hydroperoxides. Our group [26] found that freshly isolated LDL from normal
individuals always contained small amounts of lipoxygenase pathway products (e.g.,
HPODE and HPETE). These were present even when blood was collected into tubes
containing potent antioxidants. The levels of HPODE and HPETE did not increase
during in vitro incubations in the presence of these antioxidants, indicating that they
were present in LDL in vivo. However, when the freshly isolated LDL was incubated
Heart Disease and Inflammation 151
with apoA-I in the presence of antioxidants and the LDL and the apoA-I were then
rapidly separated, the LDL treated with apoA-I contained only approximately one-
third to one-half as much HPODE and HPETE as was present initially. Before these
incubations, the apoA-I contained no detectable HPODE or HPETE, but after the
incubation with LDL, one-half to two-thirds of the HPODE and HPETE that had
been present in the LDL were transferred to the apoA-I, along with some cholesterol
and phospholipid. The LDL treated with apoA-I was able to neither generate lipid
hydroperoxides nor induce monocyte adherence or monocyte chemotactic activity
when added to human artery wall cocultures. If the apoA-I that was incubated with
the LDL was subjected to lipid extraction and the extracted lipids were added back to
the LDL that had been treated with apoA-I, the reconstituted LDL was able to induce
lipid hydroperoxide formation and induce monocyte adherence and monocyte che-
motactic activity [26].
Consistent with these properties of apoA-I, it was reported [42] that HDL is a
major carrier of lipid hydroperoxides in humans. HDL appears to be the major car-
rier of lipid hydroperoxides in mice, and the concentration of lipid hydroperoxides in
HDL taken from the atherosclerosis-susceptible C57BL/6J mice either on a low-fat
chow diet or on an atherogenic diet was significantly greater than the lipid hydro-
peroxide levels found in the HDL of the atherosclerosis-resistant C3H/HeJ mice.
Six hours after the injection of human apoA-I into the tail veins of C57BL/6J mice,
their LDL was no longer able to induce lipid hydroperoxide formation or monocyte
chemotactic activity in human artery wall cocultures [26]. In contrast, injection of
human apoA-II or saline did not prevent LDL-mediated induction of lipid hydro-
peroxide formation or LDL-induced monocyte chemotactic activity. Similarly, 6 h
after infusing apoA-I and phospholipid into healthy human volunteers, there was a
dramatic decrease in the ability of their LDL to induce lipid hydroperoxide forma-
tion and monocyte chemotactic activity in the cocultures of six of six subjects. Thus,
apoA-I has the ability to remove HPODE and HPETE from human LDL and to dra-
matically reduce the inflammatory properties of LDL in both mice and humans [26].
HDL MIMETIC PEPTIDES

Studies to explain how HDL influences atherogenesis led to the realization that it
had a major role in providing antioxidant properties to dampen the proinflamma-
tory properties of oxidized LDL [26]. In turn, this led to the development of apoA-I
mimetic peptides such as the 18 amino acid peptide 4F made by our group and other
peptides made by various groups [43]. Subsequent studies showed that not only was
4F peptide able to inhibit atherosclerosis, but it potently inhibited inflammation in a
surprisingly wide variety of animal models of disease [43].
In human studies [44], 4F peptide was administered orally at doses from 0.43 to
7.14 mg/kg. The 4F plasma levels achieved were very low (Cmax 15 ng/mL). However,
peptide administration at doses of 4.3 and 7.14 mg/kg significantly improved the
HDL inflammatory index, whereas doses of 0.43 and 1.43 mg/kg did not [44]. In a
second clinical trial, it was decided to achieve high plasma levels of peptide by using
low doses (0.04–1.4 mg/kg) of peptide administered intravenously or subcutane-
ously. HDL inflammatory index is a functional assay developed by our group where
LDL is added to artery wall cells in culture and allowed to oxidize and generate
monocyte chemotactic activity. Presence of normal HDL prevents this while adding
patient HDL amplifies it. Despite achieving very high plasma levels of peptide, there
was no improvement in HDL inflammatory index [45]. This led to a reappraisal of
why peptide was so effective in mice and led to the surprising discovery that a major
site of action for the 4F peptide may be in the intestine, even when it is administered
subcutaneously [46].
Based on the information generated, we have developed several hypotheses.
Hypothesis 1 is that the oxidation of lipids by nonenzymatic means or by meta-
bolic pathways produces oxidized lipids, which trigger an inflammatory response in
many tissues including the vasculature and in the small intestine. In hypothesis 2,
we think metabolites of arachidonic acid, such as 12-HETE, can act similar to oxi-
dized phospholipids (Ox-PLs) to induce inflammation. The basis of our hypothesis 3
is that HDL contains proteins and enzymes that can inactivate and/or remove these
proinflammatory lipids, but in some circumstances, such as a systemic acute-phase
response, the proteins and enzymes associated with HDL are altered so that the
inflammatory response is either not inhibited or enhanced. In hypothesis 4, we pro-
pose that apoA-I mimetic peptides, such as 4F, reduce inflammation by binding and
removing oxidized lipids from tissues. And finally in hypothesis 5, we have come to
observe that oxidized lipids in the small intestine are important in modulating sys-
temic inflammation, and the intestine is a major site for the action of apoA-I mimetic
peptides, such as 4F, which bind these oxidized lipids.
As was stated earlier, oxidized lipids are found in the vasculature of animal models
of atherosclerosis, in human atherosclerotic lesions, and in other inflammatory
diseases. It has been clearly shown that phospholipids are the source of substrates
for multiple enzymatic pathways and an integral component of all mammalian
membranes. The role of phospholipid oxidation products in atherosclerosis has
recently been reviewed. Our group [47,48] demonstrated by liquid chromatography–
electrospray ionization/multistage mass spectrometry that Ox-PLs were present in
fatty streaks from cholesterol-fed rabbits and in lesions of apolipoprotein E–null
mice [47]. We also demonstrated [48] that the group at the sn-2 position of Ox-PL
determines the specific bioactivity and that the substitution of stearoyl for palmitoyl
at the sn-1 position or ethanolamine for choline at the sn-3 position of the phospho-
lipid did not alter bioactivity. We further showed that all parts of the phospholipid
molecules are required for these bioactivities.
The binding of oxidized LDL to the scavenger receptor CD36 in mice was
demonstrated to be attributable to Ox-PL that were associated with both lipid and
protein moieties of the lipoprotein. It was demonstrated that a variety of Ox-PLs
beyond those described earlier [47,48] are present in lesions and that they inter-
act with CD36 in the mouse [49]. A simple phospholipid, such as 1-palmitoyl-2-
arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC), when air oxidized produces
hundreds of compounds [47], and hence it is not surprising that there are a myriad
of Ox-PL found in nature.
It has been well demonstrated that oxidized lipids can initiate an inflamma-
tory response and are also formed in an inflammatory reaction. To understand the
sequence of events, the time course of the appearance of Ox-PL and monocytes in
aortas of human fetuses was followed [50]. It was found that the presence of Ox-PL
preceded the appearance of the monocytes [51]. The findings of other groups [52]
highlight the importance of Ox-PL to human disease. The vulnerability of plaques
was found to be related to the amount of LDL containing oxidized phosphatidylcho-
line in the lesion. After percutaneous angioplasty, there was a dramatic increase in
plasma levels of Ox-PL confirming the presence of Ox-PL in clinically important
lesions in humans [53]. Cardiolipin is found in bacteria, in the inner membrane of
mitochondria, and in LDL. A natural antibody to oxidized cardiolipin bound to oxi-
dized LDL, apoptotic cells, and atherosclerotic lesions has been isolated that did not
recognize native cardiolipin or native LDL, confirming that the oxidation of phos-
pholipids occurs in inflammatory conditions in vivo in rabbits and humans.
In monkey and rabbit models of atherosclerosis, it was demonstrated (54) that
during regression of lesions, Ox-PL increased in plasma and decreased in lesions,
consistent with the findings of this group in humans [53]. Interestingly, it was
found [55] that Ox-PL in human plasma is largely associated with Lp(a) lipopro-
tein and is strongly associated with angiographically documented coronary artery
disease (CAD), particularly in patients 60 years of age or younger. Ox-PL was also
shown [56] to be present in plasminogen, which is homologous to Lp(a), and affects
fibrinolysis.
It is not known precisely how diet-induced inflammation produces Ox-PL, but
still the process seems to be widespread in nature. In one study, it was demonstrated
[56,57] that Ox-PL accumulated in lesions induced by cholesterol-feeding zebra fish
larvae.
More recent work shows that the presence of Ox-PL at sites of inflammation is
not restricted to atherosclerosis. For example, Ox-PL was found [58] in the lungs
of human and animals infected with severe acute respiratory syndrome (SARS),
anthrax, or H5N1. Furthermore, pulmonary challenge with inactivated H5N1 avian
influenza virus rapidly induced acute lung injury and Ox-PL formation in mice
[58]. Consistent with these findings, after influenza infection, it was shown [59] that
interleukin-17RA–null mice had markedly better survival and less Ox-PL formation
in the lungs. Also in mice that are genetically prone to polyp formation and colon
cancer, Ox-PL has been found in the mucosa of the small intestine [60]. Ox-PL has
been found in skin lesions of patients with leprosy [61] and in brain lesions of patients
with multiple sclerosis [62]. Additionally, Ox-PL have been found in patients with
nonalcoholic fatty liver disease, and Ox-PL levels correlated with disease severity
in humans [63]. Administration of the apoA-I mimetic peptide 4F known to bind
Ox-PL with extraordinarily high affinity [64] significantly reduced hepatic fibrosis in
a mouse model of this disease [65]. The existence of Ox-PL in human eyes was seen
to increase with age and was increased in the eyes of patients with age-related macu-
lar degeneration [66]. The hearts in a mouse model of scleroderma contained higher
levels of antibody to Ox-PL as compared to controls, and with the administration of
the 4F peptide, the tissue levels of these antibodies decreased [67].
The presence of Ox-PL in a wide range of inflammatory conditions in species rang-
ing from zebra fish to humans is consistent with hypothesis 1 proposed in this review.
Some of the studies cited in this section are also consistent with hypothesis 4 pro-
posed in this review.
INFLAMMATORY REACTION IN VASCULAR AND

NONVASCULAR CELLS INITIATED BY OXIDIZED LIPIDS
Using a mixture of Ox-PL made by air oxidation of PAPC to stimulate human aortic
endothelial cells (ECs) in culture will cause them to bind monocytes and results
in the cells secreting cytokines, including the potent monocyte chemoattractant
factor monocyte chemotactic protein 1 [1]. Interestingly, individual components
of air-oxidized PAPC have different effects in vitro. For example, 1-palmitoyl-2-
(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine only stimulates monocyte binding
and strongly inhibits lipopolysaccharide-mediated induction of neutrophil binding
and the expression of E-selectin protein and mRNA [68], whereas 1-palmitoyl-2-
glutaroyl-sn-glycero-3-phosphorylcholine stimulates ECs to bind both neutrophils
and monocytes. In rabbits, the extent of vascular inflammation was seen to highly
correlate with the content of oxidized fatty acids, malondialdehyde, and Ox-PL in
lesions [69]. It was additionally shown that the levels of oxidized fatty acids derived
from oleic acid, linoleic acid, and arachidonic acid in plasma also paralleled well
with levels in lesions [69].
When Ox-PL was directly applied to carotid arteries in mice [70], the arteries
responded by inducing a set of atherosclerosis-related genes including tissue fac-
tor, interleukin 6, heme oxygenase 1, monocyte chemotactic protein 1, keratinocyte-
derived chemokine, and early growth response 1. Ox-PL triggered rolling and firm
adhesion of monocytes in a P-selectin and keratinocyte-derived chemokine-dependent
manner in isolated perfused carotid arteries [71]. The number of compounds that can
be generated by enzymatic pathways, such as the lipoxygenase pathways, is dramati-
cally increased by the crossover of multiple pathways and the ability of the lipids that
are formed to undergo nonenzymatic rearrangements [72–75]. The ability of some
enzyme systems to oxidize fatty acids to biologically active molecules while they are
still esterified to cholesterol or phospholipids further increases the complexity [76,77].
The number of biologically active oxidized lipids present in nature is very large.
The mechanism(s) by which an inflammatory response is initiated in cells by
Ox-PL is complex [78–87]. Ox-PLs appear to have oxidation-specific epitopes,
which are recognized as danger-associated molecular patterns by pattern recognition
receptors involved in innate immunity [88]. The stress response induced by Ox-PL
can be modulated by antioxidant enzymes [89]. Significantly, alteration in the pheno-
type of macrophages has been shown to occur by Ox-PL accounting for some of the
characteristics of macrophages that have been noted in atherosclerotic lesions [90].
Both hypothesis 1 and hypothesis 2 of this review therefore seem to be strengthened
by evidence on the inflammatory response of vascular cells in vitro and in vivo to a
variety of oxidized lipids and the striking similarity between the response to Ox-PL
and to metabolites of arachidonic acid, the so-called lipid mediators.
PREVENTION OF INFLAMMATORY REACTION BY HDL

Inflammatory reaction characteristics of atherosclerosis can occur by normal HDL,
normal ApoA-I, and ApoA-I mimetic peptides. This is associated with decreased
levels of oxidized lipids. Multiple components of HDL play a role in preventing
inflammatory reactions initiated by oxidized lipids [25,91]. Formation and secre-

tion of Ox-PL in response to influenza A infection was demonstrated that an apoA-I
mimetic peptide (4F) prevented in vitro studies [92]. In vivo in mice, 4F prevented
the trafficking of macrophages into the aorta in response to influenza A infection
[32]. In additional studies, normal human HDL modulated the proinflammatory
response of cultured human aortic EC to Ox-PL to a signaling cascade that was anti-
inflammatory [93].
An HDL-associated enzyme, paraoxonase 1 (PON1), inhibits the response of cul-
tured ECs to Ox-PL. Interestingly, the delivery of paraoxonase into arteries in vivo
inhibited the response to balloon injury [94]. Ox-PL was shown to be bound by
apoM, another component of normal HDL, and that increased the antioxidant effect
of HDL [95].
Induction of the formation of Ox-PL in the kidneys of LDL receptor null mice
was observed following feeding a Western diet (WD), which was accompanied by
inflammatory changes similar to those in the arteries of these mice [96]. Peptide 4F
treatment significantly reduced the inflammation in the tissues without changing
plasma lipid levels and decreased tissue levels of Ox-PL [96].
The plasma and lesion content of oxidized fatty acids was stated earlier to parallel
one another [69]. In mouse models of atherosclerosis, administration of the 4F
peptide resulted in more anti-inflammatory HDL and reduced the plasma levels of
oxidized fatty acids [97]. The observation that the work of apoA-I mimetic pep-
tides involves binding and removing Ox-PL additionally comes from the finding that
their use induces natural antibodies that recognize Ox-PL [98,99]. This seems to be
consistent with the plasma increase in Ox-PL that has been seen in animal models
of regression [53]. Hypothesis 3 and hypothesis 4 are supported by the earlier-
mentioned studies and data.
DYSFUNCTIONAL HDL
Inflammatory reaction characteristic of atherosclerosis is not prevented by HDL
obtained from animal models of atherosclerosis or from humans with atheroscle-
rosis or from mammals with other chronic inflammatory diseases, or HDL with
apoA-I modified by-products found in inflammatory reactions. Dysfunctional HDL
may even enhance inflammatory reaction.
We reported [100] that anti-inflammatory HDL becomes proinflammatory in rab-
bits and humans during an acute-phase response. Subsequently, we reported [101]
that injection of Ox-PL into mice genetically susceptible to diet-induced athero-
sclerosis (C57BL/6J mice), but not in mice resistant to diet-induced atherosclerosis
(C3H/HeJ mice), induced an acute-phase response with decreased PON1 activity
and elevations of apoJ.
Feeding an atherogenic diet to LDLR−/− mice for 3 days [102] did not decrease
hepatic PON1 mRNA but caused a dramatic decrease in plasma PON1 activity and
mass. There was a temporal relation between the decreased activity and mass of para-
oxonase and the increase in the lipid hydroperoxide content of HDL with a decrease
in HDL-cholesterol, native apoA-I, and apoA-II levels. Higher-molecular-weight
forms of apoA-I appeared as the native apoA-I disappeared from the circulation.
Some of these apoA-I species contained epitopes recognized by an antibody that

recognizes Ox-PL (EO6) [102].
Following an exposure of mice to secondhand cigarette smoke, the ability of HDL
to prevent the formation of Ox-PL was found to be reduced [103]. We have reported
the genetic control of the anti-inflammatory properties of HDL in mice and the
inverse relationship of these anti-inflammatory properties in humans with the ability
of HDL to promote cholesterol efflux from cholesterol-loaded macrophages [104].
When HDL was prepared from the plasma of patients with CAD, many acute-
phase proteins were associated with it [105]. This supported the idea that HDL is
changed in the presence of chronic inflammation. There was a favorable modifica-
tion in the effects of chronic inflammation on HDL in humans with a regimen of
combined niacin and statin treatment [106].
In a variety of inflammatory states, abnormalities in HDL have been identified
including patients with systemic lupus erythematosus [107], rheumatoid arthritis
[108], and diabetes mellitus [109,110]. The importance of HDL subpopulations has
been emphasized [111]. In humans, there is a strong relationship among the activity
of the HDL-associated enzyme PON1, systemic oxidative stress, and cardiovascular
risk [112]. Moreover, in these patients, the levels of metabolites of arachidonic acid
strongly tracked with PON1 activity and the risk for cardiovascular events [112].
In one study, modification of HDL by the enzyme myeloperoxidase released
from macrophages and neutrophils during inflammation generates a proinflam-
matory HDL particle [113]. Hemoglobin is a potent oxidant when freed from red
blood cells, and haptoglobin is an acute-phase reactant that was associated with
HDL in CAD patients. The levels of both molecules significantly predicted the
inflammatory properties and function of their HDL [114]. HDL from patients with
rheumatoid arthritis, another chronic inflammatory condition, was associated with
complement factors and acute-phase proteins. This is similar to that observed in
patients with CAD [115].
Cholesterol efflux by the ABCA1 pathway was blocked following modification
of apoA-I with malondialdehyde [116]. Moreover, HDL isolated from atheroscle-
rotic lesions contained more malondialdehyde than normal HDL [117]. Interestingly,
HDL from patients with an acute coronary syndrome or stable CAD did not have
anti-inflammatory properties [117] when presented to cultured ECs. Furthermore,
this abnormal HDL did not stimulate EC repair because it failed to induce endo-
thelial NO production. (a) HDL from these subjects activated endothelial lectin-like
oxidized LDL receptor; (b) this triggered endothelial protein kinase C βII activation.
(c) This in turn inhibited endothelial NO-activating pathways and NO production.
Reduced PON1 activity was considered to be a molecular mechanism leading to the
generation of HDL with protein kinase C βII–activating properties. This was in part
attributable to increased formation of malondialdehyde in HDL [117].
The importance of HDL in promoting cholesterol efflux independent of HDL-
cholesterol levels is well recognized [118]. This property was suggested [118] to be
a possible therapeutic target. Disassociation of apoA-I from HDL to the lipid-poor
form of apoA-I that is critical for promoting cholesterol efflux via ATP-binding cas-
sette A1 was inhibited by oxidative damage to apoA-I [119,120].
Thus, (a) modification by lipid oxidation products and enzymes produced or

released at sites of inflammation is capable of rendering HDL and its associated
proteins dysfunctional; (b) during an acute-phase response, the proteins and enzymes
associated with HDL are significantly changed to a proinflammatory phenotype; and
(c) during a chronic acute-phase reaction such as that seen in CAD patients, the ability
of HDL to be anti-inflammatory and to promote cholesterol efflux is dramatically
reduced. The data presented in this section are consistent with hypothesis 3 proposed
earlier.
SMALL INTESTINE IS IMPORTANT IN

MODULATING SYSTEMIC INFLAMMATION
Approximately 30% of the steady-state plasma HDL-cholesterol pool is derived
from the small intestine in mice [121]. A similar fraction of HDL in humans comes
from the small intestine as seen in studies in chyluric humans [122]. In both mice
and humans, it has been recently implicated that the metabolism of phospholipids
by gut bacteria can affect atherogenesis [123]. It is currently unknown what level
of oxidized lipids, phospholipids, and fatty acids is present in animals treated with
broad-spectrum antibiotics or in germ-free animals.
Mouse models of atherosclerosis studies with the 4F peptide suggested that small
intestine is a major tissue regulating systemic inflammation. The intestine therefore
might be an important site for determining the functionality of HDL [124]. To test
this hypothesis, our group administered 4F peptide at equal doses by subcutaneous
injection (subcutaneous) or orally to LDLR−/− mice on a WD. Peptide levels were
approximately 300-fold and 100-fold higher in plasma and liver, respectively, after
subcutaneous administration, whereas peptide levels in the intestine varied only by
1.7-fold. There was a significant reduction in the levels of metabolites of arachi-
donic and linoleic acids (known to bind with high affinity to the peptide) in intestine,
liver, and hepatic bile. The reduction occurred to a similar degree whether the pep-
tide was administered subcutaneously or orally. Levels of 20-HETE, however, were
unchanged. This metabolite is known to bind to the 4F peptide with low affinity.
Serum amyloid A and triglyceride levels were reduced and HDL-cholesterol levels
increased similarly after subcutaneous or oral administration of the peptide. Levels
of metabolites of arachidonic and linoleic acids in plasma significantly correlated
with serum amyloid A levels. Feeding mouse chow without the WD with the chow
containing metabolites of arachidonic acid (e.g., 12- or 15-HETE) resulted in sig-
nificantly increased plasma triglyceride and serum amyloid A levels and decreased
HDL-cholesterol and PON1 activity. Subcutaneous administration of the 4F peptide
ameliorated all of the changes [124]. Feeding LDLR−/− mice a WD in these studies
resulted in increased levels of free arachidonic acid. This suggests that phospholi-
pase activity was increased by WD. Consistent with a reduction in phospholipase
activity, treatment with the 4F peptide reduced hepatic and enterocyte levels of free
arachidonic acid [124]. The levels of free arachidonic acid were interestingly similar
in enterocytes from the small intestine and in the liver of these mice. The levels of
free metabolites of arachidonic and linoleic acids (except for 20-HETE), however,
were many fold greater in the enterocytes of the small intestine compared to hepatic
levels. The significance of intestinal phospholipase activity in regulating the metabo-
lism of arachidonic acid was demonstrated by studies of patients with an inherited
cytosolic phospholipase A2-α deficiency. These patients have been shown to have
impaired eicosanoid biosynthesis and small intestine ulceration [125]. Cytosolic
phospholipase A2 was shown to be protective against cyclooxygenase inhibitor-
induced intestinal damage [126]. Cytosolic 12-lipoxygenase and phospholipase A2
mediate monocyte adhesion to ECs in response to Ox-PL [71]. Deletion of endothe-
lial-specific cyclooxygenase-2 in mice was shown [127] to result in intestinal inflam-
mation similar to Crohn’s disease. Patients with Crohn’s disease have evidence of
altered ability of HDL to act on Ox-PL, systemic inflammation, and increased risk
for cardiovascular events [128]. In bacteria, Ox-PL have been implicated in the regu-
lation of boil formation [129]. Moreover, Ox-PL were found to be increased at an
early stage of intestinal polyp formation in a mouse model of familial adenomatous
polyposis [130]. In these mice, treatment with the 4F peptide resulted in decreased
polyp formation and reduced colon cancer. In addition, the treatment of these mice
with the 4F peptide significantly decreased plasma levels of lysophosphatidic acid
[130], which binds to the 4F peptide with an affinity of 0.000523 nmol/L. This is of
2.5-million-fold higher affinity than the binding of lysophosphatidic acid to human
apoA-I [93]. These studies suggest that in these mouse models, binding and removal
of proinflammatory lipids is a potential mechanism for the inhibition of tumor
development [130,131]. Additionally, these studies suggest that therapies targeted to
reduce inflammation in the small intestine may benefit a number of critical human
illnesses including atherosclerosis. Hypothesis 4 and hypothesis 5 proposed in this
review are consistent with the studies reviewed earlier.
CONCLUSION
The oxidation of normal lipids by metabolic pathways or by nonenzymatic means
produces oxidized lipids that trigger an inflammatory response in many tissues
including the vasculature. Metabolites of arachidonic acid, such as 12-HETE and
likely many other oxidized fatty acids including those esterified to cholesterol or
phospholipids, can act similar to Ox-PL to induce inflammation. Despite the fact that
some of the biologic activities of metabolites of arachidonic acid, such as 12-HETE
and Ox-PL, are similar, the pathways by which they exert their biologic activity may
not be similar and remain to be defined by future research. HDL contains proteins
and enzymes that can inactivate or remove these proinflammatory lipids, but in some
circumstances, such as a systemic acute-phase response, the proteins and enzymes
associated with HDL are altered so that the inflammatory response is either not
inhibited or is enhanced. ApoA-I mimetic peptides, such as 4F, reduce inflammation
by binding and removing oxidized lipids from tissues. And finally, oxidized lipids
in the small intestine are important in modulating systemic inflammation, and the
intestine is a major site for the action of apoA-I mimetic peptides, such as 4F, which
bind these oxidized lipids. Definitive proof of each of these hypotheses will require
extensive future research by many laboratories.
REFERENCES
1. Witztum JL, Steinberg D. 1991. Role of oxidized low density lipoprotein in atherogenesis.
J Clin Invest. 88:1785–1792.
2. Parthasarathy S. 1994. Modified Lipoproteins in the Pathogenesis of Atherosclerosis.
Austin, TX: R.G. Landes Co., pp. 91–119.
3. Parthasarathy S. 1994. Mechanism(s) of cell-mediated oxidation of low density
lipoprotein. In Free Radicals in the Environment, Medicine and Toxicology, H Nohl,
H Esterbauer, and CR Evans (eds.). London, U.K.: Richelieu Press, pp. 163–179.
4. Witztum JL. 1994. The oxidation hypothesis of atherosclerosis. Lancet. 344:793–795.
5. Chisolm GM. 1991. Antioxidants and atherosclerosis: A current assessment. Clin
Cardiol. 14:125–130.
6. Cyrus T, Witztum JL, Rader DJ et al. 1999. Disruption of the 12/15-lipoxygenase gene
diminishes atherosclerosis in apo E-deficient mice. J Clin Invest. 103:1597–1604.
7. Steinberg D. 1999. At last, direct evidence that lipoxygenases play a role in atherogen-
esis. J Clin Invest. 103:1487–1488.
8. Cyrus T, Pratico D, Zhao L et al. 2001. Absence of 12/15-lipoxygenase expression
decreases lipid peroxidation and atherogenesis in apolipoprotein E-deficient mice.
Circulation. 103:2277–2282.
9. George J, Afek A, Shaish A et al. 2001. 12/15-Lipoxygenase gene disruption attenuates
atherogenesis in LDL receptor- deficient mice. Circulation. 104:1646–1650.
10. Zhao L, Cuff CA, Moss E et al. 2002. Selective interleukin-12 synthesis defect in
12/15-lipoxygenase deficient macrophages associated with reduced atherosclerosis in a
mouse model of familial hypercholesterolemia. J Biol Chem. 277:35350–35356.
11. Harats D, Shaish A, George J et al. 2000. Overexpression of 15-lipoxygenase in
vascular endothelium accelerates early atherosclerosis in LDL receptor-deficient mice.
Arterioscler Thromb Vasc Biol. 20:2100–2105.
12. Reilly KB, Srinivasan S, Hatley ME et al. 2004. 12/15-Lipoxygenase activity mediates
inflammatory monocyte/endothelial interactions and atherosclerosis in vivo. J Biol
Chem. 279:9440–9450.
13. Brennan ML, Anderson MM, Shih DM et al. 2001. Increased atherosclerosis in
myeloperoxidase-deficient mice. J Clin Invest. 107:419–430.
14. Thukkani AK, McHowat J, Hsu F-F, Brennan M-L, Hazen SL, Ford DA. 2003.
Identification of alpha-chloro fatty aldehydes and unsaturated lysophosphatidylcholine
molecular species in human atherosclerotic lesions. Circulation. 108:3128–3133.
15. Shao B, Pennathur S, Heinecke JW. 2012. Myeloperoxidase targets apolipoprotein A-I,
the major high density lipoprotein protein, for site-specific oxidation in human athero-
sclerotic lesions. J Biol Chem. 287:6375–6386.
16. Carr AC, McCall MR, Frei B. 2000. Oxidation of LDL by myeloperoxidase and reactive
nitrogen species: Reaction pathways and antioxidant protection. Arterioscler Thromb
Vasc Biol. 20:1716–1723.
17. Zhang R, Brennan M-L, Shen Z et al. 2002. Myeloperoxidase functions as a major enzy-
matic catalyst for initiation of lipid peroxidation at sites of inflammation. J Biol Chem.
277:46116–46122.
18. Shishebhor MH, Aviles BJ, Brennan M-L et al. 2003. Association of nitrotyrosine
levels with cardiovascular disease and modulation by statin therapy. J Am Med Assoc.
289:1675–1680.
19. Brennan ML, Penn MS, Van Lente F et al. 2003. Prognostic value of myeloperoxidase
in patients with chest pain. N Engl J Med. 349:1595–1604.
20. Brennan M-L, Hazen SL. 2003. Emerging role of myeloperoxidase and oxidant stress
markers in cardiovascular risk assessment. Curr Opin Lipidol. 14:353–359.
21. Gaut JP, Byun J, Tran HD et al. 2002. Myeloperoxi their relevance to atherosclerosis.
Trends Cardiovasc Med. 11:124–131.
22. Zhao X, Carnevale KA, Cathcart MK. 2003. Human monocytes use Rac1, not Rac2,
in the NADPH oxidase complex. J Biol Chem. 17:40788–40792.
23. Cathcart MK. 2004. Regulation of superoxide anion production by NADPH oxidase
in monocytes/macrophages. Contributions to atherosclerosis. Arterioscler Thromb Vasc
Biol. 24:23–28.
24. Hessler JR, Robertson AL, Chisolm GM. 1979. LDL induced cytotoxicity and its
inhibition by HDL in human vascular smooth muscle and endothelial cells in culture.
Atherosclerosis. 32:213–229.
25. Navab M, Hama SY, Anantharamaiah GM et al. 2000. Normal high density lipoprotein
inhibits three steps in the formation of mildly oxidized low density lipoprotein: Steps 2
and 3. J Lipid Res. 41:1495–1508.
26. Navab M, Hama SY, Cooke CJ et al. 2000. Normal high density lipoprotein inhibits
three steps in the formation of mildly oxidized low density lipoprotein: Step 1. J Lipid
Res. 41:1481–1494.
27. Badimon JJ, Badimon L, Fuster V. 1990. Regression of atherosclerotic lesions by
high density lipoprotein plasma fraction in the cholesterol-fed rabbit. J Clin Invest.
85:1234–1241.
28. Plump AS, Scott CJ, Breslow JL. 1994. Human apolipoprotein A-I gene expression
increases high density lipoprotein and suppresses atherosclerosis in the apolipoprotein
E-deficient mouse. Proc Natl Acad Sci USA. 91:9607–9611.
29. Shah PK, Yano J, Reyes O et al. 2001. High-dose recombinant apolipoprotein A-I
Milano mobilizes tissue cholesterol and rapidly reduces plaque lipid and macrophage
content in apolipoprotein E-deficient mice: Potential implications for acute plaque sta-
bilization. Circulation. 103:3047–3050.
30. Garber DW, Datta G, Chaddha M et al. 2001. A new synthetic class A amphipa-
thic peptide analogue protects mice from diet-induced atherosclerosis. J Lipid Res.
42:545–552.
31. Navab M, Anantharamaiah GM, Hama S et al. 2002. Oral administration of an Apo A-1
mimetic peptide synthesized from D-amino acids dramatically reduces atherosclerosis
in mice independent of plasma cholesterol. Circulation. 105:290–292.
32. Van Lenten BJ, Wagner AC, Anantharamaiah GM et al. 2002. Influenza infection pro-
motes macrophage traffic into arteries of mice that is prevented by D-4F, an apolipopro-
tein A-I mimetic peptide. Circulation. 106:1127–1132.
33. Ou Z, Ou J, Ackerman A, Oldham KT, Pritchard KA. 2003. L-4F, an apolipoprotein A-1
mimetic, restores nitric oxide and superoxide anion balance in low-density lipoprotein-
treated endothelial cells. Circulation. 107:1520–1524.
34. Ou J, Ou Z, Jones DW et al. 2003. L-4F, an apolipoprotein A-1 mimetic, dramatically
improves vasodilation in hypercholesterolemia and sickle cell disease. Circulation.
107:2337–2341.
35. Nissen SE, Tsunoda T, Tuzcu EM et al. 2003. Effect of recombinant apoA-I Milano
on coronary atherosclerosis in patients with acute coronary syndromes. A randomized
controlled trial. J Am Med Assoc. 290:2292–2300.
36. Rader DJ. 2003. High-density lipoproteins as an emerging therapeutic target for athero-
sclerosis. J Am Med Assoc. 290:2322–2324.
37. Bisoendial RJ, Hovingh GK, Levels JHM et al. 2003. Restoration of endothelial func-
tion by increasing high-density lipoprotein in subjects with isolated low high-density
lipoprotein. Circulation. 107:2944–2948.
38. Zhang YZ, Zanotti I, Reilly MP, Glick JM, Rothblat GH, Rader DJ. 2003. Over expres-
sion of apolipoprotein A-I promotes reverse transport of cholesterol from macrophages
to feces in vivo. Circulation. 108:661–663.
39. Remaley AT, Thomas F, Stonik JA et al. 2003. Synthetic amphipathic helical peptides
promote lipid efflux from cells by an ABCA1-dependent and an ABCA1-independent
pathway. J Lipid Res. 44:828–836.
40. Martinez LO, Agerholm-Larsen B, Wang N, Chen W, Tall AR. 2003. Phosphorylation
of a pest sequence in ABCA1 promotes cal pain degradation and is reversed by apoA-I.
J Biol Chem. 278:37368–37374.
41. Sevanian A, Bittolo-Bon G, Cazzolato G et al. 1997. LDL- is a lipid hydroperoxide-
enriched circulating lipoprotein. J Lipid Res. 38:419–428.
42. Bowry VW, Stanley KK, Stocker R. 1992. High density lipoprotein is the major carrier
of lipid hydroperoxides in human blood plasma from fasting donors. Proc Natl Acad Sci
USA. 89:10316–10320.
43. Navab M, Shechter I, Anantharamaiah GM, Reddy ST, Van Lenten BJ, Fogelman
AM. 2010. Structure and function of HDL mimetics. Arterioscler Thromb Vasc Biol.
30:164–168.
44. Bloedon LT, Dunbar R, Duffy D et al. 2008. Safety, pharmacokinetics, and pharmaco-
dynamics of oral apo A-I mimetic peptide D-4F in high-risk cardiovascular patients.
J Lipid Res. 49:1344–1352.
45. Watson CE, Weissbach N, Kjems L et al. 2011. Treatment of patients with cardiovascu-
lar disease with L-4F, an apo-A1 mimetic, did not improve select biomarkers of HDL
function. J Lipid Res. 52:361–373.
46. Navab M, Reddy ST, Anantharamaiah GM et al. 2011. Intestine may be a major site
of action for the apoA-I mimetic peptide 4F whether administered subcutaneously or
orally. J Lipid Res. 52:1200–1210.
47. Watson AD, Leitinger N, Navab M et al. 1997. Structural identification by mass spec-
trometry of oxidized phospholipids in minimally oxidized low density lipoprotein that
induce monocyte/endothelial interactions and evidence for their presence in vivo. J Biol
Chem. 272:13597–13607.
48. Subbanagounder G, Leitinger N, Schwenke DC et al. 2000. Determinants of bioactivity
of oxidized phospholipids. Specific oxidized fatty acyl groups at the sn-2 position.
49. Boullier A, Gillotte KL, Hörkkö S et al. 2000. The binding of oxidized low density lipo-
protein to mouse CD36 is mediated in part by oxidized phospholipids that are associated
with both the lipid and protein moieties of the lipoprotein. J Biol Chem. 275:9163–9169.
50. Podrez EA, Poliakov E, Shen Z et al. 2002. A novel family of atherogenic oxidized
phospholipids promotes macrophage foam cell formation via the scavenger receptor
CD36 and is enriched in atherosclerotic lesions. J Biol Chem. 277:38517–38523.
51. Napoli C, D’Armiento FP, Mancini FP et al. 1997. Fatty streak formation occurs in
human fetal aortas and is greatly enhanced by maternal hypercholesterolemia. Intimal
accumulation of low density lipoprotein and its oxidation precede monocyte recruitment
into early atherosclerotic lesions. J Clin Invest. 100:2680–2690.
52. Nishi K, Itabe H, Uno M et al. 2002. Oxidized LDL in carotid plaques and plasma asso-
ciates with plaque instability. Arterioscler Thromb Vasc Biol. 22:1649–1654.
53. Tsimikas S, Lau HK, Han KR et al. 2004. Percutaneous coronary intervention results
in acute increases in oxidized phospholipids and lipoprotein(a): Short-term and
long-term immunologic responses to oxidized low-density lipoprotein. Circulation.
109:3164–3170.
54. Tsimikas S, Aikawa M, Miller FJ Jr et al. 2007. Increased plasma oxidized
phospholipid:apolipoprotein B-100 ratio with concomitant depletion of oxidized phos-
pholipids from atherosclerotic lesions after dietary lipid-lowering: A potential biomarker
of early atherosclerosis regression. Arterioscler Thromb Vasc Biol. 27:175–181.
55. Tsimikas S, Brilakis ES, Miller ER et al. 2005. Oxidized phospholipids, Lp(a) lipopro-
tein, and coronary artery disease. N Engl J Med. 353:46–57.
56. Leibundgut G, Arai K, Orsoni A et al. 2012. Oxidized phospholipids are present on plas-
minogen, affect fibrinolysis, and increase following acute myocardial infarction. J Am
Coll Cardiol. 59:1426–1437.
57. Fang L, Harkewicz R, Hartvigsen K et al. 2010. Oxidized cholesteryl esters and phos-
pholipids in zebrafish larvae fed a high cholesterol diet: Macrophage binding and activa-
tion. J Biol Chem. 285:32343–32351.
58. Imai Y, Kuba K, Neely GG et al. 2008. Identification of oxidative stress and toll-like
receptor 4 signaling as a key pathway of acute lung injury. Cell. 133:235–249.
59. Crowe CR, Chen K, Pociask DA et al. 2009. Critical role of IL-17RA in immunopathol-
ogy of influenza infection. J Immunol. 183:5301–5310.
60. Ikeda K, Mutoh M, Teraoka N, Nakanishi H, Wakabayashi K, Taguchi R. 2011. Increase
of oxidant-related triglycerides and phosphatidylcholines in serum and small intestinal
mucosa during development of intestinal polyp formation in Min mice. Cancer Sci.
102:79–87.
61. Cruz D, Watson AD, Miller CS et al. 2008. Host-derived oxidized phospholipids and
HDL regulate innate immunity in human leprosy. J Clin Invest. 118:2917–2928.
62. Haider L, Fischer MT, Frischer JM et al. 2011. Oxidative damage in multiple sclerosis
lesions. Brain. 134(Pt 7):1914–1924.
63. Ikura Y, Ohsawa M, Suekane T et al. 2006. Localization of oxidized phosphatidylcho-
line in nonalcoholic fatty liver disease: Impact on disease progression. Hepatology.
43:506–514.
64. Van Lenten BJ, Wagner AC, Jung CL et al. 2008. Anti-inflammatory apoA-I-mimetic
peptides bind oxidized lipids with much higher affinity than human apoA-I. J Lipid Res.
49:2302–2311.
65. DeLeve LD, Wang X, Kanel GC, Atkinson RD, McCuskey RS. 2008. Prevention of
hepatic fibrosis in a murine model of metabolic syndrome with nonalcoholic steatohepa-
titis. Am J Pathol. 173:993–1001.
66. Suzuki M, Kamei M, Itabe H et al. 2007. Oxidized phospholipids in the macula increase
with age and in eyes with age-related macular degeneration. Mol Vis. 13:772–778.
67. Weihrauch D, Xu H, Shi Y et al. 2007. Effects of D-4F on vasodilation, oxidative stress,
angiostatin, myocardial inflammation, and angiogenic potential in tight-skin mice. Am J
Physiol Heart Circ Physiol. 293:H1432–H1441.
68. Leitinger N, Tyner TR, Oslund L et al. 1999. Structurally similar oxidized phospholipids
differentially regulate endothelial binding of monocytes and neutrophils. Proc Natl Acad
Sci USA. 96:12010–12015.
69. Napoli C, Witztum JL, Calara F, de Nigris F, Palinski W. 2000. Maternal hypercholester-
olemia enhances atherogenesis in normocholesterolemic rabbits, which is inhibited by
antioxidant or lipid-lowering intervention during pregnancy: An experimental model of
atherogenic mechanisms in human fetuses. Circ Res. 87:946–952.
70. Furnkranz A, Schober A, Bochkov VN et al. 2005. Oxidized phospholipids trigger
atherogenic inflammation in murine arteries. Arterioscler Thromb Vasc Biol.
25:633–638.
71. Huber J, Fürnkranz A, Bochkov VN et al. 2006. Specific monocyte adhesion to endothe-
lial cells induced by oxidized phospholipids involves activation of cPLA2 and lipoxy-
genase. J Lipid Res. 47:1054–1062.
72. Honda HM, Leitinger N, Frankel M et al. 1999. Induction of monocyte binding to endo-
thelial cells by MM-LDL: Role of lipoxygenase metabolites. Arterioscler Thromb Vasc
Biol. 19:680–686.
73. Patricia MK, Kim JA, Harper CM et al. 1999. Lipoxygenase products increase mono-
cyte adhesion to human aortic endothelial cells. Arterioscler Thromb Vasc Biol.
19:2615–2622.
74. Griesser M, Suzuki T, Tejera N et al. 2011. Biosynthesis of hemiketal eicosanoids by

cross-over of the 5-lipoxygenase and cyclooxygenase-2 pathways. Proc Natl Acad Sci
USA. 108:6945–6950.
75. Griesser M, Pistis V, Suzuki T, Tejera N, Pratt DA, Schneider C. 2011. Autoxidative
and cyclooxygenase-2 catalyzed transformation of the dietary chemo preventive agent
curcumin. J Biol Chem. 14:1114–1124.
76. Tejera N, Boeglin WE, Suzuki T, Schneider C. 2012. COX-2-dependent and-independent
biosynthesis of dihydroxy-arachidonic acids in activated human leukocytes. J Lipid Res.
53:87–94.
77. Harkewicz R, Hartvigsen K, Almazan F, Dennis EA, Witztum JL, Miller YI. 2008.
Cholesteryl ester hydroperoxides are biologically active components of minimally
oxidized low density lipoprotein. J Biol Chem. 283:10241–10251.
78. Berliner JA, Gharavi NM. 2008. Endothelial cell regulation by phospholipid oxidation
products. Free Radic Biol Med. 45:119–123.
79. Berliner JA, Leitinger N, Tsimikas S. 2009. The role of oxidized phospholipids in
atherosclerosis. J Lipid Res. 50(Suppl):S207–S212.
80. Lee S, Gharavi NM, Honda H et al. 2009. A role for NADPH oxidase 4 in the activation of
vascular endothelial cells by oxidized phospholipids. J Free Radic Biol Med. 47:145–151.
81. Zimman A, Chen SS, Komisopoulou E et al. 2010. Activation of aortic endothe-
lial cells by oxidized phospholipids: A phosphoproteomic analysis. J Proteome Res.
9:2812–2824.
82. Romanoski CE, Che N, Yin F et al. 2011. Network for activation of human endothe-
lial cells by oxidized phospholipids: A critical role of heme oxygenase 1. Circ Res.
109:e27–e41.
83. Kadl A, Sharma PR, Chen W et al. 2011. Oxidized phospholipid-induced inflammation
is mediated by Toll-like receptor 2. J Free Radic Biol Med. 51:1903–1909.
84. Birukova AA, Lee S, Starosta V et al. 2012. A role for VEGFR2 activation in endothelial
responses caused by barrier disruptive OxPAPC concentrations. PLoS ONE. 7:e30957.
85. Starosta V, Wu T, Zimman A et al. 2012. Differential regulation of endothelial cell per-
meability by high and low doses of oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-
phosphocholine. Am J Respir Cell Mol Biol. 46:331–341.
86. Lee S, Springstead JR, Parks BW et al. 2012. Metalloproteinase processing of HBEGF
is a proximal event in the response of human aortic endothelial cells to oxidized phos-
pholipids. Arterioscler Thromb Vasc Biol. 32:1246–1254.
87. Springstead JR, Gugiu BG, Lee S, Cha S, Watson AD, Berliner JA. 2012. Evidence
for the importance of OxPAPC interaction with cysteines in regulating endothelial cell
function. J Lipid Res. 53:1304–1315.
88. Miller YI, Choi SH, Wiesner P et al. 2011. Oxidation-specific epitopes are danger-
associated molecular patterns recognized by pattern recognition receptors of innate
immunity. Circ Res. 108:235–248.
89. Kim JB, Xia YR, Romanoski CE et al. 2011. Paraoxonase-2 modulates stress response
of endothelial cells to oxidized phospholipids and a bacterial quorum-sensing molecule.
90. Kadl A, Meher AK, Sharma PR et al. 2010. Identification of a novel macrophage phe-
notype that develops in response to atherogenic phospholipids via Nrf2. Circ Res.
107:737–746.
91. Navab M, Ananthramaiah GM, Reddy ST et al. 2004. The oxidation hypothesis of ath-
erogenesis: The role of oxidized phospholipids and HDL. J Lipid Res. 45:993–1007.
92. Van Lenten BJ, Wagner AC, Navab M et al. 2004. D-4F, an apolipoprotein A-I mimetic
peptide, inhibits the inflammatory response induced by influenza A infection of human
type II pneumocytes. Circulation. 110:3252–3258.
93. Gharavi NM, Gargalovic PS, Chang I et al. 2007. High-density lipoprotein modulates
oxidized phospholipid signaling in human endothelial cells from proinflammatory to
anti-inflammatory. Arterioscler Thromb Vasc Biol. 27:1346–1353.
94. Miyoshi M, Nakano Y, Sakaguchi T et al. 2007. Gene delivery of paraoxonase-1 inhib-
its neointimal hyperplasia after arterial balloon-injury in rabbits fed a high-fat diet.
Hypertens Res. 30:85–91.
95. Elsøe S, Ahnström J, Christoffersen C et al. 2012. Apolipoprotein M binds oxidized
phospholipids and increases the antioxidant effect of HDL. Atherosclerosis. 221:91–97.
96. Buga GM, Frank JS, Mottino GA et al. 2008. D-4F reduces EO6 immunoreactivity,
SREBP-1c mRNA levels, and renal inflammation in LDL receptor-null mice fed a
Western diet. J Lipid Res. 49:192–205.
97. Imaizumi S, Grijalva V, Navab M et al. 2010. L-4F differentially alters plasma levels of
oxidized fatty acids resulting in more anti-inflammatory HDL in mice. Drug Metab Lett.
4:139–148.
98. Wool GD, Cabana VG, Lukens J et al. 2011. 4F Peptide reduces nascent atherosclero-
sis and induces natural antibody production in apolipoprotein E-null mice. FASEB J.
25:290–300.
99. Getz GS, Reardon CA. 2011. Apolipoprotein A-I and A-I mimetic peptides: A role in
atherosclerosis. J Inflamm Res. 4:83–92.
100. Van Lenten BJ, Hama SY, de Beer FC et al. 1995. Anti-inflammatory HDL becomes
pro-inflammatory during the acute phase response. Loss of protective effect of HDL
against LDL oxidation in aortic wall cell cocultures. J Clin Invest. 96:2758–2767.
101. Navab M, Hama-Levy S, Van Lenten BJ et al. 1997. Mildly oxidized LDL induces an
increased apolipoprotein J/paraoxonase ratio. J Clin Invest. 99:2005–2019.
102. Hedrick CC, Hassan K, Hough GP et al. 2000. Short-term feeding of atherogenic diet to
mice results in reduction of HDL and paraoxonase that may be mediated by an immune
mechanism. Arterioscler Thromb Vasc Biol. 20:1946–1952.
103. Navab M, Hama SY, Hough GP, Subbanagounder G, Reddy ST, Fogelman AM. 2001.
A cell-free assay for detecting HDL that is dysfunctional in preventing the formation of
or inactivating oxidized phospholipids. J Lipid Res. 42:1308–1317.
104. Navab M, Ananthramaiah GM, Reddy ST et al. 2005. The double jeopardy of HDL. Ann
Med. 37:173–178.
105. Vaisar T, Pennathur S, Green PS et al. 2007. Shotgun proteomics implicates protease
inhibition and complement activation in the antiinflammatory properties of HDL. J Clin
Invest. 117:746–756.
106. Green PS, Vaisar T, Pennathur S et al. 2008. Combined statin and niacin therapy
remodels the high-density lipoprotein proteome. Circulation. 118:1259–1267.
107. McMahon M, Skaggs BJ, Sahakian L et al. 2011. High plasma leptin levels confer
increased risk of atherosclerosis in women with systemic lupus erythematosus, and are
associated with inflammatory oxidised lipids. Ann Rheum Dis. 70:1619–1624.
108. Charles-Schoeman C, Lee YY, Grijalva V et al. 2012. Cholesterol efflux by high density
lipoproteins is impaired in patients with active rheumatoid arthritis. Ann Rheum Dis.
71:1157–1162.
109. Mastorikou M, Mackness M, Mackness B. 2006. Defective metabolism of oxidized
phospholipid by HDL from people with type 2 diabetes. Diabetes. 55:3099–3103.
110. Morgantini C, Natali A, Boldrini B et al. 2011. Anti-inflammatory and antioxidant
properties of HDLs are impaired in type 2 diabetes. Diabetes. 60:2617–2623.
111. Kontush A, Chapman MJ. 2010. Antiatherogenic function of HDL particle subpopula-
tions: Focus on antioxidative activities. Curr Opin Lipidol. 21:312–318.
112. Bhattacharyya T, Nicholls SJ, Topol EJ et al. 2008. Relationship of paraoxonase 1
(PON1) gene polymorphisms and functional activity with systemic oxidative stress and
cardiovascular risk. JAMA. 299:1265–1276.
113. Undurti A, Huang Y, Lupica JA, Smith JD, DiDonato JA, Hazen SL. 2009. Modification
of high density lipoprotein by myeloperoxidase generates a pro-inflammatory particle.
J Biol Chem. 284:30825–30835.
114. Watanabe J, Grijalva V, Hama S et al. 2009. Hemoglobin and its scavenger protein hap-
toglobin associate with apoA-1-containing particles and influence the inflammatory
properties and function of high density lipoprotein. J Biol Chem. 284:18292–18301.
115. Watanabe J, Charles-Schoeman C, Miao Y et al. 2012. Proteomic profiling following
immunoaffinity capture of high-density lipoprotein: Association of acute-phase proteins
and complement factors with proinflammatory high-density lipoprotein in rheumatoid
arthritis. Arthritis Rheum. 64:1828–1837.
116. Shao B, Pennathur S, Pagani I et al. 2010. Modifying apolipoprotein A-I by malondial-
dehyde, but not by an array of other reactive carbonyls, blocks cholesterol efflux by the
ABCA1 pathway. J Biol Chem. 285:18473–18484.
117. Besler C, Heinrich K, Rohrer L et al. 2011. Mechanisms underlying adverse effects
of HDL on eNOS-activating pathways in patients with coronary artery disease. J Clin
Invest. 121:2693–2708.
118. Khera AV, Cuchel M, de la Llera-Moya M et al. 2011. Cholesterol efflux capacity, high-
density lipoprotein function, and atherosclerosis. N Engl J Med. 364:127–135.
119. Heinecke J. 2011. HDL and cardiovascular-disease risk–time for a new approach?
N Engl J Med. 364:170–171.
120. Cavigiolio G, Geier EG, Shao B, Heinecke JW, Oda MN. 2010. Exchange of apolipo-
protein A-I between lipid-associated and lipid-free states: A potential target for oxidative
generation of dysfunctional high density lipoproteins. J Biol Chem. 285:18847–18857.
121. Brunham LR, Kruit JK, Iqbal J et al. 2006. Intestinal ABCA1 directly contributes to
HDL biogenesis in vivo. J Clin Invest. 116:1052–1062.
122. Green PH, Glickman RM, Saudek CD, Blum CB, Tall AR. 1979. Human intestinal
lipoproteins. Studies in chyluric subjects. J Clin Invest. 64:233–242.
123. Wang Z, Klipfell E, Bennett BJ et al. 2011. Gut flora metabolism of phosphatidylcholine
promotes cardiovascular disease. Nature. 472:57–63.
124. Navab M, Reddy ST, Anantharamaiah GM et al. 2012. D-4F-mediated reduction in
metabolites of arachidonic and linoleic acids in the small intestine is associated with
decreased inflammation in low-density lipoprotein receptor-null mice. J Lipid Res.
53:437–445.
125. Adler DH, Cogan JD, Phillips JA 3rd et al. 2008. Inherited human cPLA(2alpha) defi-
ciency is associated with impaired eicosanoid biosynthesis, small intestinal ulceration,
and platelet dysfunction. J Clin Invest. 118:2121–2131.
126. Montrose DC, Kadaveru K, Ilsley JN et al. 2010. cPLA2 is protective against COX
inhibitor-induced intestinal damage. Toxicol Sci. 117:122–132.
127. Watanabe J, Lin JA, Narasimha AJ et al. 2010. Novel anti-inflammatory functions for
endothelial and myeloid cyclooxygenase-2 in a new mouse model of Crohn’s disease.
Am J Physiol Gastrointest Liver Physiol. 298:G842–G850.
128. van Leuven SI, Hezemans R, Levels JH et al. 2007. Enhanced atherogenesis and altered
high density lipoprotein in patients with Crohn’s disease. J Lipid Res. 48:2640–2646.
129. Rao J, DiGiandomenico A, Artamonov M, Leitinger N, Amin AR, Goldberg JB. 2011.
Host derived inflammatory phospholipids regulate rahU (PA0122) gene, protein, and
biofilm formation in Pseudomonas aeruginosa. Cell Immunol. 270:95–102.
130. Su F, Grijalva V, Navab K et al. 2012. HDL mimetics inhibit tumor development
in both induced and spontaneous mouse models of colon cancer. Mol Cancer Ther.
11:1311–1319.
131. Su F, Kozak KR, Imaizumi S et al. 2010. Apolipoprotein A-I (apoA-I) and apoA-I
mimetic peptides inhibit tumor development in a mouse model of ovarian cancer. Proc
Natl Acad Sci USA. 107:19997–20002.
11 Chronic Kidney Disease
and Inflammation
Karl J. Neff and Carel Le Roux
CONTENTS
Introduction............................................................................................................. 167
Injury and the Initiation of Renal Inflammation..................................................... 167
Kidney Disease and Inflammation: The Role of Macrophages.............................. 168
Kidney Disease and Inflammation: The Role of T-Cells........................................ 170
Kidney Disease and Inflammation: Proinflammatory
and Proresolution Molecules................................................................................... 171
Course of Inflammation and CKD.......................................................................... 172
Nutrition and Renal Inflammation: Potential Interactions...................................... 174
Conclusion.............................................................................................................. 175
References............................................................................................................... 175
INTRODUCTION
Inflammation is a key mechanism in the development of chronic kidney disease
(CKD). Severe acute or chronic inflammation can initiate, maintain, and promote
progression of CKD. Inflammation in CKD results from an imbalance of proin-
flammatory and proresolving mediators that determine the extent of activity of the
inflammatory response. The regulation of this system is complex, but several key
immune cells, with macrophages being of particular importance, are major modula-
tors of the inflammatory response in CKD.
This balance can be disrupted by any number of insults including ischemia and
hyperglycemia. Once injured, the renal cells become part of an inflammatory pro-
cess regulated by the injured cells themselves and the immune system. Several pro-
inflammatory and proresolving mediators are involved.
INJURY AND THE INITIATION OF RENAL INFLAMMATION

Renal epithelium can be injured by factors such as hyperglycemia-derived advanced
glycation end products (AGEs), hypertension-associated oxidative stress, or nephro-
toxic agents. Ischemia or ureteric obstruction can also contribute to injury. Animal
models that simulate these injuries and studies involving patients with diabetes or
hypertension allow us to investigate the effect of these factors on the initiation, main-
tenance, and progression of renal inflammation.
167
When renal epithelium encounters an insult, adhesion molecules are upregulated,

and leukocytes infiltrate the interstitium beginning an inflammatory process that
either resolves or perpetuates depending on signals from the renal epithelial, mesan-
gial, and infiltrating immune cells. If the inflammatory process persists, glomerular
injury follows with the onset and progression of CKD [1]. CKD is characterized by
progressive loss of functional nephrons and therefore reduced glomerular filtration
rates (GFR). Histological markers include glomerulosclerosis and tubulointerstitial
fibrosis (TIF), which develop in association with a profibrotic extracellular matrix [2].
Renal injury precipitates the production of proinflammatory chemokines and
cytokines including monocyte-chemoattractant protein-1 (MCP-1), interleukin-8
(IL-8), and interferon gamma (INFγ) from local renal epithelial and immune cells.
These signals attract leukocytes to the renal glomeruli and interstitium and initiate the
adhesion of leukocytes to the renal epithelium. Adhesion of leukocytes to the epithe-
lium is mediated by selectins and integrins, which also facilitate the migration of acti-
vated leukocytes across the epithelial cell membrane. Intracellular adhesion molecule
1 (ICAM-1) is a key facilitator of leukocyte migration across the cell membrane [3].
ICAM-1 expression can be upregulated by AGEs, oxidative stress, and proinflamma-
tory cytokines [4]. Inhibition of ICAM-1 in animal models is renoprotective by restrict-
ing the transport of leukocytes across renal cell membranes [3,5]. While similar data
on ICAM-1 are not available in humans, vascular cell adhesion molecule-1 (VCAM-1)
is upregulated in humans with diabetic kidney disease (DKD) [6]. VCAM-1 is cen-
tral in the transport of leukocytes from the circulation into the renal interstitium, and
upregulation facilitates greater migration of leukocytes to the site of injury.
Once within the cell, chemokines from inflamed renal cells and activated immune
cells attract further leukocytes, and the ongoing leukocyte influx maintains the
inflammatory cycle. Leukocytes produce IL-1, tumor necrosis factor alpha (TNF-α),
and INFγ, which stimulate further proinflammatory cytokine and chemokine pro-
duction within the renal cells. INFγ and macrophage migration inhibitory factor
(MIF) promote transmigration of leukocytes across the cell membrane. The accu-
mulating immune cell colony is the key driver of renal inflammation and maintains
its mass via ongoing signaling and recruitment of circulating leukocytes. Unless this
is interrupted, chronic inflammation ensues.
KIDNEY DISEASE AND INFLAMMATION:

THE ROLE OF MACROPHAGES
Macrophages are the key immune cells in the inflammatory process in CKD and
can mediate several central inflammatory pathways [7]. Macrophages can present as
proinflammatory (classically activated; designated M1) or proresolution phenotypes
(prorepair; designated M2 and related subsets). There is significant plasticity within
the macrophage population, and macrophages can switch phenotypes from M1 to
M2 and vice versa depending on the predominant milieu, that is, proresolving or
proinflammatory. Macrophages are found in both acute and chronic kidney disease
and are detected in the glomerulus and the interstitium of kidney cortex and medulla.
Acute renal insult activates proinflammatory macrophages that move to the
site of injury. Macrophages can be activated by proinflammatory cytokines and
Chronic Kidney Disease and Inflammation 169
chemokines, as well as pathogen-associated molecular patterns (e.g., bacterial anti-

gens such as flagellin). Pattern recognition receptors, including Toll-like receptors,
are the target for many of these molecules. Through intracellular signaling pathways
including nuclear factor kappa beta (NF-κβ) macrophages produce multiple proin-
flammatory cytokines and chemokines and generate reactive oxygen and nitrogen
species. This further augments the inflammatory process and stimulates migration
of activated leukocytes to the site of injury.
Therefore, activated proinflammatory macrophages are potent proinflammatory
cells, which generate further inflammation via the promotion of leukocytic infiltra-
tion and activation. There is also some associated direct cell injury and dysregula-
tion of the cell cycle, which can result in cell malfunction or apoptosis [8]. A change
from proresolution to proinflammatory macrophage phenotypes can advance exist-
ing renal disease and is likely to be an initiating event in CKD [7,9]. The number
of proinflammatory macrophages infiltrating the renal tissue negatively correlates
with renal outcomes. Chronic stress from AGEs in DKD or from oxidative stress in
hypertensive renal disease could produce a state of chronic activity in the macro-
phage population and a preponderance of activated proinflammatory macrophages.
This could alter the balance of inflammation and resolution to the proinflammatory
state, thereby initiating and maintaining the inflammation of CKD.
The mechanism by which macrophage action within the kidney is mediated
involves profibrotic proinflammatory chemokines and cytokines including IL-1,
TNF-α, transforming growth factor beta (TGFβ), and proresolving lipid mediators.
Typical insults such as hyperglycemia are associated with macrophage infiltration
and with increased chemokine production and renal fibrosis [10,11]. However, renal
epithelial cells themselves can also secrete proinflammatory molecules such as
MCP-1, therefore contributing directly to the maintenance of inflammation. The
active inflammation results in permeability of the cell membrane and migration
of activated immune cells across the membrane. Activated proinflammatory mac-
rophages produce profibrotic molecules such as INFγ, TGFβ, and TGFα and dys-
regulate NF-κβ and stress-activated protein kinase pathways. The end-result of this
process is accumulating TIF, which is a hallmark of CKD. The degree of macrophage
accumulation correlates with the degree of fibrosis, proteinuria, and markers of GFR.
However, proresolving macrophages are potent agents of resolution and can
improve outcomes. This is partially achieved by phagocytic clearance of proinflam-
matory cells and cellular debris. This clearance occurs without further immune cell
activation and is an effective means of resolving the inflammatory process. Inhibition
of proinflammatory macrophage infiltration in animal models reduces fibrosis,
improves histological appearance, and downregulates TGFβ [12,13]. Transfection
of a proresolving macrophage (M2) phenotype can reduce renal disease in rodent
models, whereas transfection of a proinflammatory (M1) macrophage accelerates
renal injury [14]. In this model, macrophages were isolated from BALB/c mice and
stimulated with lipopolysaccharide to induce a proinflammatory (M1) macrophage
phenotype or treated with ILs 4 and 13 to induce a M2 phenotype. The treated mac-
rophages were then infused into an adriamycin-induced CKD mouse model. Those
infused with M2 macrophages had less severe histological and functional disease as
compared to the M1-treated animals.
Of the proinflammatory signals, MCP-1 is of particular importance in macro-

phage activity. Expression of MCP-1 is correlated with the degree of macrophage
infiltration, and in human studies, urinary MCP-1 correlates with urinary albuminuria
[15]. In mice without MCP-1, renal injury is more difficult to initiate, and CKD
does not develop despite repetitive chemical insult [16]. This can be therapeutically
exploited in the experimental setting at the receptor level. Antagonism of the MCP-1
receptor in animal models improves histological outcomes [16–18]. Human studies
on antagonism of this pathway are ongoing.
Proresolving mediators promote the modulation of the macrophage population
in the renal interstitium from proinflammatory to proresolving. Lipoxins induce
macrophages to clear apoptotic cells without releasing proinflammatory mediators,
so-called nonphlogistic phagocytosis [19]. This is a key effect of the proresolution
pathway in renal disease and enhances resolution. Resolvins and protectins inhibit
the secretion of TNF-α from stimulated proinflammatory macrophages and reduce
migration of further proinflammatory cells into the inflamed area in ischemic mod-
els of renal disease [20].
Why macrophages become chronically proinflammatory or why the proresolving
population is unable to remediate chronically activated pathways in not yet fully
understood. However, the answer is likely to be related to key epithelial pathways and
intracellular signaling between immune cells. T-cells may have the most important
role in this latter category.
KIDNEY DISEASE AND INFLAMMATION: THE ROLE OF T-CELLS

T-cells are recognized in the pathogenesis of specific glomerulonephritides, but
are now also found to be of importance in DKD and CKD [11,21] Renal injuries
such as those induced by AGEs can stimulate T-cell action. Activated T-cells
produce INFγ that promotes proinflammatory pathways and further macrophage
accumulation within renal cells [22]. Specific chemokines such as regulated
on activation normal on T-cell expressed and secreted (RANTES or CCL5)
regulate T-cell infiltration, and blockade or knockout of this pathway reduces
proinflammatory renal chemokine expression and T-cell infiltration and improves
histological outcomes [23].
The role of the T-cell depends on the pathology. In DKD, T-cells are proin-
flammatory and proinjury, and inhibition of T-cell action improves outcomes [24].
However, in ischemic injury, T-cell inhibition can promote further damage, indicat-
ing a prorepair or proresolution role in this scenario [25,26]. Therefore, proresolution
may be the most important function for the T-cell population.
Regulatory T-cells (TRegs) are dysfunctional in diabetic animal models [26]. In
TReg-depleted models, renal histological outcomes are worse, and when TRegs are
transfected into these models, renal disease is attenuated [27]. TRegs suppress the
production of proinflammatory chemokines and cytokines by macrophages, which
in turn protect against macrophage-dependent renal injury. Therefore, they are key
regulators of the inflammatory cycle in CKD.
Other T-cell populations including CD4+ cells and Th1 cells modulate the activity
and function of TRegs. Specific chemokines mediate renal recruitment of both
T-cells and can determine the balance between a proinflammatory and proresolv-
ing milieu [28]. Therefore, multiple T-cell subsets are involved in the inflammatory
process in CKD. Understanding the relationship and activating mechanisms between
each subset should be of particular interest, as it may be that TReg modulation could
become a viable therapeutic target for CKD in the future.
KIDNEY DISEASE AND INFLAMMATION: PROINFLAMMATORY

AND PRORESOLUTION MOLECULES
The predominant paradigm of inflammation in general has been that of a proinflam-
matory state that is initiated by insult and which resolves spontaneously. However,
several molecules are now recognized to actively promote and regulate the resolution
of inflammation. It is the balance of these molecules that determines if inflammation
will be perpetuated or resolved.
There are particular proinflammatory cytokines that can be correlated with the
development of renal disease [17]. Monocyte chemoattractant protein-1 (MCP-1),
also known as CC chemokine ligand 2 (CCL2), is one that is strongly associated with
the major forms of CKD including both obesity-associated and diabetic nephropathy
[29,30]. MCP-1 is understood to cause renal injury via macrophage accumulation
and renal inflammation [30]. As a major mediator of macrophage and monocyte acti-
vation and migration, MCP-1 is key in the pathogenesis of CKD, and in particular of
DKD [31]. It is found on most renal cells and is associated with markers of progres-
sion of DKD in animal and human models [10,32,33]. Inhibition or blockade of the
MCP-1 pathway delays the progression of renal disease in diabetic animal models
and is associated with improved human renal cell function in vitro [18,34,35].
Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine
involved in macrophage activation and the recruitment of macrophages to the site
of inflammation. Increased MIF expression is detected in some subtypes of human
glomerulonephritis [36]. It is also increased in obesity [37]. MIF is involved in che-
moattraction of macrophages and in transmigration of activated immune cells across
cell membranes. Treatment with anti-MIF antibodies reduces proinflammatory cyto-
kine production and the severity of glomerulonephritis [38].
TNF-α stimulates the production of reactive oxygen species, which inhibits effec-
tive cellular repair. This promotes renal cell dysfunction and apoptosis [33]. In CKD,
TNF-α is mainly produced by macrophages and T-cells, but can also be produced
by monocytes and renal cells. TNF-α enhances renal cell endothelial permeability
and is a chemoattractant for macrophages. Therefore, it is important in perpetuating
the influx of macrophages and the ongoing cycle of inflammation [39]. In models of
DKD, TNF-α is associated with proteinuria and progression of disease [40]. It is also
associated with reduced GFR [39].
Several ILs have important roles in the progression and maintenance of inflam-
mation. IL-1 has a major role in promoting endothelial permeability, in part by
enhancing ICAM-1 expression [41–43]. It also contributes to increased activity of
the profibrotic TGFβ pathway [42–44]. IL18 is another proinflammatory cytokine,
which promotes IL-1, INFγ, and TGFα production [45,46]. It also enhances epithelial
cell permeability at least partially through upregulation of ICAM-1. Elevated IL-18
is associated with increased renal cell apoptosis [47,48]. Renal tubular epithelium is
the major source, but macrophages and T-cells can also produce IL-18 [49].
Adiponectin is produced by adipocytes and is a key agent in the antiinflammatory
response. It acts by countering the effect of TNF-α on leukocyte transport across
epithelial membranes and on macrophage proinflammatory signaling [50–52].
This can impede the migration of macrophages into the renal interstitium and
therefore could potentially minimize renal inflammation. Additional effects on
TGFb and endothelial function could improve renal histological outcomes and
reduce proteinuria [53].
Active proresolution pathways are also central to the development and pathogenesis
of inflammation in CKD. Defective inflammatory resolution can result in the
interstitial fibrosis that is a central feature of CKD [54,55]. Localized production of
lipid mediators, such as prostaglandins and leukotrienes, result in vasodilatation and
chemoattraction of proinflammatory immune cells. Prostaglandin formation also
induces the production of proresolving lipid mediators via lipoxygenase-catalyzed
pathways [56]. These mediators, including lipoxins, protectins, and resolvins, reduce
vascular permeability, inhibit immune cell recruitment, and promote the conversion
of proinflammatory macrophage phenotypes to proresolving. Therefore, this mecha-
nism affects an inflammatory break while inflammation is developing. If this break
is defective, then inflammation can continue unabated leading to CKD.
Lipoxins are proresolution agents in glomerulonephritis and acute kidney injury
that can reduce proteinuria and mesangial cell proliferation in human. The activity
of lipoxins may be partially mediated by a reduction in proinflammatory cytokines
and chemokines [57]. However, they also reduce immune cell migration and can
promote the development of a proresolving macrophage phenotype [58].
Resolvins, protectins, and maresins are other proresolving mediators that are
important in the resolution of inflammation. These act by inhibiting neutrophil
migration and modulating macrophage activity [20,59,60]. Resolvins and protec-
tins can both attenuate renal injury in animal models of ischemia–reperfusion [20].
Introducing these agents once inflammation has been initiated by ischemia can result
in improved resolution, demonstrating the importance of their role not only in deter-
mining the onset or degree of inflammatory activity, but also their active role in
resolving the inflammatory process.
COURSE OF INFLAMMATION AND CKD

The kidney can completely recover from an ischemic or toxic injury under nor-
mal circumstances. However, when injured, tubular epithelial cells produce mul-
tiple proinflammatory cytokines and chemokines, including MCP-1 and TNF-α.
Epithelial cells also express regulatory molecules and Toll-like receptors that stimu-
late proinflammatory T-cell activity. The ongoing inflammation results in the classic
pathological features of CKD, which include TIF, glomerulosclerosis, and loss of
parenchyma.
Fibrosis is irreversible and causes end-stage kidney disease. The pathological
features of CKD are all associated with leukocyte infiltration and inflammation.
Infiltrating leukocytes act on mesangial cells, interstitial fibroblasts, and tubular
epithelial cells to produce fibrosis [61]. Activation of mesangial cells and fibroblasts
is an early event. After this process has developed, epithelial-to-mesenchymal transi-
tion (EMT) occurs.
Activated fibroblasts are termed myofibroblasts and are characterized by de novo
production of alpha SMA and production of excessive amounts of profibrotic extra-
cellular matrix. These cells can be derived from residing fibroblasts or from other
mesenchymal cells [62]. Several of the proinflammatory cytokines promote fibro-
blast activation, but TGFb is one of the most consistently implicated molecules [63].
In rodent models, overexpression of TGFβ is associated with marked glomeruloscle-
rosis and TIF [64]. Administration of a TGFβ–neutralizing antibody attenuates the
development of fibrotic renal disease [65,66]. There is also an effect to reduce the
onset of renal impairment [66].
However, TGFβ should not be considered a purely profibrotic agent, as it can
also inhibit NF-κβ via SMAD pathways, thereby modulating renal inflammation
[67]. This can implicate TGFβ in proresolution pathways via intermediaries such as
SMAD 7 [67]. TGFβ seems to induce phenotype-switching of macrophages to the
M2 proresolving phenotype, and overexpression of TGFβ is anti-inflammatory and
reduces renal fibrosis [68]. Therefore, the role of TGFβ is important but complex,
and the mechanisms underlying the multiple interactions need to be fully elucidated.
EMT normally terminates when inflammation is resolved as a part of the repair
process after tissue injury. If inflammation persists, as is the case in CKD, then
EMT continues, and the activated mesenchymal cells promote fibrosis [69]. In this
scenario, the chronic exposure of renal tubular epithelial cells to proinflammatory
profibrotic signals produces a mesenchymal phenotype giving rise to fibroblasts and
myofibroblasts. EMT is characterized by reorganization of the actin cytoskeleton,
disruption of tubular basement membrane, a loss of epithelial cell adhesion, and
enhanced cell migration and invasion.
The development of EMT is associated with chronic inflammation and may
involve glomerular podocytes resulting in functional impairment. This can result
in proteinuria and glomerulosclerosis. EMT is regulated by intracellular signal
transduction pathways involving TGFβ/SMAD, integrin-linked kinase, and Wnt/β-
catenin signaling. TGFβ also promotes this process. Markers of EMT such as vimen-
tin are found in humans with CKD [69,70]. These markers correlate with declining
renal function.
In severe sustained or repetitive kidney insult, the cell cycle can become dysregu-
lated, and in concert with profibrotic TGFβ production from renal epithelial cells,
epigenetic changes in resident fibroblasts can result with subsequent myofibroblast
formation and activation [8]. Therefore, EMT conversion of epithelial cells to myo-
fibroblasts may not be the only source of this cell population, although this remains
a controversial concept.
To date, demonstrating a clear relationship between EMT and CKD has been
difficult as fibroblastic conversion has been incompletely defined due to a paucity of
specific markers. Most of the available markers such as vimentin are not specific for
fibroblasts because they are also present in other inflammatory and endothelial cells.
Injured renal tubular endothelial cells in vivo can undergo partial EMT in which
only one or two markers are altered. Therefore, conclusive evidence demonstrating
the presence of EMT in CKD and determining its contribution is elusive. This must
be understood when considering the role EMT plays in the course of inflammation
in CKD.
NUTRITION AND RENAL INFLAMMATION:

POTENTIAL INTERACTIONS
Proresolving lipid mediators are derived from long-chain polyunsaturated fatty acids
(LC-PUFAs). Therefore, there is a potential role for PUFA supplementation as many
patients with renal disease do not have an adequate intake of dietary PUFA [71]. The
main form of PUFA delivery in clinical practice is omega-3 in fish oil. However,
supplemented soya milk is also available for vegans.
PUFAs are associated with reduced mortality in end-stage renal disease and are
implicated in reduced levels of renal fibrosis by modulation of the TGFb pathways
and regulating cell growth factors [72,73]. The supplementation of these compounds
to modify the inflammatory process has been investigated in a variety of conditions
[71]. There are few data in CKD cohorts specifically, but in the available data, PUFA
supplementation can reduce inflammation as measured by C-reactive peptide and
can result in a decrease in IL-6 and TNF-α levels [74,75].
In arthrosclerosis, PUFAs can modify the inflammatory milieu within arthro-
sclerotic plaques by reducing the infiltration of foam cells and proinflammatory
T-cells [76]. Similar data on immune cell modulation are not available in renal
disease. However, doses of LC-PUFA of between 1.5 and 2.4 g/day can reduce
C-reactive protein, IL-6, and TNF-α levels [74,75]. Other nutritional compounds are
of increasing interest in modifying renal inflammation. Dairy products can modify
TNF-α and IL-6 levels, although the evidence base is not fully developed, and there
are no studies specifically investigating renal disease [77].
There are early data suggesting an effect on inflammation from l-carnitine, lipoic
acids, and plant sterols [78]. Vitamin supplementation also remains to be investigated
in renal inflammation. Addition of supplemental vitamin D to the diet, for example,
could potentially act to modulate macrophages in the inflammatory response by
modifying TNF-α and NFkb pathways [79]. However, focused research is needed to
clarify any potential role for these compounds, and at this time, there is no proven
role for these agents in modulating renal inflammation.
Flavonoids, phenolic acids, and isoflavones have potent antioxidant properties that
could be of use in renal inflammation. Many of these agents have been investigated
in various disease models including cardiovascular disease. Genistein is an isofla-
vone found in legumes, which is currently being tested in renal disease. The supple-
mentation of this agent in animal models can inhibit TGFb-mediated renal fibrosis
and can downregulate cell adhesion molecules [80,81]. This could have significant
effects on macrophage transmigration, although this remains to be proven. Genistein
is effective in attenuating the effect of hyperglycemia-associated fibrotic pathways,
including NFkb-mediated pathways, in the short term [82]. This could indicate a
future role for genistein or a derived agent in DKD.
Dietary protein can modify renal disease both in terms of histological and
functional markers [83–87]. Generally, it is considered that low-protein diets are
beneficial in CKD as it may improve functional outcomes [85,86]. Part of this

effect may be mediated via reducing inflammation [83]. However, high-protein
diets are associated with mesangial expansion and the increased expression of sev-
eral proinflammatory cytokines in animal models [88]. This is particularly rel-
evant in obesity or bariatric surgery, where protein is proportionately increased in
the diet. While casein or other proteins may be deleterious to renal function, soy
protein is beneficial in animal models of obesity-associated DKD [87]. These data
would suggest that soy protein is preferred in dietary therapy in obese cohorts,
especially in those with CKD.
Therefore, while research into the use of nutritional supplements in remediating
renal inflammation and optimizing renal outcomes remains at an early stage, it is a
field full of possibility. Further evidence is awaited to clarify the potential role of
nutrition in treating renal inflammation specifically. However, PUFA supplementa-
tion would appear to be likely to produce benefit, but evidence from a randomized
controlled trial using clinical outcome measures would be very helpful.
CONCLUSION
Inflammation is a critical mechanism, which can initiate CKD and which can pro-
mote the progression of CKD to end-stage renal disease. This is mainly due to the
macrophage-mediated migration of activated proinflammatory immune cells into
the renal interstitium. The renal tubular endothelium and the T-cell populations are
also key participants in this process. However, the overarching mechanism relies on
a balance between the proinflammatory and proresolving milieu. Further therapeutic
strategies, including nutritional supplementation, focused on modifying this balance
to a proresolving environment by modulating immune cell activity is likely to be of
importance in the future treatment of CKD.
REFERENCES
1. Schieppati A, Remuzzi G. 2005. Chronic renal diseases as a public health problem:
Epidemiology, social, and economic implications. Kidney Int Suppl 98:S7–S10.
2. Liu Y. 2004. Epithelial to mesenchymal transition in renal fibrogenesis: Pathologic
significance, molecular mechanism, and therapeutic intervention. J Am Soc Nephrol
15:1–12.
3. Chow FY, Nikolic-Paterson DJ, Ozols E, Atkins RC, Tesch GH. 2005. Intercellular
adhesion molecule-1 deficiency is protective against nephropathy in type 2 diabetic
db/db mice. J Am Soc Nephrol 16:1711–1722.
4. Galkina E, Ley K. 2006. Leukocyte recruitment and vascular injury in diabetic nephrop-
athy. J Am Soc Nephrol 17:368–377.
5. Okada S, Shikata K, Matsuda M, Ogawa D, Usui H, Kido Y et al. 2003. Intercellular
adhesion molecule-1-deficient mice are resistant against renal injury after induction of
diabetes. Diabetes 52:2586–2593.
6. Seron D, Cameron JS, Haskard DO. 1991. Expression of VCAM-1 in the normal and
diseased kidney. Nephrol Dial Transplant 6:917–922.
7. Wang Y, Harris DC. 2011. Macrophages in renal disease. J Am Soc Nephrol 22:21–27.
8. Yang L, Besschetnova TY, Brooks CR, Shah JV, Bonventre JV. 2010. Epithelial cell
cycle arrest in G2/M mediates kidney fibrosis after injury. Nat Med 16:535–543.
9. Duffield JS. 2011. Macrophages in kidney repair and regeneration. J Am Soc Nephrol
22:199–201.
10. Chow F, Ozols E, Nikolic-Paterson DJ, Atkins RC, Tesch GH. 2004. Macrophages in
mouse type 2 diabetic nephropathy: Correlation with diabetic state and progressive renal
injury. Kidney Int 65:116–128.
11. Chow FY, Nikolic-Paterson DJ, Atkins RC, Tesch GH. 2004. Macrophages in strep-
tozotocin-induced diabetic nephropathy: Potential role in renal fibrosis. Nephrol Dial
Transplant 19:2987–2996.
12. Wada T, Furuichi K, Sakai N, Iwata Y, Kitagawa K, Ishida Y et al. 2004. Gene therapy
via blockade of monocyte chemoattractant protein-1 for renal fibrosis. J Am Soc Nephrol
15:940–948.
13. Sung SA, Jo SK, Cho WY, Won NH, Kim HK. 2007. Reduction of renal fibrosis as a
result of liposome encapsulated clodronate induced macrophage depletion after unilat-
eral ureteral obstruction in rats. Nephron Exper Nephrol 105:e1–e9.
14. Wang Y, Wang YP, Zheng G, Lee VW, Ouyang L, Chang DH et al. 2007. Ex vivo
programmed macrophages ameliorate experimental chronic inflammatory renal disease.
Kidney Int 72:290–299.
15. Eardley KS, Zehnder D, Quinkler M, Lepenies J, Bates RL, Savage CO et al. 2006.
The relationship between albuminuria, MCP-1/CCL2, and interstitial macrophages in
chronic kidney disease. Kidney Int 69:1189–1197.
16. Chow FY, Nikolic-Paterson DJ, Ozols E, Atkins RC, Rollin BJ, Tesch GH. 2006.
Monocyte chemoattractant protein-1 promotes the development of diabetic renal injury
in streptozotocin-treated mice. Kidney Int 69:73–80.
17. Chow FY, Nikolic-Paterson DJ, Ma FY, Ozols E, Rollins BJ, Tesch GH. 2007. Monocyte
chemoattractant protein-1-induced tissue inflammation is critical for the development of
renal injury but not type 2 diabetes in obese db/db mice. Diabetologia 50:471–480.
18. Kanamori H, Matsubara T, Mima A, Sumi E, Nagai K, Takahashi T et al. 2007. Inhibition
of MCP-1/CCR2 pathway ameliorates the development of diabetic nephropathy.
Biochem Biophys Res Commun 360:772–777.
19. Godson C, Mitchell S, Harvey K, Petasis NA, Hogg N, Brady HR. 2000. Cutting edge:
Lipoxins rapidly stimulate nonphlogistic phagocytosis of apoptotic neutrophils by
monocyte-derived macrophages. J Immunol 164:1663–1667.
20. Duffield JS, Hong S, Vaidya VS, Lu Y, Fredman G, Serhan CN et al. 2006. Resolvin D
series and protectin D1 mitigate acute kidney injury. J Immunol 177:5902–5911.
21. Xiao X, Ma B, Dong B, Zhao P, Tai N, Chen L et al. 2009. Cellular and humoral immune
responses in the early stages of diabetic nephropathy in NOD mice. J Autoimmun
32:85–93.
22. Imani F, Horii Y, Suthanthiran M, Skolnik EY, Makita Z, Sharma V et al. 1993. Advanced
glycosylation end-product-specific receptors on human and rat T-lymphocytes mediate
synthesis of interferon gamma: Role in tissue remodeling. J Exp Med 178:2165–2172.
23. Turner JE, Paust HJ, Steinmetz OM, Peters A, Meyer-Schwesinger C, Heymann F et al.
2008. CCR5 deficiency aggravates crescentic glomerulonephritis in mice. J Immunol
181:6546–6556.
24. Lim AK, Ma FY, Nikolic-Paterson DJ, Kitching AR, Thomas MC, Tesch GH. 2010.
Lymphocytes promote albuminuria, but not renal dysfunction or histological damage in
a mouse model of diabetic renal injury. Diabetologia 53:1772–1782.
25. Kinsey GR, Sharma R, Huang L, Li L, Vergis AL, Ye H et al. 2009. Regulatory T cells
suppress innate immunity in kidney ischemia-reperfusion injury. J Am Soc Nephrol
20:1744–1753.
26. Zhen Y, Sun L, Liu H, Duan K, Zeng C, Zhang L et al. 2012. Alterations of peripheral
CD4+CD25+Foxp3+ T regulatory cells in mice with STZ-induced diabetes. Cell Mol
Immunol 9:75–85.
27. Eller K, Kirsch A, Wolf AM, Sopper S, Tagwerker A, Stanzl U et al. 2011. Potential role
of regulatory T cells in reversing obesity-linked insulin resistance and diabetic nephrop-
athy. Diabetes November; 60(11):2954–2962.
28. Turner JE, Paust HJ, Steinmetz OM, Peters A, Riedel JH, Erhardt A et al. 2010. CCR6
recruits regulatory T cells and Th17 cells to the kidney in glomerulonephritis. J Am Soc
Nephrol 21:974–985.
29. Tesch GH. 2008. MCP-1/CCL2: A new diagnostic marker and therapeutic target for pro-
gressive renal injury in diabetic nephropathy. Am J Physiol Renal Physiol 294:F697–F701.
30. Wang X, Chen H, Zhang M, Liu Z. 2012. Roles of mast cells and monocyte chemoat-
tractant protein-1 in the renal injury of obesity-related glomerulopathy. Am J Med Sci
346:295–301, December 18, 2012.
31. Gu L, Tseng SC, Rollins BJ. 1999. Monocyte chemoattractant protein-1. Chem Immunol
72:7–29.
32. Kato S, Luyckx VA, Ots M, Lee KW, Ziai F, Troy JL et al. 1999. Renin-angiotensin
blockade lowers MCP-1 expression in diabetic rats. Kidney Int 56:1037–1048.
33. Morii T, Fujita H, Narita T, Shimotomai T, Fujishima H, Yoshioka N et al. 2003.
Association of monocyte chemoattractant protein-1 with renal tubular damage in dia-
betic nephropathy. J Diabetes Complications 17:11–15.
34. Tarabra E, Giunti S, Barutta F, Salvidio G, Burt D, Deferrari G et al. 2009. Effect of the
monocyte chemoattractant protein-1/CC chemokine receptor 2 system on nephrin expres-
sion in streptozotocin-treated mice and human cultured podocytes. Diabetes 58:2109–2118.
35. Darisipudi MN, Kulkarni OP, Sayyed SG, Ryu M, Migliorini A, Sagrinati C et al. 2011.
Dual blockade of the homeostatic chemokine CXCL12 and the proinflammatory che-
mokine CCL2 has additive protective effects on diabetic kidney disease. Am J Pathol
179:116–124.
36. Brown FG, Nikolic-Paterson DJ, Hill PA, Isbel NM, Dowling J, Metz CM et al. 2002.
Urine macrophage migration inhibitory factor reflects the severity of renal injury in
human glomerulonephritis. J Am Soc Nephrol 13(Suppl 1):S7–S13.
37. Church TS, Willis MS, Priest EL, Lamonte MJ, Earnest CP, Wilkinson WJ et al. 2005.
Obesity, macrophage migration inhibitory factor, and weight loss. Int J Obes (Lond)
29:675–681.
38. Lan HY, Bacher M, Yang N, Mu W, Nikolic-Paterson DJ, Metz C et al. 1997. The patho-
genic role of macrophage migration inhibitory factor in immunologically induced kid-
ney disease in the rat. J Exp Med 185:1455–1465.
39. Baud L, Ardaillou R. 1995. Tumor necrosis factor in renal injury. Miner Electrolyte
Metab 21:336–341.
40. Navarro JF, Mora C, Muros M, Garcia J. 2006. Urinary tumour necrosis factor-alpha
excretion independently correlates with clinical markers of glomerular and tubulointer-
stitial injury in type 2 diabetic patients. Nephrol Dial Transplant 21:3428–3434.
41. Park CW, Kim JH, Lee JH, Kim YS, Ahn HJ, Shin YS et al. 2000. High glucose-induced
intercellular adhesion molecule-1 (ICAM-1) expression through an osmotic effect in rat
mesangial cells is PKC-NF-kappa B-dependent. Diabetologia 43:1544–1553.
42. Royall JA, Berkow RL, Beckman JS, Cunningham MK, Matalon S, Freeman BA. 1989.
Tumor necrosis factor and interleukin 1 alpha increase vascular endothelial permeability.
Am J Physiol 257:L399–L410.
43. Vesey DA, Cheung C, Cuttle L, Endre Z, Gobe G, Johnson DW. 2002. Interleukin-
1beta stimulates human renal fibroblast proliferation and matrix protein production by
means of a transforming growth factor-beta-dependent mechanism. J Lab Clin Med
140:342–350.
44. Pfeilschifter J, Pignat W, Vosbeck K, Marki F. 1989. Interleukin 1 and tumor necrosis
factor synergistically stimulate prostaglandin synthesis and phospholipase A2 release
from rat renal mesangial cells. Biochem Biophys Res Commun 159:385–394.
45. Macconi D, Chiabrando C, Schiarea S, Aiello S, Cassis L, Gagliardini E et al. 2009.

Proteasomal processing of albumin by renal dendritic cells generates antigenic peptides.
J Am Soc Nephrol 20:123–130.
46. Yang D, Chen Q, Le Y, Wang JM, Oppenheim JJ. 2001. Differential regulation of formyl
peptide receptor-like 1 expression during the differentiation of monocytes to dendritic
cells and macrophages. J Immunol 166:4092–4098.
47. Dai SM, Matsuno H, Nakamura H, Nishioka K, Yudoh K. 2004. Interleukin-18 enhances
monocyte tumor necrosis factor alpha and interleukin-1beta production induced by
direct contact with T lymphocytes: Implications in rheumatoid arthritis. Arthritis Rheum
50:432–443.
48. Marino E, Cardier JE. 2003. Differential effect of IL-18 on endothelial cell apoptosis
mediated by TNF-alpha and Fas (CD95). Cytokine 22:142–148.
49. Melnikov VY, Ecder T, Fantuzzi G, Siegmund B, Lucia MS, Dinarello CA et al. 2001.
Impaired IL-18 processing protects caspase-1-deficient mice from ischemic acute renal
failure. J Clin Invest 107:1145–1152.
50. Ouedraogo R, Gong Y, Berzins B, Wu X, Mahadev K, Hough K et al. 2007. Adiponectin
deficiency increases leukocyte-endothelium interactions via upregulation of endothelial
cell adhesion molecules in vivo. J Clin Invest 117:1718–1726.
51. Yokota T, Oritani K, Takahashi I, Ishikawa J, Matsuyama A, Ouchi N et al. 2000.
Adiponectin, a new member of the family of soluble defense collagens, negatively reg-
ulates the growth of myelomonocytic progenitors and the functions of macrophages.
Blood 96:1723–1732.
52. Ouchi N, Kihara S, Arita Y, Nishida M, Matsuyama A, Okamoto Y et al. 2001.
Adipocyte-derived plasma protein, adiponectin, suppresses lipid accumulation and class
A scavenger receptor expression in human monocyte-derived macrophages. Circulation
103:1057–1063.
53. Nakamaki S, Satoh H, Kudoh A, Hayashi Y, Hirai H, Watanabe T. 2011. Adiponectin
reduces proteinuria in streptozotocin-induced diabetic Wistar rats. Exp Biol Med
(Maywood) 236:614–620.
54. Maderna P, Godson C. 2009. Lipoxins: Resolutionary road. Br J Pharmacol 158:947–959.
55. Serhan CN, Yacoubian S, Yang R. 2008. Anti-inflammatory and proresolving lipid medi-
ators. Annu Rev Pathol 3:279–312.
56. Levy BD, Clish CB, Schmidt B, Gronert K, Serhan CN. 2001. Lipid mediator class
switching during acute inflammation: Signals in resolution. Nat Immunol 2:612–619.
57. Leonard MO, Hannan K, Burne MJ, Lappin DW, Doran P, Coleman P et al. 2002.
15-Epi-16-(para-fluorophenoxy)-lipoxin A(4)-methyl ester, a synthetic analogue of
15-epi-lipoxin A(4), is protective in experimental ischemic acute renal failure. J Am Soc
Nephrol 13:1657–1662.
58. Mitchell S, Thomas G, Harvey K, Cottell D, Reville K, Berlasconi G et al. 2002.
Lipoxins, aspirin-triggered epi-lipoxins, lipoxin stable analogues, and the resolution of
inflammation: Stimulation of macrophage phagocytosis of apoptotic neutrophils in vivo.
J Am Soc Nephrol 13:2497–2507.
59. Hassan IR, Gronert K. 2009. Acute changes in dietary omega-3 and omega-6 polyunsat-
urated fatty acids have a pronounced impact on survival following ischemic renal injury
and formation of renoprotective docosahexaenoic acid-derived protectin D1. J Immunol
182:3223–3232.
60. Dalli J, Zhu M, Vlasenko NA, Deng B, Haeggstrom JZ, Petasis NA et al. 2013. The
novel 13S,14S-epoxy-maresin is converted by human macrophages to maresin1 (MaR1),
inhibits leukotriene A4 hydrolase (LTA4H), and shifts macrophage phenotype. FASEB J
27:2573–2583.
61. Liu Y. 2006. Renal fibrosis: New insights into the pathogenesis and therapeutics. Kidney
Int 69:213–217.
62. Hewitson TD. 2009. Renal tubulointerstitial fibrosis: Common but never simple. Am J
Physiol Renal Physiol 296:F1239–F1244.
63. Phillips A. 2007. The role of proximal tubular cells in interstitial fibrosis: Understanding
TGF-beta1. Chang Gung Med J 30:2–6.
64. Kopp JB, Factor VM, Mozes M, Nagy P, Sanderson N, Bottinger EP et al. 1996.
Transgenic mice with increased plasma levels of TGF-beta 1 develop progressive renal
disease. Lab Invest 74:991–1003.
65. Chen S, Iglesias-de la Cruz MC, Jim B, Hong SW, Isono M, Ziyadeh FN. 2003.
Reversibility of established diabetic glomerulopathy by anti-TGF-beta antibodies in
db/db mice. Biochem Biophys Res Commun 300:16–22.
66. Ziyadeh FN, Hoffman BB, Han DC, Iglesias-De La Cruz MC, Hong SW, Isono M
et al. 2000. Long-term prevention of renal insufficiency, excess matrix gene expression,
and glomerular mesangial matrix expansion by treatment with monoclonal antitrans-
forming growth factor-beta antibody in db/db diabetic mice. Proc Natl Acad Sci USA
97:8015–8020.
67. Wang W, Koka V, Lan HY. 2005. Transforming growth factor-beta and Smad signalling
in kidney diseases. Nephrology (Carlton) 10:48–56.
68. Martinez FO, Sica A, Mantovani A, Locati M. 2008. Macrophage activation and polar-
ization. Front Biosci 13:453–461.
69. Rastaldi MP, Ferrario F, Giardino L, Dell’Antonio G, Grillo C, Grillo P et al. 2002.
Epithelial-mesenchymal transition of tubular epithelial cells in human renal biopsies.
Kidney Int 62:137–146.
70. Hertig A, Anglicheau D, Verine J, Pallet N, Touzot M, Ancel PY et al. 2008. Early
epithelial phenotypic changes predict graft fibrosis. J Am Soc Nephrol 19:1584–1591.
71. Rangel-Huerta OD, Aguilera CM, Mesa MD, Gil A. 2012. Omega-3 long-chain polyun-
saturated fatty acids supplementation on inflammatory biomakers: A systematic review
of randomised clinical trials. Br J Nutr 107(Suppl 2):S159–S170.
72. Priante G, Musacchio E, Valvason C, Clari G, Bordin L, Sartori L et al. 2012. Further
insights about the beneficial effects of n-3 fatty acids in the early molecular events of
renal fibrosis in vitro. J Nephrol 9:0.
73. Huang X, Stenvinkel P, Qureshi AR, Riserus U, Cederholm T, Barany P et al. 2012.
Essential polyunsaturated fatty acids, inflammation and mortality in dialysis patients.
Nephrol Dial Transplant 27:3615–3620.
74. Bowden RG, Wilson RL, Deike E, Gentile M. 2009. Fish oil supplementation lowers
C-reactive protein levels independent of triglyceride reduction in patients with end-stage
renal disease. Nutr Clin Pract 24:508–512.
75. Perunicic-Pekovic GB, Rasic ZR, Pljesa SI, Sobajic SS, Djuricic I, Maletic R et al. 2007.
Effect of n-3 fatty acids on nutritional status and inflammatory markers in haemodialysis
patients. Nephrology (Carlton) 12:331–336.
76. Cawood AL, Ding R, Napper FL, Young RH, Williams JA, Ward MJ et al. 2010.
Eicosapentaenoic acid (EPA) from highly concentrated n-3 fatty acid ethyl esters is
incorporated into advanced atherosclerotic plaques and higher plaque EPA is associ-
ated with decreased plaque inflammation and increased stability. Atherosclerosis
212:252–259.
77. Labonte ME, Couture P, Richard C, Desroches S, Lamarche B. 2013, Impact of dairy
products on biomarkers of inflammation: A systematic review of randomized con-
trolled nutritional intervention studies in overweight and obese adults. Am J Clin Nutr
97:706–717.
78. Rosa FT, Zulet MA, Marchini JS, Martinez JA. 2012. Bioactive compounds with effects
on inflammation markers in humans. Int J Food Sci Nutr 63:749–765.
79. Chagas CE, Borges MC, Martini LA, Rogero MM. 2012. Focus on vitamin D, inflam-
mation and type 2 diabetes. Nutrients 4:52–67.
80. Yuan WJ, Jia FY, Meng JZ. 2009. Effects of genistein on secretion of extracellular
matrix components and transforming growth factor beta in high-glucose-cultured rat
mesangial cells. J Artif Organs 12:242–246.
81. Elmarakby AA, Ibrahim AS, Faulkner J, Mozaffari MS, Liou GI, Abdelsayed R. 2011.
Tyrosine kinase inhibitor, genistein, reduces renal inflammation and injury in strepto-
zotocin-induced diabetic mice. Vascul Pharmacol 55:149–156.
82. Kim MJ, Lim Y. 2013. Protective effect of short-term genistein supplementation on the
early stage in diabetes-induced renal damage. Mediators Inflamm 2013:510212.
83. Fanti P, Asmis R, Stephenson TJ, Sawaya BP, Franke AA. 2006. Positive effect of
dietary soy in ESRD patients with systemic inflammation—Correlation between blood
levels of the soy isoflavones and the acute-phase reactants. Nephrol Dial Transplant
21:2239–2246.
84. Fair DE, Ogborn MR, Weiler HA, Bankovic-Calic N, Nitschmann EP, Fitzpatrick-Wong
SC et al. 2004. Dietary soy protein attenuates renal disease progression after 1 and
3 weeks in Han: SPRD-cy weanling rats. J Nutr 134:1504–1507.
85. Levey AS, Greene T, Beck GJ, Caggiula AW, Kusek JW, Hunsicker LG et al. 1999.
Dietary protein restriction and the progression of chronic renal disease: What have all
of the results of the MDRD study shown? Modification of Diet in Renal Disease Study
group. J Am Soc Nephrol 10:2426–2439.
86. Pedrini MT, Levey AS, Lau J, Chalmers TC, Wang PH. 1996. The effect of dietary
protein restriction on the progression of diabetic and nondiabetic renal diseases: A meta-
analysis. Ann Intern Med 124:627–632.
87. Trujillo J, Cruz C, Tovar A, Vaidya V, Zambrano E, Bonventre JV et al. 2008.
Renoprotective mechanisms of soy protein intake in the obese Zucker rat. Am J Physiol
Renal Physiol 295:F1574–F1582.
88. Tovar-Palacio C, Tovar AR, Torres N, Cruz C, Hernandez-Pando R, Salas-Garrido G et al.
2011. Proinflammatory gene expression and renal lipogenesis are modulated by dietary
protein content in obese Zucker fa/fa rats. Am J Physiol Renal Physiol 300:F263–F271.
12 Alzheimer’s Disease
and Inflammation
Stephen T. Chen and Gary W. Small
CONTENTS
Introduction............................................................................................................. 181
Overview of Pathogenesis of Alzheimer’s Disease................................................. 182
Overview of the Inflammatory System and AD...................................................... 183
Anti-inflammatory Treatments................................................................................ 186
Antioxidants............................................................................................................ 188
Ginkgo biloba.......................................................................................................... 190
Estrogens................................................................................................................. 191
Curcumin................................................................................................................. 192
Fatty Acids.............................................................................................................. 193
Immunotherapy....................................................................................................... 194
Additional Aβ-Lowering Therapies........................................................................ 195
Summary................................................................................................................. 196
References............................................................................................................... 197
INTRODUCTION
Just three years after Alois Alzheimer published the clinical and histopathological
features of the first case of presenile dementia in 1907 [1], Oskar Fischer hypoth-
esized that inflammation was present in the brains of patients with dementia [2];
however, only in the last two decades has this hypothesis been systematically stud-
ied and confirmed. Thanks to this relatively recent consideration, inflammatory
pathways are now essential to the discussion of the pathogenesis and progression
of Alzheimer’s disease (AD). As in heart disease, diabetes, cancer, arthritis, and
numerous other diseases, inflammation plays a central role in the pathophysiology of
AD, the most common neurodegenerative disorder of aging.
Scientists have identified several inflammatory pathways and substances that con-
tribute to the neurodegenerative process. These findings have translated to numerous
treatment and prevention studies that may eventually mitigate the impact of AD,
which afflicts an estimated 5.4 million people in the United States—one in eight
Americans over age 65—and costs approximately $200 billion in direct healthcare
costs and $210 billion in unpaid caregiving each year [3].
Under homeostatic physiological conditions, the inflammatory system can aid
the brain with tissue remodeling, neurogenesis, neural plasticity, and long-term
181
potentiation. However, when activated by pathogens related to injury or infec-

tion, inflammatory molecules and processes can have deleterious effects on brain
functioning.
OVERVIEW OF PATHOGENESIS OF ALZHEIMER’S DISEASE

Our current understanding of the pathogenesis of AD is based upon a multitude of
neuropathological, neurochemical, genetic, and neuroimaging studies. Alzheimer’s
disease was first characterized by its neuropathological changes, which include the
hallmark intracellular neurofibrillary tangles (NFTs) consisting of hyperphosphory-
lated tau protein and extracellular senile plaques formed by aggregates of the beta-
amyloid (Aβ) protein. Extensive study of the events that result in these changes has
led to several hypotheses to explain the mechanisms that lead to AD, with two of
them currently prevailing: the cholinergic and the amyloid cascade hypotheses.
The cholinergic hypothesis posits that a dysfunctional cholinergic system causes
the symptoms of AD. Clinical evidence in the early 1970s showed that medica-
tions with anticholinergic activity impaired cognitive function in older adults [4].
Biochemical evidence shortly followed, indicating neocortical deficits in the enzyme
responsible for the synthesis of acetylcholine (ACh)—choline acetyltransferase
(ChAT)—in AD brains [5–7]. Subsequent findings of reduced choline uptake (8) and
ACh release [9] and selective neurodegeneration in the nucleus basalis of Meynert
[10], a major source of cholinergic innervation, confirmed a presynaptic cholinergic
deficit in AD.
Although some studies observed a decrease in cholinergic markers only in the
more severe stages of AD [11,12], other evidence points to cholinergic deficits early
in the course of the disease. Age-related decline in cholinergic synaptic transmis-
sion has been identified [13]. In addition, imaging studies using positron emission
tomography (PET) have shown glucose metabolic deficits in brain regions receiving
cholinergic projections (frontal, parietal, temporal) in nondemented middle-aged
and older persons with the apolipoprotein E (APOE) ε4 allele [14,15]. Together,
these findings suggest that cholinergic deficits begin early and persist throughout the
course of the disease.
The amyloid hypothesis states that AD is caused by the excess accumulation and
deposition of Aβ to form neuritic plaques. Aβ is a polypeptide derived from the
proteolytic cleavage of amyloid precursor protein (APP), which is normally cleaved
by three proteases, α-, β-, and γ-secretases. The production of Aβ is orchestrated by
an initial cleavage by β-secretase, with further processing by γ-secretase to yield Aβ
polypeptides consisting of 40 (Aβ1–40) versus 42 (Aβ1–42) residues. Normally, Aβ1–40
is the predominant species of Aβ. However, under pathological conditions such as
AD, there is an accumulation of Aβ1–42, which aggregates more readily than Aβ1–40
and is believed to lead to the deposition of amyloid plaques. Neuronal dystrophy, tau
accumulation and hyperphosphorylation, and neurofibrillary tangles are believed to
result from downstream effects of Aβ accumulation [16]. These pathological cas-
cades are not completely elucidated, but Aβ can be directly neurotoxic, induce oxida-
tive stress, initiate an inflammatory response and vascular damage, and alter calcium
homeostasis [17–19].
Alzheimer’s Disease and Inflammation 183
Genetic studies suggest that amyloid and its precursors are causative in AD and
not disease markers. Mutations in the APP, presenilin 1, and presenilin 2 genes that
increase total Aβ are linked to early-onset familial AD [20–23]. APOE4, which
increases Aβ deposition, is a major risk factor for late-onset AD [24].
Results from several investigations have posed challenges to the amyloid hypoth-
esis. Perhaps the strongest opposition comes from evidence that Aβ plaque load cor-
relates poorly with cognitive impairment in AD patients [25,26]. Moreover, severity
and duration of AD correlate with neurofibrillary tangles, but not with senile plaques
[27]. Amyloid deposition is poorly correlated with other pathological markers of AD,
including synaptic and neuronal loss and cytoskeleton abnormalities [26]. Opponents
of the amyloid hypothesis argue that not only is amyloid not responsible for AD, but it
is actually protective against cellular stress and oxidative damage [28,29]. However,
individually or collectively, these perceived weaknesses are not sufficient to under-
mine the broad framework of data that supports the amyloid hypothesis, though they
do illustrate some deficiencies in our knowledge of AD.
Rather than compete with one another, the amyloid and cholinergic hypotheses
may be integrated by evidence that the two pathways converge. Acetylcholine and
Aβ can have reciprocal neuromodulatory effects. Muscarinic agonists increase the
secretion of nonamyloidogenic APP derivatives and reduce the production of amy-
loidogenic Aβ peptides [30–32]. Lesions of the basal forebrain cholinergic neurons or
transient inhibition of cortical ACh release can elevate local APP synthesis [33–35].
Insults that reduce cholinergic transmission may make cholinergic neurons more
vulnerable to the direct toxicity of Aβ [36,37]. Amyloidogenic Aβ is toxic to cholin-
ergic enzymes and neurons [38,39] and can induce a strong inflammatory response
that is accompanied by a decrease in the number of cholinergic neurons around
the amyloid deposits and hypofunction of the cortical cholinergic system [40]. The
destabilization of neuronal calcium homeostasis and the production of toxic and
inflammatory mediators are mechanisms that could explain Aβ-induced cholinergic
dysfunction and degeneration [41–43].
OVERVIEW OF THE INFLAMMATORY SYSTEM AND AD

The inflammatory system involves many components and pathways that are impli-
cated in AD, largely centered on the presence of Aβ. Pathological Aβ can activate
central nervous system (CNS) phagocytic microglia and astrocytes to secrete cyto-
kines, chemokines, and other proinflammatory molecules, such as prostaglandins
and free radicals [44–47], which can have both protective and deleterious CNS func-
tions. A summary of inflammatory mediators and their roles in AD is presented in
Table 12.1.
Microglia are the resident macrophages of the CNS. Under homeostatic condi-
tions, microglia perform surveillance for pathological changes, such as unfamiliar
microbes and protein aggregates, which then induce microglia to become activated
and perform other functions. Activated microglia can release cytokines and other
molecules that are both pro- and anti-inflammatory [48]. Certain modes of microglial
activation may be protective against AD through increasing Aβ clearance, reducing
glutamate-mediated neurotoxicity, and promoting neurogenesis [48], while strong
TABLE 12.1
Inflammatory Mediators and Their Effects on Pathophysiology and
Clinical Features of Alzheimer’s Disease
Inflammatory
Mediator Protective Effects ProInflammatory Effects References
Microglia Increases beta-amyloid (Aβ) Decreases Aβ clearance [48–51]
clearance Releases cytokines
Interleukin-1 Facilitates long-term potentiation Increases amyloid [31,52–56]
(LTP) precursor protein (APP)
synthesis
Increases
acetylcholinesterase
Decreases LTP
Interleukin-6 Anti-inflammatory Increases APP [60–65]
Immunosuppressive transcription
Tumor necrosis Neurotrophic and neuroprotective Increases expression of [68–73]
factor-α against Aβ, glutamate, reactive complement and
oxygen species cyclooxygenases (COX)
Induces expression of protective
manganese superoxide
dismutase and calbindin
Tumor growth Protects against glutamate, Aβ Stimulates prostaglandin [77–79]
factor-β E2 synthesis, expression
of COX-1 and COX-2
Chemokines Inhibits apoptosis Associated with lower [82–85]
Increases brain-derived cognitive scores and
neurotrophic factor faster cognitive decline in
Suppresses expression of humans
inflammatory mediators
Complement Unknown Activates microglia and [95–98]
astrocytes
activation might impede clearance of Aβ and increase production of proinflamma-

tory cytokines, which can further decrease the microglia’s ability to scavenge and
degrade Aβ [49–51]. Microglia are highly activated in brains with AD and surround
Aβ plaques [18], but exactly how microglia contribute to the formation of senile
plaques remains unknown.
Interleukin (IL)-1 is a cytokine that, at physiological levels, can facilitate long-term
potentiation (LTP), which is believed to be necessary for learning and memory [52].
However, in AD brains, IL-1 overexpression early in the disease promotes the syn-
thesis [53,54] and processing [31] of APP and thus of amyloid production and plaque
deposition. Excess IL-1 also induces the activity and mRNA expression of acetyl-
cholinesterase [55], which may further contribute to the cholinergic deficit in the AD
brain. Overexpression of the IL-1 receptor antagonist further impairs hippocampal
memory and LTP [56]. In transgenic mice, the deletion of the IL-1 receptor antagonist
gene increases susceptibility to infusion of Aβ and neuroinflammation [57], while

chronic overexpression of IL-1β ameliorates Aβ fibrillar plaque formation [58].
Interleukin-6 is normally barely detectable in the CNS, though it is strongly
induced under pathological conditions to be a proinflammatory cytokine that induces
destructive inflammatory and immunological responses. Animal models of overex-
pressed IL-6 demonstrate its association with CNS damage and deficits in learn-
ing and behavior [18,59]. IL-6 may modulate APP synthesis [60] and enhance APP
transcription and expression [61]. Although the overexpression of IL-6 is generally
detrimental and associated with CNS pathology, IL-6 may also have antiinflamma-
tory, immunosuppressive, and other beneficial properties under certain experimental
conditions [62–65].
Although tumor necrosis factor-alpha (TNF-α) is elevated in AD serum [66], cere-
brospinal fluid (CSF), and cortex [67] and has been shown to be a potent proinflam-
matory, cytotoxic cytokine in other CNS disorders, its role in AD remains debated.
TNF-α has been reported to be neurotrophic [68] and neuroprotective against gluta-
mate, free radicals, and Aβ toxicity [69]. TNF-α induces the expression of protective
molecules such as manganese superoxide dismutase [70] and calbindin [71], as well as
proinflammatory molecules such as complement and cyclooxygenase (COX) [72,73].
Transforming growth factor-betas (TGF-βs) modulate processes implicated in
AD, including brain injury and inflammatory response, production and distribution
of amyloid, regulation of APP, APOE, and COX-2, and inhibition of cell death [18].
TGF-βs have been found in AD plaques [74], AD brains [75], and CSF [76]. TGF-βs
stimulate prostaglandin-E2 synthesis [77] and increase the expression of COX-1 and
COX-2 [78]. On the other hand, TGF-βs have also been shown to protect against Aβ
and glutamate neurotoxicity [79].
Chemokines are cytokines that induce chemotaxis, tissue extravasation, and func-
tional modulation of leukocytes during inflammation [80]. Chemokines and their
receptors are upregulated in resident CNS cells in the AD brain and may contrib-
ute to plaque-associated inflammation and neurodegeneration [81]. Raised chemo-
kine (C-C motif) receptor 2 (CCR2) expression was strongly associated with lower
minimental state exam (MMSE) scores in older adults [82]. In transgenic mice
expressing APP and presenilin 1, CCR2 deficiency aggravates memory deficits and
amyloid pathology and stimulates expression of inflammatory mediators [83]. Levels
of CCR2’s main ligand, CCL2, in CSF was found to be associated with a faster
cognitive decline in patients with mild cognitive impairment (MCI) [84]. However,
chemokines may also have roles that protect against AD. In AD brain tissue, che-
mokine CXCL8 inhibited Aβ-induced neuronal apoptosis and increased neuronal
brain-derived neurotrophic factor production [85]. Ablation of chemokine CX3CR1
in mice overexpressing human APP enhanced the neurotoxic effects of inflammatory
cytokines and impaired memory retention [86].
The classical complement pathway is made up of more than 20 components that
can be sequentially activated as an amplifying cascade. Neuropathological mark-
ers of AD, including Aβ [87–90], tau-containing neurofibrillary tangles [91], and
neurodegeneration by-products, extracellular DNA [92,93], neurofilaments [94], and
myelin fragments [93], are all potential sources of complement activation. The activa-
tion of complement produces anaphylatoxins and opsonins that provide chemotactic
and activating signals to microglia and astrocytes [95–98], thereby stimulating fur-
ther inflammatory changes. In the AD brain, complement activation fragments,
reactive astrocytes, and activated microglia are all highly colocalized with plaques
containing aggregated Aβ [89,99–101].
While not a hallmark of AD, vascular pathology is present in 30%–60% of AD
patients [102]. Moreover, AD pathology is present in 40%–80% of vascular dementia
patients [103,104]. A link between heart disease and AD pathology has also been
described: increased Aβ deposits have been observed in the brains of nondemented
heart disease patients [105,106]. A common pathway may be chronic inflamma-
tion, which increases risk of atherosclerosis and has been documented in AD brains
[107–109]. APOE4 is a risk factor for developing both atherosclerosis and late-onset
AD [110]. These relationships suggest that neurovascular damage is a primary occur-
rence and that subsequent injuries, including Aβ deposition, amplifies and/or exacer-
bates vascular damage that then leads to neurodegenerative processes and ultimately
cognitive decline [111].
ANTI-INFLAMMATORY TREATMENTS
While we do not know whether inflammation in AD might be a cause and/or effect
of the disease, we do know that the inflammatory response is localized around the
Aβ plaques [112]. Regardless of whether Aβ deposition precedes inflammation, once
that deposition begins, it is thought to activate proinflammatory glial cells to pro-
duce inflammatory molecules [18], which may lead to a continuous cycle of Aβ
deposition and inflammation. Nonsteroidal anti-inflammatory drugs (NSAIDs) may
disrupt this pathogenic cycle by inhibiting COX, which converts arachidonic acid
to several prostaglandins, hormones that help recruit and organize inflammatory
responses [113,114]. Some NSAIDs have the capability of reducing plaque bur-
den independently of COX by modulating the activity of γ-secretase to cleave APP
to yield more benign Aβ1–40 and less toxic Aβ1–42 [115]. These NSAIDS are also
referred to as selective Aβ-lowering agents (SALAs). Ibuprofen and indomethacin
are SALAs, while aspirin, naproxen, and celecoxib are not.
Epidemiological studies during the 1990s reported that arthritis or anti-
inflammatory drug use was associated with reduced prevalence of AD. A 1996
meta-analysis of seven of these studies suggested that anti-inflammatory drugs have
a protective effect against AD, with an odds ratio of 0.556 (p = 0.0001) [116]. Later
nonprospective studies focused specifically on NSAID use, with the majority report-
ing that greater NSAID use was associated with lower AD prevalence [117]. A meta-
analysis of these data, which included 1,833 AD cases and 13,780 controls, yielded an
overall odds ratio of 0.47, 95% confidence interval (CI) = 0.36–0.62 [117]. Prospective
studies later reported favorable, though not as robust, results for NSAIDs. A meta-
analysis of five prospective studies [118–122], which included 836 incident AD cases
and 16,294 controls, yielded an overall risk ratio (RR) for any lifetime use of nonaspi-
rin NSAIDs and AD of 0.71 (CI 0.58–0.87) [117]. In the three studies in which duration
of use was available [118,119,121], the combined RR for two or more years of NSAID
use was 0.42 (CI 0.26–0.66). The overall RR for any lifetime use of aspirin was 0.83
(CI 0.59–1.17) and for aspirin use greater than two years was 0.73 (CI 0.55–0.97) [117].
Subsequent studies have reported mixed results, two showing that NSAIDs may pro-
tect against AD [123,124], and two showing no protective effect [125,126].
Randomized controlled trials of NSAIDs for the prevention of AD are few in
number. The Alzheimer’s disease anti-inflammatory prevention trial (ADAPT),
a randomized controlled study of naproxen, celecoxib, and placebo in over 2000
elderly asymptomatic individuals with a family history suggesting increased risk
of AD [127] began in 2001, but was suspended in 2004 by the US Food and Drug
Administration (FDA) because of an apparent increase in cardiovascular and cere-
brovascular events with naproxen, but not with celecoxib, compared to placebo. At
that point, after an average 24 months of treatment, the analysis suggested a possible
increase in risk of AD with either NSAID versus placebo, with hazard ratios (HR)
of 1.99 (CI 0.80–4.97, p = 0.14) for celecoxib and 2.35 (CI 0.95–5.77, p = 0.06) for
naproxen [128]. After treatment was suspended, investigators continued to follow
subjects for incident AD cases, the primary outcome of the study. Results from the
extended observation period of 18–24 months showed that the early NSAID-related
harm was no longer evident, though secondary analyses showed that increased risk
remained notable in the first 2.5 years of observations, especially in subjects enrolled
with cognitive impairment but no dementia (CIND) [127]. These subjects had HRs of
3.2 (CI 0.72–13.8) for naproxen and 4.0 (CI 1.00–15.6) for celecoxib. Secondary anal-
yses excluding CIND subjects yielded higher HRs in the first 2.5 years of the study,
2.50 (CI 0.72–8.7) for naproxen and 3.11 (CI 0.92–11) for celecoxib, but lower HRs
during the extended observation period, 0.33 (CI 0.11–0.98) for naproxen and 0.64
(CI 0.28–1.5) for celecoxib. The results of these secondary analyses indicate that
asymptomatic individuals treated with NSAIDs have a reduced risk of developing
AD, but only after an interval of two to three years, consistent with findings from
some of the earlier studies discussed. The authors hypothesized that subjects who
developed dementia early in the study, most of whom had CIND or lower baseline
cognitive scores, probably had substantial AD pathology at enrollment and that the
NSAIDs had an adverse effect on AD pathogenesis in its later stages. This hypoth-
esis was based on findings that inhibition of COX-2 and its role in the transduction
of postsynaptic signs from N-methyl d-aspartate-type glutamate receptors decreases
the efficiency of such signaling [129] and could provoke increased presynaptic
stimulation and possibly produce a deleterious effect on already dysfunctional neu-
rons, as in individuals with early or presymptomatic AD and CIND. Heavy NSAID
use was also found to be associated with greater neuritic plaque accumulation in a
population-based study [130].
In another AD prevention study, Thal and colleagues conducted a randomized,
double-blind study of 1457 patients with MCI to investigate whether rofecoxib could
delay conversion to clinical AD [131]. The estimated annual AD incidence rates were
lower than the anticipated 10%–15% for MCI, but actually higher in the rofecoxib
group (6.4%) than in the placebo group (4.5%). The treatment groups did not differ
in measures of cognition and global function. The authors concluded that COX-2
inhibition is not a useful therapeutic approach in AD.
In a study of the effects of the COX-2 inhibitor, celecoxib (200 or 400 mg), on
cognitive performance and regional cerebral glucose metabolism in nondemented
volunteers with mild age-related memory decline, the investigators randomized
88 subjects, aged 40–81 years (mean: 58.7, SD: 8.9 years) to 18 months of exposure to
active drug or placebo. Forty subjects completed the study. Subjects in the celecoxib
group showed benefits in executive functioning and language/semantic memory
compared with the placebo group. Concomitantly, positron emission tomography
(PET) scans of regional glucose metabolism demonstrated significant bilateral meta-
bolic increases in prefrontal cortex in the celecoxib group but not in the placebo
group. Results from this small study suggest that daily celecoxib use may improve
cognitive performance and increase regional brain metabolism in people with age-
associated memory decline [132].
Among patients with a diagnosis of AD, randomized controlled trials provide weak
support for NSAIDs, delaying the progression of AD (Table 12.2). Animal models
have demonstrated that NSAIDs prevent early AD-related pathogenic events before the
onset of Aβ deposition, but fail to reverse existing pathogenic changes [133]. This find-
ing suggests that NSAID use must begin very early in the disease process, well before
the onset of symptoms, to be effective in AD prevention or delay and may offer an
explanation for studies not demonstrating any benefit from NSAID use. Another possi-
ble explanation for the disappointing results is the relatively short duration of treatment,
two years or less in each study. The one group that appeared to benefit from an NSAID
was treated for two years [134]. Data from the observational and prevention studies
reviewed suggest that longer duration of treatment yields more favorable outcomes.
ANTIOXIDANTS
Oxidative stress may play a key role in the pathogenesis of AD and other neurode-
generative conditions via inflammatory mediators. Reactive oxygen species (ROS)
are chemically unstable molecules that are formed through oxidative processes and
efficiently scavenged by endogenous antioxidants under physiological conditions.
However, in conditions that induce an inflammatory response, activated microglia
and Aβ peptides can activate oxidative processes and generate excess ROS that can-
not be destroyed, resulting in oxidative stress [135,136]. Oxidative stress may mani-
fest as DNA, RNA, protein oxidation, or lipid peroxidation, all of which have been
described in AD [137]. Antioxidants have been studied as a possible treatment or
preventive strategy for AD on the premise that they reduce oxidative damage to cel-
lular components.
Early evidence for the utility of antioxidants in AD came from epidemiologi-
cal studies. In a study of over 4000 elderly individuals, lower vitamin E serum
levels were associated with decreasing memory, though levels of vitamins A and C,
β-carotene, and selenium were not [138]. In the Honolulu–Asia aging study, use of
vitamins C and E was not protective against onset of AD, and use of either vitamin
was protective against vascular and other dementias and associated with better cogni-
tive performance in nondemented men [139]. However, longitudinal data from that
same study did not find that the dietary intake of antioxidants modified the risk of
developing dementia [140]. Long-term use of vitamins C and E, but not either alone,
was associated with better cognitive performance in a large study of elderly women
in the Nurses’ Health Study [141]. The Chicago Health and Aging Project questioned
elderly community residents about dietary antioxidant intake and found that high
TABLE 12.2
Published Randomized Controlled Studies on Efficacy of Nonsteroidal Anti-Inflammatory Drugs in Patients with Alzheimer’s
Disease (AD)
Lead Author Findings (Treatment
[Reference] Year Agent, Daily Dose N Study Group Duration Outcome Measures vs. Placebo)
Scharf et al. [276] 1999 Diclofenac/Misoprostol 41 Mild-moderate AD 25 weeks ADAS-Cog, MMSE No significant difference
Aisen et al. [275] 2003 Rofecoxib 25 mg daily 351 Mild-moderate AD 1 year ADAS-Cog No significant difference
Naproxen 220 mg twice daily
Alzheimer’s Disease and Inflammation
Reines et al. [274] 2004 Rofecoxib 25 mg 481 Mild-moderate AD 1 year ADAS-Cog No significant difference
Wilcock et al. [134] 2008 Tarenflurbil 400 mg or 800 mg 210 Mild-moderate AD 1 year + 1 year ADAS-Cog,ADCS- Mild AD/800 mg/2 years:
twice daily extension ADL, CDR-sb lower rates of decline in
all measures than placebo
1 year + tarenflurbil 1 year
De Jong et al. [277] 2008 Indomethacin 100 mg 51 Mild-moderate AD 1 year ADAS-Cog, MMSE No significant difference
Green et al. [262] 2009 Tarenflurbil 800 mg twice daily 1046 Mild-moderate AD 18 months ADAS-Cog No significant difference
Pasqualetti et al. [278] 2009 Ibuprofen 400 mg twice daily 132 Mild-moderate AD 1 year ADAS-Cog No significant difference
Aisen et al. [279] 2002 Nimesulide 100 mg twice daily 40 Probable AD 12 weeks ADAS-Cog No significant difference
Note: ADAS-Cog, cognitive subscale of the AD assessment scale; MMSE, mini-mental state exam; CDR-sb, clinical dementia rating sum of boxes.
189
vitamin E intake, but not vitamin C or carotene, was associated with lower rates of
cognitive decline [142]. A separate analysis from this study found, among the high
vitamin E intake group, only non-APOE4 carriers had a lower risk of developing AD
[143]. Similarly, in the Rotterdam study, an unexpected subgroup, current smokers
who had a high intake of antioxidants, had the lowest risk of developing AD, though
overall, high intake of vitamins C and E was associated with a lower risk of AD [144].
Three randomized controlled trials of vitamin E and cognitive decline have been
published. In a two-year study of patients with moderate AD, the group that received
2000 IU/day of vitamin E survived 230 days longer than the placebo group in delaying
one of the following: death, institutionalization, loss of ability to perform basic activi-
ties of daily living, or severe dementia [145]. Vitamin E intake, however, did not influ-
ence the rate of decline based on cognitive testing. Among subjects with MCI, vitamin
E did not reduce the probability of converting to dementia during the 3-year treatment
[146]. A recent study of mild to moderate AD reported that vitamin E, taken with other
antioxidants for 16 weeks, not only provided no benefit, but actually accelerated cogni-
tive decline, though it did reduce a CSF oxidative stress biomarker [147].
The positive clinical trials using vitamin E led many clinicians to prescribe high
doses until an analysis of existing studies pointed to increased mortality associated
with high-dose vitamin E use. A meta-analysis of 19 randomized controlled trials
that included over 135,000 patients with a variety of medical conditions cautioned
that high-dose vitamin E (> = 400 IU/day) may increase mortality [148], particularly
in older patients and those with pre-existing cardiac conditions.
Explanations for the lack of benefit from vitamin E in clinical trials have been
proposed [149]. Incorrect dosing could accelerate cognitive decline [147], increase
mortality [148], or result in a suboptimal redox potential that would not reduce oxi-
dative stress [150]. Studies involving MCI and/or AD may be too late in the disease
process for antioxidants to significantly influence cognitive outcomes. Vitamin E,
a lipophilic compound, may need to be coupled with a water-soluble antioxidant such
as vitamin C to protect against oxidation of aqueous-phase nucleic acids and pro-
teins. Perhaps more important than eliminating ROS, which at physiological levels
perform vital cellular functions, is maintaining redox potential that may be disturbed
by excess antioxidants [151]. Also, the particular form of vitamin E (e.g., α-tocopherol
vs. γ-tocopherol) used in clinical trials could influence results.
Other readily available antioxidants include polyphenols, which have demon-
strated neuroprotective effects across different model systems. Resveratrol, quer-
cetin, and (+)-catechin are compounds in red wine that have been shown to prevent
hippocampal cell death and intracellular ROS accumulation [152]. In cultured rat
pheochromocytoma cells, resveratrol attenuated Aβ-induced cytotoxicity, apoptotic
features, and intracellular ROS accumulation [153]. Phase II clinical trials examin-
ing the effects of resveratrol on neurodegenerative diseases are ongoing [154].
GINKGO BILOBA
Ginkgo biloba extract (EGb 761) is one of the most widely used and studied herbal
remedies for dementia and cognitive impairment [155] and appears to have anti-
inflammatory and antioxidant effects. EGb 761 has been shown to reduce tissue
levels of ROS and inhibit membrane lipid peroxidation [156]. Ginkgolide B, a bio-
logically active constituent of EGb 761, ameliorated the neurological injury and
expression of inflammatory mediators in the brain tissue of rats subjected to cerebral
ischemia–reperfusion [157].
Despite over 750 publications of clinical trials involving ginkgo products and cogni-
tive decline or dementia, the use of ginkgo to treat cognitive disorders remains a subject
of controversy [155]. Many of these studies had methodological limitations, such as very
small samples [158], acute administration [159–161], brief treatment durations [162],
combinations of agents [163], or inclusion of younger healthy volunteers [160,163].
The Ginkgo Evaluation of Memory (GEM) study is the largest completed ran-
domized, double-blind, placebo-controlled dementia prevention trial to date. The
GEM randomized 3069 community-dwelling participants aged 72–96 years with no
or mild cognitive impairment to EGb 761 120 mg twice daily or placebo and admin-
istered annual comprehensive neuropsychological test batteries for median period of
6.1 years. Results showed that the incidence of AD and the rate of cognitive decline
were no different between EGb 761 and placebo [164,165].
A recent meta-analysis of patients with dementia yielded more favorable results
for EGb 761. Only nine clinical trials met its study criteria, which included a diag-
nosis of AD, vascular or mixed dementia, use of the standardized extract EGb 761,
a minimum treatment duration of 12 weeks, a minimum number of participants of
ten per group, and the availability of a full-text publication [155]. All trials included
2372 patients with mild to moderate dementia and were randomized, double blinded,
and, with one exception, placebo controlled. Cognitive outcomes for patients treated
with EGb 761 were significantly better than for those treated with placebo for all
patients with dementia, as well as for the subgroup of patients with AD. Among all
patients with dementia, standardized change scores were greater for ginkgo than for
placebo, with the standardized mean difference (SMD) = −0.58 (95% CI −1.14 −0.01,
z = 2.01, N = 7, p = 0.04), indicating a moderate treatment effect, though heterogene-
ity, or extent of differences between individual studies, was substantial (χ2 = 178.92,
I2 = 97%). Separate analyses for the AD subgroup also yielded greater standardized
change scores for ginkgo than for placebo, with SMD = −0.63 (95% CI −1.16 −0.10,
z = 2.35, N = 6, p = 0.02), but also revealed high heterogeneity (χ2 = 95.96, I2 = 95%).
The authors of the meta-analysis stated that none of the studies sufficiently con-
sidered criteria for external validity [166]. Some studies tried to assure high internal
validity by excluding patients with somatic or psychiatric comorbidity and not allow-
ing concomitant medications, thereby limiting generalizability, although the setting
in most of the studies included patients being treated by outpatient clinics or practice-
based physicians. The study excluded over 95% of publications on ginkgo and dementia
or cognitive decline, indicating probable further limitation of generalizability. Other
review publications similarly excluded the vast majority of related studies [167,168].
ESTROGENS
Estrogens protect against Aβ neurotoxicity through anti-inflammatory mechanisms.
Estradiol can downregulate inflammatory gene expression in the brain [169–171]
and reduce hippocampal neuronal loss and microglial activation surrounding Aβ
plaques in ovariectomized mice [172]. Estradiol increases APP expression in neu-

rons [173–175] and reduces Aβ peptide production while enhancing its clearance
[175–177]. In AD transgenic mouse models, ovariectomy significantly increases Aβ
accumulation and worsens memory performance, while chronic estradiol treatment
prevents these effects [178].
Efficacy data from clinical trials of estrogen therapy are mixed. Among the five
placebo-controlled studies that examined clinical outcomes in women with AD, three
showed favorable results for estrogen therapy, including superior visual and semantic
memory [179], better mood and word learning in patients without APOE-4 [180],
and greater attention, verbal, semantic, and visual memory [181]. By contrast, two
studies showed no significant treatment effect on cognition, function [182,183], or
disease progression [183] The five studies were relatively small, the largest enrolling
120 subjects [183]. An open comparison to tacrine, an acetylcholinesterase inhibitor,
showed no differences in cognition or mood, but greater function with estrogen in
postmenopausal women with AD [184]. However, another study of women with AD
showed that estrogen therapy may enhance the cognitive and overall clinical effects
of tacrine [185]. Two epidemiological studies of elderly postmenopausal women also
showed contrasting results: one study found that estrogen plus progestin increased
the risk of dementia and did not prevent MCI [186], while another found that fre-
quency of estrogen use was higher among those without AD than those with AD,
even after adjusting for age, education, ages at menarche and menopause, smoking
and alcohol use, body weight, and number of children (odds ratio 0.28, CI 0.08–0.98)
[187]. However, the effects of estrogen therapy on risk of dementia may depend on
its timing: use of estrogen in midlife may protect against dementia, whereas estrogen
initiation in late life could raise the risk of dementia [188].
CURCUMIN
Curcumin, derived from the plant Curcuma longa and found in the Asian spice
turmeric, has demonstrated anti-inflammatory effects through various mechanisms
that may have a beneficial effect in AD. Anti-inflammatory effects are based on the
inhibition of transcription of cytokines, nitric oxide synthase (NOS), and COX-2.
Curcumin has an inhibitory effect on Aβ aggregation [189] and Aβ-induced DNA
damage, tau hyperphosphorylation, increase in intracellular calcium, reduction of
antioxidant levels [190], and generation of ROS [191]. Curcuminoids have been
shown to repair immune defects in AD patients [192] and to inhibit acetylcho-
linesterase, the primary enzyme that breaks down acetylcholine [193]. Most in
vivo animal studies show positive effects with curcumin in reducing Aβ, plaque
burden, and tau phosphorylation [194–198]. To date, there have been only five
clinical trials involving curcumin and AD patients. The only study with published
data showed no significant difference in MMSE scores and Aβ1–40 levels between
groups of possible or probable AD patients after receiving 0, 1, or 4 g of curcumin
daily for six months [199]. The absence of a treatment effect may relate to the
particular form of curcumin used, its bioavailability and dosing, duration of treat-
ment, and stage of illness. Curcumin may be more effective in protecting the brain
from neurodegeneration if ingested in mild stages of illness and for longer periods.
Also, it is not clear whether curcumin extract from supplements is as effectively

absorbed compared to when it is ingested when mixed in oils used in cooked Asian
dishes. However, curcumin may not need to penetrate the blood–brain barrier to
exert a systemic anti-inflammatory effect, which could be triggered by reaction
with gastrointestinal immune cells. Questions regarding the effects of curcumin on
AD warrant further study.
FATTY ACIDS
The omega-3 long-chain polyunsaturated fatty acids (n-3 LC PUFAs)
eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are crucial to nor-
mal brain development and function and play important roles in neuronal growth,
development of synaptic processing of neural cell interaction, and expression of
genes regulating cell differentiation and growth [200]. Later in life, n-3 LC PUFAs
enhance brain function by promoting synaptic activity, neurogenesis, and dendritic
spine density [201–203]. n-3 LC PUFAs also have antioxidative stress and anti-
inflammation effects, protecting against age-related neuronal damage [201]. In aged
rats, EPA attenuates inflammatory changes associated with the age-related deficit in
hippocampal long-term potentiation [204]. In a mouse model of ischemic stroke, a
bioactive DHA derivative inhibited two major steps in post-stroke neuronal injury,
lipid peroxidation, and leukocyte infiltration [205]. These findings indicate potential
mechanisms by which n-3 LC PUFAs help maintain neuronal health by reversing
age-related inflammation changes.
Studies suggest that dietary n-3 LC PUFAs can influence age-related cognitive
changes. DHA concentration in the brain decreases with age in humans [206] and
rats [207]. Supplementation with n-3 LC PUFAs improves memory or spatial task
performance in aged mice [208] and in rats depleted of n-3 LC PUFAs [209,210]. In
humans, studies consistently demonstrate that higher intake of fish, the major source
of n-3 LC PUFAs, is related to less cognitive decline [211–214], lower incidence of
dementia [212,215,216], and better cognitive performance [217–219]. However, vari-
able associations have been found between dietary intake levels of n-3 LC PUFAs
and cognitive outcomes; only a handful of the aforementioned studies that also
examined relationships between cognitive outcomes and dietary intake levels of n-3
LC PUFAs have found significant positive relationships [211,216,217].
Higher concentrations of n-3 LC PUFAs in plasma or erythrocytes have been
associated with better cognitive function, less cognitive decline, or lower risk of
developing dementia in cognitively normal older adults in both cross-sectional and
prospective studies [220–223]. By contrast, the study by Laurin et al. found no sig-
nificant difference in n-3 LC PUFA concentrations between controls and both preva-
lent cases of cognitive impairment and dementia in its cross-sectional analysis. In the
prospective analysis, a higher EPA concentration was found in cognitively impaired
cases compared to controls, while higher DHA, omega-3, and total PUFA concentra-
tions were found in dementia cases [224].
Randomized controlled trials of n-3 LC PUFA supplementation on cognitive
functioning in the elderly have yielded less positive results. Patients with mild to
moderate AD who took n-3 LC PUFAs did not experience different rates of cognitive
decline than those who took placebo [225–227]. The smallest of those studies
included 23 subjects with MCI, who did show significant improvement in cognitive
performance after 24 weeks on n-3 LC PUFAs compared to placebo [225]. Among
the three studies that enrolled cognitively healthy individuals, two studies found no
overall effect of DHA and EPA supplementation on cognitive performance despite
higher plasma levels [228,229]. The third study showed that DHA supplementation
did improve immediate and delayed verbal memory scores, but not working memory
or executive function tests [230].
IMMUNOTHERAPY
Immunotherapies are in development to target reducing Aβ and subsequent plaque for-
mation and possible downstream effects of Aβ such as inflammation. Immunizations
are either active, with full-length Aβ or Aβ analogues together with an adjuvant,
or passive, with humanized anti-Aβ antibodies or intravenous immunoglobulins. In
1999, Schenk and colleagues reported the first promising results of Aβ immuno-
therapy, showing that active vaccination with Aβ1–42 and Freund’s adjuvant not only
prevented Aβ accumulation in younger transgenic mice that overexpress APP, the Aβ
precursor, but also cleared pre-existing amyloid plaques in older animals [231]. The
first human immunotherapy trial used active immunization, aggregated Aβ1–42 and
the QS-21 adjuvant in patients with probable AD and demonstrated good safety and
tolerability and a high antibody response in Phase I [232], but was halted in Phase II
after 6% of the treatment group developed meningoencephalitis [233], perhaps due
to a T-cell response against Aβ [234]. However, the clinical outcomes were positive:
antibody responders performed better in the neuropsychological test battery at the
end of the study [235] and demonstrated less functional decline several years after
the study [236] than the placebo group.
Because of the adverse effects of active vaccination, attention turned to pas-
sive immunization with humanized monoclonal antibodies or immunoglobulins
[237,238], which bind to either Aβ plaques and other Aβ aggregates in the brain and
thereby induce Aβ clearance by microglia or to soluble Aβ in the periphery, which
clears Aβ before ever reaching the brain [239]. Preclinical studies of transgenic AD
mice have shown that passive administration of antibodies directed against Aβ enter
the brain, reduce amyloid burden in brain parenchyma and vasculature [240–243],
and improve cognition [244].
Bapineuzumab, a humanized monoclonal antibody to Aβ, has demonstrated
effects on both clinical and pathological markers. When administered to patients
with mild to moderate AD, bapineuzumab resulted in less cognitive decline among
study completers and APOE4 noncarriers than placebo [245]. Bapineuzumab-
treated patients had decreases in CSF tau, which may indicate downstream effects on
the degenerative process [246]. Treatment with bapineuzumab for 78 weeks reduced
cortical retention of Carbon-11-labeled Pittsburgh compound B, a marker of corti-
cal fibrillar Aβ load in vivo, compared with both baseline and placebo, but did not
demonstrate cognitive benefits [247].
Similar to active immunization, passive immunization has not been without diffi-
culties. Microhemorrhages associated with cerebral amyloid angiopathy (CAA) were
increased in APP transgenic mice treated with some antibodies [248,249]. Amyloid-
related imaging abnormalities (ARIA) suggestive of microhemorrhages, vasogenic
edema, sulcal effusions, and hemosiderin deposits have been reported in AD patients
treated with bapineuzumab [250]. Several mechanisms may be responsible for these
microhemorrhages, including increased T-cell activation [251], the matrix metallo-
proteinase protein degradation system [252], and interactions of anti-Aβ antibodies
with vascular amyloid causing structural fragility of degenerated vessel walls [248].
Another possible explanation might be related to the interaction between antibodies
and effector cells such as macrophages and microglia. Deglycosylated antibodies,
which reduce this interaction, remain effective in clearing amyloid plaques while
reducing microhemorrhages in transgenic mice [253]. Not limited to passive immu-
nization, increased vascular amyloid and microhemorrhage have also been observed
with active immunization [254].
Until recently, immunotherapy trials have only included patients with clinical
signs of AD. In May 2012, crenezumab was selected for the first trial of a
humanized monoclonal antibody against Aβ1–40 and Aβ1–42 on individuals with
no signs of dementia to investigate whether early intervention can help prevent or
slow the disease. The drug will be tested among members of an extended family
of about 5000 people from the Antioquia region of Colombia, about one-third of
whom carry the presenilin 1 gene and may experience symptoms of AD as early as
the fourth decade of life. The drug manufacturer hopes to enroll the first patients
in early 2013 and have the first interim analysis in early 2017. (Huffington Post,
May 15, 2012)
ADDITIONAL Aβ-LOWERING THERAPIES

Therapies aimed at curbing the production of Aβ, and thus its proinflammatory
effects, are being developed. Inhibition of β- and γ-secretases, which convert APP
into Aβ, has been the primary focus of intervention. Certain γ-secretase inhibi-
tors have been shown to decrease soluble Aβ levels and Aβ accumulation in ani-
mal studies [255–257]. In human clinical trials, γ-secretase inhibitor semagacestat
(LY450139) reduces Aβ concentrations in plasma [258–260], but showed Aβ reduc-
tion in the CNS in only one study, conducted on healthy male volunteers [261]. In its
only trial of clinical efficacy, semagacestat had no effect on cognitive or functional
decline among patients with mild to moderate AD [260,262]. The development of
β-secretase inhibitors has been limited by loss of potency in cellular systems, low
oral bioavailability/high metabolic clearance, inadequate CNS penetrance, and toxi-
cology findings [263].
Additional targets of interventions along the amyloid cascade are the inhibition of
Aβ aggregate formation and the removal of Aβ. Tramiprosate is a glycosaminoglycan
mimetic that reduces brain and plasma levels of Aβ and prevents the formation of
neurotoxic aggregates that lead to amyloid plaque deposition in the brain [264,265].
In limited clinical trials, patients with mild to moderate AD treated with tramipro-
sate trended toward slower cognitive decline [266,267] and suffered less hippocam-
pal volume loss than those who were given placebo [266,268]. Aβ catabolism may
be facilitated by agents that inhibit plasminogen activator inhibitor-1 and thereby
generate more plasmin, a protease that degrades Aβ oligomers and monomers [269].
One such agent significantly lowered plasma and brain Aβ levels, restored long-term
potentiation deficits in hippocampal slices, and reversed cognitive deficits in trans-
genic Aβ-producing mice [270]. Facilitators of Aβ degradation remain in the early
stages of clinical testing.
SUMMARY
The study of inflammation demonstrates its increasing importance in the pathogen-
esis of AD and may hold the key to developing effective prevention and treatment
strategies for those who are at risk of and who suffer from the disease. Preclinical
studies have shown that inflammatory pathways are intimately connected to the pres-
ence of Aβ, which aggregates and is deposited to form neuritic plaques, one of the
pathological hallmarks of AD. Despite elucidating these inflammatory mechanisms,
more than a century after Alois Alzheimer first defined its clinical and histopatho-
logical features, AD continues to confound scientists in discovering a satisfactory
remedy. At present, the only drugs approved by the FDA for the treatment of AD,
acetylcholinesterase inhibitors and memantine, are not known to directly affect the
inflammatory pathways that are involved in AD and demonstrate only modest tran-
sient symptomatic benefit and no evidence of significantly modifying disease pro-
gression [271]. Unfortunately, among the many pharmacological agents reviewed
here that do affect these inflammatory pathways, none has shown adequate promise
in clinical trials for the prevention or treatment of AD.
The difficulty in observing a robust treatment effect in AD may lie in the natural
course of the disease, which begins years before the onset of symptoms or signs
and typically progresses slowly. Deleterious inflammatory responses can occur at
all ages, and which ones may impact diseases such as AD is unknown. The esti-
mated time frame for pathological Aβ to accumulate to levels found in patients with
clinical AD is 10–15 years [272]. The current prevailing methodology of testing anti-
inflammatory or anti-Aβ therapies in patients with dementia or even MCI, in whom
the neurodegenerative process has already begun, for no more than a few years,
does not conform to what is known from preclinical data, has yielded disappointing
results, and will likely continue to result in no observable treatment benefit. The most
likely successful therapies will need to intervene well before symptoms are evident,
thus requiring sensitive early detection methods, and be monitored for many years,
with a minimum time frame of 15–20 years proposed by some [272]. Remarkable
advances in AD biomarker techniques have been made in the past decade [273] and
must continue to further techniques to reliably predict who will most likely develop
AD and who will respond to which treatments at what stages of disease and to moni-
tor the biochemical and clinical responses. Clearly, advancing these biomarker tech-
niques and designing and implementing such protracted clinical trials will require
monumental efforts to overcome substantial financial, regulatory, and scientific
hurdles. However, the costs of not developing more effective therapies cannot be sus-
tained. The successful development of such therapies will almost certainly depend
on the further exploration of resident mediators and therapeutic modifiers of brain
inflammation.
REFERENCES
1. Alzheimer A. 1907. Über eine eigenartige Erkrankung der Hirnrinde. Allgemeine
Zeitschrift für Psychiatrie und psychisch-gerichtliche Medizin 64:146–148.
2. Fischer O. 1910. Die presbyophrene Demenz, deren anatomische Grundlage und
klinische Abgrenzung. Zeitschrift für die gesamte Neurologie und Psychiatrie 3:371–71.
3. Alzheimer’s Association. 2012. 2012 Alzheimer’s disease facts and figures. Alzheimers
Dementia 8:131–168.
4. Drachman DA, Leavitt J. 1974. Human memory and the cholinergic system. A relation-
ship to aging? Arch Neurol 30:113–121.
5. Davies P, Maloney AJ. 1976. Selective loss of central cholinergic neurons in Alzheimer’s
disease. Lancet 2:1403.
6. Bowen DM, Smith CB, White P et al. 1976. Neurotransmitter-related enzymes and indi-
ces of hypoxia in senile dementia and other abiotrophies. Brain J Neurol 99:459–496.
7. Perry EK, Perry RH, Blessed G et al. 1977. Necropsy evidence of central cholinergic
deficits in senile dementia. Lancet 1:189.
8. Rylett RJ, Ball MJ,Colhoun EH. 1983. Evidence for high affinity choline transport in
synaptosomes prepared from hippocampus and neocortex of patients with Alzheimer’s
disease. Brain Res 289:169–175.
9. Nilsson L, Nordberg A, Hardy J et al. 1986. Physostigmine restores 3H-acetylcholine
efflux from Alzheimer brain slices to normal level. J Neural Transm 67:275–285.
10. Whitehouse PJ, Price DL, Struble RG et al. 1982. Alzheimer’s disease and senile
dementia: Loss of neurons in the basal forebrain. Science 215:1237–1239.
11. Davis KL, Mohs RC, Marin D et al. 1999. Cholinergic markers in elderly patients with
early signs of Alzheimer disease. JAMA 281:1401–1406.
12. DeKosky ST, Ikonomovic MD, Styren SD et al. 2002. Upregulation of choline
acetyltransferase activity in hippocampus and frontal cortex of elderly subjects with
mild cognitive impairment. Ann Neurol 51:145–155.
13. Shen J, Barnes CA. 1996. Age-related decrease in cholinergic synaptic transmission in
three hippocampal subfields. Neurobiol Aging 17:439–451.
14. Reiman EM, Caselli RJ, Yun LS et al. 1996. Preclinical evidence of Alzheimer’s disease
in persons homozygous for the epsilon 4 allele for apolipoprotein E. N Engl J Med
334:752–758.
15. Small GW, Ercoli LM, Silverman DH et al. 2000. Cerebral metabolic and cognitive decline
in persons at genetic risk for Alzheimer’s disease. Proc Natl Acad Sci USA 97:6037–6042.
16. Price DL, Tanzi RE, Borchelt DR et al. 1998. Alzheimer’s disease: Genetic studies and
transgenic models. Annu Rev Genet 32:461–493.
17. Small DH, Mok SS, Bornstein JC. 2001. Alzheimer’s disease and Abeta toxicity: From
top to bottom. Nat Rev Neurosci 2:595–598.
18. Akiyama H, Barger S, Barnum S et al. 2000. Inflammation and Alzheimer’s disease.
Neurobiol Aging 21:383–421.
19. Varadarajan S, Yatin S, Aksenova M et al. 2000. Review: Alzheimer’s amyloid beta-
peptide-associated free radical oxidative stress and neurotoxicity. J Struct Biol 30:184–208.
20. Nilsberth C, Westlind-Danielsson A, Eckman CB et al. 2001. The ‘Arctic’ APP muta-
tion (E693G) causes Alzheimer’s disease by enhanced Abeta protofibril formation. Nat
Neurosci 4:887–893.
21. Scheuner D, Eckman C, Jensen M et al. 1996. Secreted amyloid beta-protein similar to
that in the senile plaques of Alzheimer’s disease is increased in vivo by the presenilin
1 and 2 and APP mutations linked to familial Alzheimer’s disease. Nat Med 2:864–870.
22. Eckman CB, Mehta ND, Crook R et al. 1997. A new pathogenic mutation in the APP
gene (I716V) increases the relative proportion of A beta 42(43). Hum Mol Genet
6:2087–2089.
23. Tamaoka A, Kondo T, Odaka A et al. 1994. Biochemical evidence for the long-tail form
(A beta 1–42/43) of amyloid beta protein as a seed molecule in cerebral deposits of
Alzheimer’s disease. Biochem Biophys Res Commun 205:834–842.
24. Holtzman DM, Fagan AM, Mackey B et al. 2000. Apolipoprotein E facilitates neuritic
and cerebrovascular plaque formation in an Alzheimer’s disease model. Ann Neurol
47:739–747.
25. Terry RD, Masliah E, Salmon DP et al. 1991. Physical basis of cognitive alterations in
Alzheimer’s disease: Synapse loss is the major correlate of cognitive impairment. Ann
Neurol 30:572–580.
26. Neve RL, Robakis NK. 1998. Alzheimer’s disease: A re-examination of the amyloid
hypothesis. Trends Neurosci 21:15–19.
27. Arriagada PV, Growdon JH, Hedley-Whyte ET et al. 1992. Neurofibrillary tangles but
not senile plaques parallel duration and severity of Alzheimer’s disease. Neurology
42:631–639.
28. Atwood CS, Robinson SR, Smith MA. 2002. Amyloid-beta: Redox-metal chelator and
antioxidant. J Alzheimers Dis 4:203–214.
29. Smith MA, Casadesus G, Joseph JA et al. 2002. Amyloid-beta and tau serve antioxidant
functions in the aging and Alzheimer brain. J Free Radic Biol Med 33:1194–1199.
30. Roberson MR, Harrell LE. 1997. Cholinergic activity and amyloid precursor protein
metabolism. Brain Res Rev 25:50–69.
31. Buxbaum JD, Oishi M, Chen HI et al. 1992. Cholinergic agonists and interleukin
1 regulate processing and secretion of the Alzheimer beta/A4 amyloid protein precursor.
Proc Natl Acad Sci USA 89:10075–10078.
32. Pittel Z, Heldman E, Barg J et al. 1996. Muscarinic control of amyloid precursor protein
secretion in rat cerebral cortex and cerebellum. Brain Res 742:299–304.
33. Wallace W, Ahlers ST, Gotlib J et al. 1993. Amyloid precursor protein in the cerebral
cortex is rapidly and persistently induced by loss of subcortical innervation. Proc Natl
Acad Sci USA 90:8712–8716.
34. Lin L, Georgievska B, Mattsson A et al. 1999. Cognitive changes and modified pro-
cessing of amyloid precursor protein in the cortical and hippocampal system after
cholinergic synapse loss and muscarinic receptor activation. Proc Natl Acad Sci USA
96:12108–12113.
35. Iverfeldt K, Walaas SI, Greengard P. 1993. Altered processing of Alzheimer amy-
loid precursor protein in response to neuronal degeneration. Proc Natl Acad Sci USA
90:4146–4150.
36. Pike CJ, Burdick D, Walencewicz AJ et al. 1993. Neurodegeneration induced by
beta-amyloid peptides in vitro: the role of peptide assembly state. J Neurosci 13:
1676–1687.
37. Harkany T, Lengyel Z, Soos K et al. 1995. Cholinotoxic effects of beta-amyloid (1-42)
peptide on cortical projections of the rat nucleus basalis magnocellularis. Brain Res
695:71–75.
38. Auld DS, Kornecook TJ, Bastianetto S et al. 2002. Alzheimer’s disease and the basal
forebrain cholinergic system: Relations to beta-amyloid peptides, cognition, and treat-
ment strategies. Prog Neurobiol 68:209–245.
39. Blusztajn JK, Berse B. 2000. The cholinergic neuronal phenotype in Alzheimer’s dis-
ease. Metab Brain Dis 15:45–64.
40. Giovannini MG, Scali C, Prosperi C et al. 2002. Beta-amyloid-induced inflammation
and cholinergic hypofunction in the rat brain in vivo: Involvement of the p38MAPK
pathway. Neurobiol Dis 11:257–274.
41. Mattson MP, Cheng B, Davis D et al. 1992. Beta-amyloid peptides destabilize calcium
homeostasis and render human cortical neurons vulnerable to excitotoxicity. J Neurosci
12:376–389.
42. Hensley K, Carney JM, Mattson MP et al. 1994. A model for beta-amyloid aggregation
and neurotoxicity based on free radical generation by the peptide: Relevance to
Alzheimer disease. Proc Natl Acad Sci USA 91:3270–3274.
43. Behl C, Davis J, Cole GM et al. 1992. Vitamin E protects nerve cells from amyloid beta
protein toxicity. Biochem Biophys Res Commun 186:944–950.
44. Dickson DW, Lee SC, Mattiace LA et al. 1993. Microglia and cytokines in neurological
disease, with special reference to AIDS and Alzheimer’s disease. Glia 7:75–83.
45. Griffin WS, Sheng JG, Roberts GW et al. 1995. Interleukin-1 expression in different
plaque types in Alzheimer’s disease: Significance in plaque evolution. J Neuropathol
Exp Neurol 54:276–281.
46. Meda L, Baron P, Scarlato G. 2001. Glial activation in Alzheimer’s disease: The role of
Abeta and its associated proteins. Neurobiol Aging 22:885–893.
47. Pachter JS. 1997. Inflammatory mechanisms in Alzheimer disease: The role of beta-
amyloid/glial interactions. Mol Psychiatry 2:91–5.
48. Hanisch UK, Kettenmann H. 2007. Microglia: Active sensor and versatile effector cells
in the normal and pathologic brain. Nat Neurosci 10:1387–1394.
49. Boissonneault V, Filali M, Lessard M et al. 2009. Powerful beneficial effects of
macrophage colony-stimulating factor on beta-amyloid deposition and cognitive impair-
ment in Alzheimer’s disease. Brain 132:1078–1092.
50. Chen K, Iribarren P, Hu J et al. 2006. Activation of Toll-like receptor 2 on microg-
lia promotes cell uptake of Alzheimer disease-associated amyloid beta peptide. J Biol
Chem 281:3651–3659.
51. Arnaud L, Robakis NK, Figueiredo-Pereira ME. 2006. It may take inflammation, phos-
phorylation and ubiquitination to ‘tangle’ in Alzheimer’s disease. Neurodegener Dis
3:313–319.
52. Yirmiya R, Goshen I. 2011. Immune modulation of learning, memory, neural plasticity
and neurogenesis. Brain Behav Immun 25:181–213.
53. Goldgaber D, Harris HW, Hla T et al. 1989. Interleukin 1 regulates synthesis of amy-
loid beta-protein precursor mRNA in human endothelial cells. Proc Natl Acad Sci USA
86:7606–7610.
54. Mackenzie IR. 2000. Activated microglia in dementia with Lewy bodies. Neurology
55:132–134.
55. Li Y, Liu L, Kang J et al. 2000. Neuronal-glial interactions mediated by interleukin-1
enhance neuronal acetylcholinesterase activity and mRNA expression. J Neurosci
20:149–155.
56. Goshen I, Avital A, Kreisel T et al. 2009. Environmental enrichment restores memory
functioning in mice with impaired IL-1 signaling via reinstatement of long-term poten-
tiation and spine size enlargement. J Neurosci 29:3395–3403.
57. Craft JM, Watterson DM, Hirsch E et al. 2005. Interleukin 1 receptor antagonist knock-
out mice show enhanced microglial activation and neuronal damage induced by intra-
cerebroventricular infusion of human beta-amyloid. J Neuroinflamm 2:15.
58. Shaftel SS, Kyrkanides S, Olschowka JA et al. 2007. Sustained hippocampal IL-1 beta
overexpression mediates chronic neuroinflammation and ameliorates Alzheimer plaque
pathology. J Clin Invest 117:1595–1604.
59. Heyser CJ, Masliah E, Samimi A et al. 1997. Progressive decline in avoidance learning
paralleled by inflammatory neurodegeneration in transgenic mice expressing interleukin
6 in the brain. Proc Natl Acad Sci USA 94:1500–1505.
60. Vandenabeele P, Fiers W. 1991. Is amyloidogenesis during Alzheimer’s disease due to an
IL-1-/IL-6-mediated ‘acute phase response’ in the brain? Immunol Today 12:217–219.
61. Ringheim GE, Szczepanik AM, Petko W et al. 1998. Enhancement of beta-amyloid
precursor protein transcription and expression by the soluble interleukin-6 receptor/
interleukin-6 complex. Brain Res 55:35–44.
62. Gadient RA, Otten UH. 1997. Interleukin-6 (IL-6)—A molecule with both beneficial
and destructive potentials. Prog Neurobiol 52:379–390.
63. Gruol DL, Nelson TE. 1997. Physiological and pathological roles of interleukin-6 in the
central nervous system. Mol Neurobiol 15:307–339.
64. Kushima Y, Hatanaka H. 1992. Interleukin-6 and leukemia inhibitory factor promote the
survival of acetylcholinesterase-positive neurons in culture from embryonic rat spinal
cord. Neurosci Lett 143:110–114.
65. Marz P, Cheng JG, Gadient RA et al. 1998. Sympathetic neurons can produce and
respond to interleukin 6. Proc Natl Acad Sci USA 95:3251–3256.
66. Fillit H, Ding WH, Buee L et al. 1991. Elevated circulating tumor necrosis factor levels
in Alzheimer’s disease. Neurosci Lett 129:318–320.
67. Tarkowski E, Blennow K, Wallin A et al. 1999. Intracerebral production of tumor
necrosis factor-alpha, a local neuroprotective agent, in Alzheimer disease and vascular
dementia. J Clin Immunol 19:223–230.
68. Barger SW, Harmon AD. 1997. Microglial activation by Alzheimer amyloid precursor
protein and modulation by apolipoprotein E. Nature 388:878–881.
69. Barger SW, Horster D, Furukawa K et al. 1995. Tumor necrosis factors alpha and beta
protect neurons against amyloid beta-peptide toxicity: Evidence for involvement of a
kappa B-binding factor and attenuation of peroxide and Ca2+ accumulation. Proc Natl
Acad Sci USA 92:9328–9332.
70. Bruce-Keller AJ, Geddes JW, Knapp PE et al. 1999. Anti-death properties of TNF against
metabolic poisoning: Mitochondrial stabilization by MnSOD. J Neuroimmunol 93:53–71.
71. Mattson MP, Cheng B, Baldwin SA et al. 1995. Brain injury and tumor necrosis factors
induce calbindin D-28k in astrocytes: Evidence for a cytoprotective response. J Neurosci
Res 42:357–370.
72. Cardinaux JR, Allaman I, Magistretti PJ. 2000. Pro-inflammatory cytokines induce the
transcription factors C/EBPbeta and C/EBPdelta in astrocytes. Glia 29:91–97.
73. Yamamoto K, Arakawa T, Ueda N et al. 1995. Transcriptional roles of nuclear factor
kappa B and nuclear factor-interleukin-6 in the tumor necrosis factor alpha-dependent
induction of cyclooxygenase-2 in MC3T3-E1 cells. J Biol Chem 270:31315–31320.
74. van der Wal EA, Gomez-Pinilla F, Cotman CW. 1993. Transforming growth factor-
beta 1 is in plaques in Alzheimer and Down pathologies. Neuroreport 4:69–72.
75. Flanders KC, Lippa CF, Smith TW et al. 1995. Altered expression of transforming
growth factor-beta in Alzheimer’s disease. Neurology 45:1561–1569.
76. Chao CC, Hu S, Frey WH, 2nd et al. 1994. Transforming growth factor beta in
Alzheimer’s disease. Clin Diagn Lab Immunol 1:109–110.
77. Minghetti L, Polazzi E, Nicolini A et al. 1998. Opposite regulation of prostaglandin E2
synthesis by transforming growth factor-beta1 and interleukin 10 in activated microglial
cultures. J Neuroimmunol 82:31–39.
78. Luo J, Lang JA, Miller MW. 1998. Transforming growth factor beta1 regulates the
expression of cyclooxygenase in cultured cortical astrocytes and neurons. J Neurochem
71:526–534.
79. Flanders KC, Ren RF, Lippa CF. 1998. Transforming growth factor-betas in neurode-
generative disease. Prog Neurobiol 54:71–85.
80. Luster AD. 1998. Chemokines—Chemotactic cytokines that mediate inflammation.
N Engl J Med 338:436–445.
81. Xia MQ, Hyman BT. 1999. Chemokines/chemokine receptors in the central nervous
system and Alzheimer’s disease. J Neurovirol 5:32–41.
82. Harries LW, Bradley-Smith RM, Llewellyn DJ et al. 2012. Leukocyte CCR2 expression
is associated with mini-mental state examination score in older adults. Rejuvenation Res
15:395–405.
83. Naert G, Rivest S. 2011. CC chemokine receptor 2 deficiency aggravates cognitive

impairments and amyloid pathology in a transgenic mouse model of Alzheimer’s
disease. J Neurosci 31:6208–6220.
84. Westin K, Buchhave P, Nielsen H et al. 2012. CCL2 is associated with a faster rate of
cognitive decline during early stages of Alzheimer’s disease. PloS One 7:e30525
85. Ashutosh, Kou W, Cotter R et al. 2011. CXCL8 protects human neurons from
amyloid-beta-induced neurotoxicity: Relevance to Alzheimer’s disease. Biochem
Biophys Res Commun 412:565–571.
86. Cho SH, Sun B, Zhou Y et al. 2011. CX3CR1 protein signaling modulates microglial
activation and protects against plaque-independent cognitive deficits in a mouse model
of Alzheimer disease. J Biol Chem 286:32713–32722.
87. Afagh A, Cummings BJ, Cribbs DH et al. 1996. Localization and cell association of C1q
in Alzheimer’s disease brain. Exp Neurol 138:22–32.
88. Chen S, Frederickson RC, Brunden KR. 1996. Neuroglial-mediated immunoinflam-
matory responses in Alzheimer’s disease: Complement activation and therapeutic
approaches. Neurobiol Aging 17:781–787.
89. Rogers J, Cooper NR, Webster S et al. 1992. Complement activation by beta-amyloid in
90. Webster S, Glabe C, Rogers J. 1995. Multivalent binding of complement protein C1Q to
the amyloid beta-peptide (A beta) promotes the nucleation phase of A beta aggregation.
Biochem Biophys Res Commun 217:869–875.
91. Shen Y, Lue L, Yang L et al. 2001. Complement activation by neurofibrillary tangles in
Alzheimer’s disease. Neurosci Lett 305:165–168.
92. Geula C, Wu CK, Saroff D et al. 1998. Aging renders the brain vulnerable to amyloid
beta-protein neurotoxicity. Nat Med 4:827–831.
93. Johns TG, Bernard CC. 1997. Binding of complement component Clq to myelin oligo-
dendrocyte glycoprotein: A novel mechanism for regulating CNS inflammation. Mol
Immunol 34:33–38.
94. Linder E, Lehto VP, Stenman S. 1979. Activation of complement by cytoskeletal inter-
mediate filaments. Nature 278:176–178.
95. Gasque P, Neal JW, Singhrao SK et al. 2002. Roles of the complement system in human
neurodegenerative disorders: Pro-inflammatory and tissue remodeling activities. Mol
Neurobiol 25:1–17.
96. Mukherjee P, Pasinetti GM. 2000. The role of complement anaphylatoxin C5a in
neurodegeneration: Implications in Alzheimer’s disease. J Neuroimmunol 105:
124–130.
97. Gasque P, Singhrao SK, Neal JW et al. 1997. Expression of the receptor for complement
C5a (CD88) is up-regulated on reactive astrocytes, microglia, and endothelial cells in
the inflamed human central nervous system. Am J Pathol 150:31–41.
98. Lacy M, Jones J, Whittemore SR et al. 1995. Expression of the receptors for the
C5a anaphylatoxin, interleukin-8 and FMLP by human astrocytes and microglia.
J Neuroimmunol 61:71–78.
99. Ishii T, Haga S. 1984. Immuno-electron-microscopic localization of complements in
amyloid fibrils of senile plaques. Acta Neuropathol 63:296–300.
100. Rogers J, Luber-Narod J, Styren SD et al. 1988. Expression of immune system-
associated antigens by cells of the human central nervous system: Relationship to the
pathology of Alzheimer’s disease. Neurobiol Aging 9:339–49.
101. McGeer PL, Akiyama H, Itagaki S et al. 1989. Activation of the classical complement
pathway in brain tissue of Alzheimer patients. Neurosci Lett 107:341–346.
102. Jellinger KA. 2007. The enigma of vascular cognitive disorder and vascular dementia.
Acta Neuropathol 113:349–388.
103. Formichi P, Parnetti L, Radi E et al. 2010. CSF biomarkers profile in CADASIL-A
model of pure vascular dementia: Usefulness in differential diagnosis in the dementia
disorder. Int J Alzheimer’s Dis. August 18, 2010. doi:pii: 959257.
104. Kalaria RN, Ballard C. 1999. Overlap between pathology of Alzheimer disease and
vascular dementia. Alzheimer Dis Assoc Disord 13(Suppl 3):S115–S123.
105. Sparks DL, Hunsaker JC, 3rd, Scheff SW et al. 1990. Cortical senile plaques in coronary
artery disease, aging and Alzheimer’s disease. Neurobiol Aging 11:601–607.
106. Sparks DL, Liu H, Scheff SW et al. 1993. Temporal sequence of plaque formation in the
cerebral cortex of non-demented individuals. J Neuropathol Exp Neurol 52:135–42.
107. Duong T, Nikolaeva M, Acton PJ. 1997. C-reactive protein-like immunoreactivity in the
neurofibrillary tangles of Alzheimer’s disease. Brain Res 749:152–156.
108. Cleland SJ, Sattar N, Petrie JR et al. 2000. Endothelial dysfunction as a possible link
between C-reactive protein levels and cardiovascular disease. Clin Sci 98:531–535.
109. Fay WP. 2010. Linking inflammation and thrombosis: Role of C-reactive protein. World
J Cardiol 2:365–369.
110. Strittmatter WJ, Saunders AM, Schmechel D et al. 1993. Apolipoprotein E: High-avidity
binding to beta-amyloid and increased frequency of type 4 allele in late-onset familial
111. Grammas P. 2011. Neurovascular dysfunction, inflammation and endothelial activation:
Implications for the pathogenesis of Alzheimer’s disease. J Neuroinflammation 8:26.
112. Tan J, Town T, Paris D et al. 1999. Activation of microglial cells by the CD40 pathway:
Relevance to multiple sclerosis. J Neuroimmunol 97:77–85.
113. Vane JR. 1971. Inhibition of prostaglandin synthesis as a mechanism of action for
aspirin-like drugs. Nature 231:232–235.
114. Meade EA, Smith WL, De Witt DL. 1993. Differential inhibition of prostaglandin endo-
peroxide synthase (cyclooxygenase) isozymes by aspirin and other non-steroidal anti-
inflammatory drugs. J Biol Chem 268:6610–6614.
115. Eriksen JL, Sagi SA, Smith TE et al. 2003. NSAIDs and enantiomers of flurbiprofen
target gamma-secretase and lower Abeta 42 in vivo. J Clin Invest 112:440–449.
116. McGeer PL, Schulzer M, McGeer EG. 1996. Arthritis and anti-inflammatory agents as
possible protective factors for Alzheimer’s disease: A review of 17 epidemiologic stud-
ies. Neurology 47:425–432.
117. Szekely CA, Town T, Zandi PP. 2007. NSAIDs for the chemoprevention of Alzheimer’s
disease. Subcell Biochem 42:229–248. Dordrecht, the Netherlands, Springer.
118. Stewart WF, Kawas C, Corrada M et al. 1997. Risk of Alzheimer’s disease and duration
of NSAID use. Neurology 48:626–632.
119. in t’ Veld BA, Ruitenberg A, Hofman A et al. 2001. Nonsteroidal antiinflammatory
drugs and the risk of Alzheimer’s disease. N Engl J Med 345:1515–1521.
120. Lindsay J, Laurin D, Verreault R et al. 2002. Risk factors for Alzheimer’s disease:
A prospective analysis from the Canadian Study of Health and Aging. Am J Epidemiol
156:445–453.
121. Zandi PP, Anthony JC, Hayden KM et al. 2002. Reduced incidence of AD with NSAID
but not H2 receptor antagonists: The Cache County Study. Neurology 59:880–886.
122. Cornelius C, Fastbom J, Winblad B et al. 2004. Aspirin, NSAIDs, risk of demen-
tia, and influence of the apolipoprotein E epsilon 4 allele in an elderly population.
Neuroepidemiology 23:135–143.
123. Vlad SC, Miller DR, Kowall NW et al. 2008. Protective effects of NSAIDs on the
development of Alzheimer disease. Neurology 70:1672–1677.
124. Szekely CA, Breitner JC, Fitzpatrick AL et al. 2008. NSAID use and dementia risk in
the Cardiovascular Health Study: Role of APOE and NSAID type. Neurology 70:17–24.
125. Arvanitakis Z, Grodstein F, Bienias JL et al. 2008. Relation of NSAIDs to incident AD,
change in cognitive function, and AD pathology. Neurology 70:2219–2225.
126. Breitner JC, Haneuse SJ, Walker R et al. 2009. Risk of dementia and AD with prior
exposure to NSAIDs in an elderly community-based cohort. Neurology 72:1899–1905.
127. Breitner JC, Baker LD, Montine TJ et al. 2011. Extended results of the Alzheimer’s
disease anti-inflammatory prevention trial. Alzheimers Dement 7:402–411.
128. Lyketsos CG, Breitner JC, Green RC et al. 2007. Naproxen and celecoxib do not prevent
AD in early results from a randomized controlled trial. Neurology 68:1800–1808.
129. Manabe Y, Anrather J, Kawano T et al. 2004. Prostanoids, not reactive oxygen species,
mediate COX-2-dependent neurotoxicity. Ann Neurol 55:668–675.
130. Sonnen JA, Larson EB, Walker RL et al. 2010. Nonsteroidal anti-inflammatory drugs are
associated with increased neuritic plaques. Neurology 75:1203–1210.
131. Thal LJ, Ferris SH, Kirby L et al. 2005. A randomized, double-blind, study of rofecoxib
in patients with mild cognitive impairment. Neuropsychopharmacology 30:1204–1215.
132. Small GW, Siddarth P, Silverman DH et al. 2008. Cognitive and cerebral metabolic
effects of celecoxib versus placebo in people with age-related memory loss: Randomized
controlled study. Am J Geriatr Psychiatry 16:999–1009.
133. Varvel NH, Bhaskar K, Kounnas MZ et al. 2009. NSAIDs prevent, but do not reverse,
neuronal cell cycle reentry in a mouse model of Alzheimer disease. J Clin Invest
119:3692–3702.
134. Wilcock GK, Black SE, Hendrix SB et al. 2008. Efficacy and safety of tarenflurbil
in mild to moderate Alzheimer’s disease: A randomised phase II trial. Lancet Neurol
7:483–493.
135. Klegeris A, McGeer PL. 1997. beta-amyloid protein enhances macrophage production
of oxygen free radicals and glutamate. J Neurosci Res 49:229–235.
136. Bianca VD, Dusi S, Bianchini E et al. 1999. beta-amyloid activates the O-2 form-
ing NADPH oxidase in microglia, monocytes, and neutrophils. A possible inflam-
matory mechanism of neuronal damage in Alzheimer’s disease. J Biol Chem 274:
15493–15499.
137. Nunomura A, Castellani RJ, Zhu X et al. 2006. Involvement of oxidative stress in
Alzheimer disease. J Neuropathol Exp Neurol 65:631–641.
138. Perkins AJ, Hendrie HC, Callahan CM et al. 1999. Association of antioxidants with
memory in a multiethnic elderly sample using the Third National Health and Nutrition
Examination Survey. Am J Epidemiol 150:37–44.
139. Masaki KH, Losonczy KG, Izmirlian G et al. 2000. Association of vitamin E and C
supplement use with cognitive function and dementia in elderly men. Neurology
54:1265–1272.
140. Laurin D, Masaki KH, Foley DJ et al. 2004. Midlife dietary intake of antioxidants and
risk of late-life incident dementia: The Honolulu-Asia Aging Study. Am J Epidemiol
159:959–967.
141. Grodstein F, Chen J, Willett WC. 2003. High-dose antioxidant supplements and cogni-
tive function in community-dwelling elderly women. Am J Clin Nutr 77:975–984.
142. Morris MC, Evans DA, Bienias JL et al. 2002. Vitamin E and cognitive decline in older
persons. Arch Neurol 59:1125–1132.
143. Morris MC, Evans DA, Bienias JL et al. 2002. Dietary intake of antioxidant nutri-
ents and the risk of incident Alzheimer disease in a biracial community study. JAMA
287:3230–3237.
144. Engelhart MJ, Geerlings MI, Ruitenberg A et al. 2002. Dietary intake of antioxidants
and risk of Alzheimer disease. JAMA 287:3223–3239.
145. Sano M, Ernesto C, Thomas RG et al. 1997. A controlled trial of selegiline, alpha-
tocopherol, or both as treatment for Alzheimer’s disease. The Alzheimer’s Disease
Cooperative Study. N Engl J Med 336:1216–1222.
146. Petersen RC, Thomas RG, Grundman M et al. 2005. Vitamin E and donepezil for the
treatment of mild cognitive impairment. N Engl J Med 352:2379–2388.
147. Galasko DR, Peskind E, Clark CM et al. 2012. Antioxidants for Alzheimer Disease:
A randomized clinical trial with cerebrospinal fluid biomarker measures. Arch Neurol
69:836–841.
148. Miller ER, 3rd, Pastor-Barriuso R, Dalal D et al. 2005. Meta-analysis: High-dosage vita-
min E supplementation may increase all-cause mortality. Ann Intern Med 142:37–46.
149. Brewer GJ. 2010. Why vitamin E therapy fails for treatment of Alzheimer’s disease.
J Alzheimers Dis 19:27–30.
150. Lloret A, Badia MC, Mora NJ et al. 2009. Vitamin E paradox in Alzheimer’s disease:
It does not prevent loss of cognition and may even be detrimental. J Alzheimers Dis
17:143–149.
151. Perry G, Zhu X, Moreira PI et al. 2009. Altered redox balance in disease: Can we change
the new equilibria? Ann Neurol 65:121–123.
152. Bastianetto S, Zheng WH, Quirion R. 2000. Neuroprotective abilities of resveratrol and
other red wine constituents against nitric oxide-related toxicity in cultured hippocampal
neurons. Br J Pharmacol 131:711–720.
153. Jang JH, Surh YJ. 2003. Protective effect of resveratrol on beta-amyloid-induced oxida-
tive PC12 cell death. J Free Radic Biol Med 34:1100–1110.
154. Pogacic Kramp V, Herrling P. 2011. List of drugs in development for neurodegenerative
diseases: Update June 2010. Neurodegener Dis 8:44–94.
155. Weinmann S, Roll S, Schwarzbach C et al. 2010. Effects of Ginkgo biloba in dementia:
Systematic review and meta-analysis. BMC Geriatr 10:14.
156. DeFeudis FV, Drieu K. 2000. Ginkgo biloba extract (EGb 761) and CNS functions:
Basic studies and clinical applications. Curr Drug Targets 1:25–58.
157. Liu YG, Li FJ, Wang J et al. 2010. Effects of Ginkgolide B on inflammation induced by
cerebral ischemia-reperfusion in rats. Zhong yao cai 33:578–580.
158. Kaschel R. 2009. Ginkgo biloba: Specificity of neuropsychological improvement—
A selective review in search of differential effects. Hum Psychopharmacol 24:345–370.
159. Allain H, Raoul P, Lieury A et al. 1993. Effect of two doses of Ginkgo biloba extract
(EGb 761) on the dual-coding test in elderly subjects. Clin Ther 15:549–558.
160. Kennedy DO, Scholey AB, Wesnes KA. 2000. The dose-dependent cognitive effects of
acute administration of Ginkgo biloba to healthy young volunteers. Psychopharmacology
151:416–423.
161. Hindmarch I. 1986. Activity of Ginkgo biloba extract on short-term memory. Presse
Med 5:1592–1594.
162. van Dongen M, van Rossum E, Kessels A et al. 2003. Ginkgo for elderly people with
dementia and age-associated memory impairment: A randomized clinical trial. J Clin
Epidemiol 56:367–376.
163. Wesnes KA, Ward T, McGinty A et al. 2000. The memory enhancing effects of
a Ginkgo biloba/Panax ginseng combination in healthy middle-aged volunteers.
Psychopharmacology 152:353–361.
164. Snitz BE, O’Meara ES, Carlson MC et al. 2009. Ginkgo biloba for preventing cognitive
decline in older adults: A randomized trial. JAMA 302:2663–2670.
165. DeKosky ST, Williamson JD, Fitzpatrick AL et al. 2008. Ginkgo biloba for prevention
of dementia: A randomized controlled trial. JAMA 300:2253–2262.
166. Bornhoft G, Maxion-Bergemann S, Wolf U et al. 2006. Checklist for the qualitative
evaluation of clinical studies with particular focus on external validity and model valid-
ity. BMC Med Res Methodol 6:56.
167. Bornhoft G, Maxion-Bergemann S, Matthiessen PF. 2008. External validity of clini-
cal trials for treatment of dementia with Ginkgo biloba extracts. Z Gerontol Geriatr
41:298–312.
168. Janssen IM, Sturtz S, Skipka G et al. 2010. Ginkgo biloba in Alzheimer’s disease:
A systematic review. Wien Med Wochenschr 160:539–546.
169. Vegeto E, Bonincontro C, Pollio G et al. 2001. Estrogen prevents the lipopolysaccha-
ride-induced inflammatory response in microglia. J Neurosci 21:1809–1818.
170. Vegeto E, Belcredito S, Etteri S et al. 2003. Estrogen receptor-alpha mediates the brain
antiinflammatory activity of estradiol. Proc Natl Acad Sci USA 100:9614–9619.
171. Vegeto E, Belcredito S, Ghisletti S et al. 2006. The endogenous estrogen status reg-
ulates microglia reactivity in animal models of neuroinflammation. Endocrinology
147:2263–2272.
172. Manaye KF, Allard JS, Kalifa S et al. 2011. 17alpha-estradiol attenuates neuron loss in
ovariectomized Dtg AbetaPP/PS1 mice. J Alzheimers Dis 23:629–639.
173. Chang D, Kwan J, Timiras PS. 1997. Estrogens influence growth, maturation, and amy-
loid beta-peptide production in neuroblastoma cells and in a beta-APP transfected kid-
ney 293 cell line. Adv Exp Med Biol 429:261–271.
174. Jaffe AB, Toran-Allerand CD, Greengard P et al. 1994. Estrogen regulates metabolism
of Alzheimer amyloid beta precursor protein. J Biol Chem 269:13065–13068.
175. Xu H, Gouras GK, Greenfield JP et al. 1998. Estrogen reduces neuronal generation of
Alzheimer beta-amyloid peptides. Nat Med 4:447–451.
176. Chao HM, Spencer RL, Frankfurt M et al. 1994. The effects of aging and hormonal
manipulation on amyloid precursor protein APP695 mRNA expression in the rat hip-
pocampus. J Neuroendocrinol 6:517–521.
177. Li R, Shen Y, Yang LB et al. 2000. Estrogen enhances uptake of amyloid beta-protein by
microglia derived from the human cortex. J Neurochem 75:1447–1454.
178. Carroll JC, Rosario ER, Chang L et al. 2007. Progesterone and estrogen regulate
Alzheimer-like neuropathology in female 3xTg-AD mice. J Neurosci 27:13357–13365.
179. Wharton W, Baker LD, Gleason CE et al. 2011. Short-term hormone therapy with trans-
dermal estradiol improves cognition for postmenopausal women with Alzheimer’s dis-
ease: Results of a randomized controlled trial. J Alzheimers Dis 26:495–505.
180. Valen-Sendstad A, Engedal K, Stray-Pedersen B et al. 2010. Effects of hormone therapy
on depressive symptoms and cognitive functions in women with Alzheimer disease:
A 12 month randomized, double-blind, placebo-controlled study of low-dose estradiol
and norethisterone. Am J Geriatr Psychiatry 18:11–20.
181. Asthana S, Baker LD, Craft S et al. 2001. High-dose estradiol improves cognition for
women with AD: Results of a randomized study. Neurology 57:605–612.
182. Henderson VW, Paganini-Hill A, Miller BL et al. 2000. Estrogen for Alzheimer’s disease
in women: Randomized, double-blind, placebo-controlled trial. Neurology 54:295–301.
183. Mulnard RA, Cotman CW, Kawas C et al. 2000. Estrogen replacement therapy for treat-
ment of mild to moderate Alzheimer disease: A randomized controlled trial. Alzheimer’s
Disease Cooperative Study. JAMA 283:1007–1015.
184. Yoon BK, Kim DK, Kang Y et al. 2003. Hormone replacement therapy in postmeno-
pausal women with Alzheimer’s disease: A randomized, prospective study. Fertil Steril
79:274–280.
185. Schneider LS, Farlow M. 1997. Combined tacrine and estrogen replacement therapy in
patients with Alzheimer’s disease. Ann NY Acad Sci 826:317–322.
186. Shumaker SA, Legault C, Rapp SR et al. 2003. Estrogen plus progestin and the inci-
dence of dementia and mild cognitive impairment in postmenopausal women: The
Women’s Health Initiative Memory Study: A randomized controlled trial. JAMA
289:2651–2662.
187. Baldereschi M, Di Carlo A, Lepore V et al. 1998. Estrogen-replacement therapy and
Alzheimer’s disease in the Italian Longitudinal Study on Aging. Neurology 50:996–1002.
188. Whitmer RA, Quesenberry CP, Zhou J et al. 2011. Timing of hormone therapy and
dementia: The critical window theory revisited. Ann Neurol 69:163–169.
189. Ono K, Hasegawa K, Naiki H et al. 2004. Curcumin has potent anti-amyloidogenic
effects for Alzheimer’s beta-amyloid fibrils in vitro. J Neurosci Res 75:742–750.
190. Park SY, Kim HS, Cho EK et al. 2008. Curcumin protected PC12 cells against beta-
amyloid-induced toxicity through the inhibition of oxidative damage and tau hyperphos-
phorylation. Food Chem Toxicol 46:2881–2887.
191. Shimmyo Y, Kihara T, Akaike A et al. 2008. Epigallocatechin-3-gallate and curcumin
suppress amyloid beta-induced beta-site APP cleaving enzyme-1 upregulation.
Neuroreport 19:1329–1333.
192. Fiala M, Liu PT, Espinosa-Jeffrey A et al. 2007. Innate immunity and transcription
of MGAT-III and Toll-like receptors in Alzheimer’s disease patients are improved by
bisdemethoxycurcumin. Proc Natl Acad Sci USA 104:12849–12854.
193. Alter MD, Gilani AI, Champagne FA et al. 2009. Paternal transmission of complex
phenotypes in inbred mice. Biol Psychol 66:1061–1066.
194. Lim GP, Chu T, Yang F et al. 2001. The curry spice curcumin reduces oxidative damage
and amyloid pathology in an Alzheimer transgenic mouse. J Neurosci 21:8370–8377.
195. Begum AN, Jones MR, Lim GP et al. 2008. Curcumin structure-function, bioavailability,
and efficacy in models of neuroinflammation and Alzheimer’s disease. J Pharmacol Exp
Ther 326:196–208.
196. Yang F, Lim GP, Begum AN et al. 2005. Curcumin inhibits formation of amyloid
beta oligomers and fibrils, binds plaques, and reduces amyloid in vivo. J Biol Chem
280:5892–5901.
197. Frautschy SA, Hu W, Kim P et al. 2001. Phenolic anti-inflammatory antioxidant reversal
of Abeta-induced cognitive deficits and neuropathology. Neurobiol Aging 22:993–1005.
198. Ma QL, Yang F, Rosario ER et al. 2009. Beta-amyloid oligomers induce phosphorylation
of tau and inactivation of insulin receptor substrate via c-Jun N-terminal kinase signal-
ing: Suppression by omega-3 fatty acids and curcumin. J Neurosci 29:9078–9089.
199. Baum L, Lam CW, Cheung SK et al. 2008. Six-month randomized, placebo-controlled,
double-blind, pilot clinical trial of curcumin in patients with Alzheimer disease. J Clin
Psychopharmacol 28:110–113.
200. Uauy R, Hoffman DR, Peirano P et al. 2001. Essential fatty acids in visual and brain
development. Lipids 36:885–895.
201. Su HM. 2010. Mechanisms of n-3 fatty acid-mediated development and maintenance of
learning memory performance. J Nutr Biochem 21:364–373.
202. Neuringer M, Anderson GJ, Connor WE. 1988. The essentiality of n-3 fatty acids for the
development and function of the retina and brain. Annu Rev Nutr 8:517–541.
203. Uauy R, Dangour AD. 2006. Nutrition in brain development and aging: Role of essential
fatty acids. Nutr Rev 64:S24–S33.
204. Lynch AM, Loane DJ, Minogue AM et al. 2007. Eicosapentaenoic acid confers neu-
roprotection in the amyloid-beta challenged aged hippocampus. Neurobiol Aging
28:845–855.
205. Marcheselli VL, Hong S, Lukiw WJ et al. 2003. Novel docosanoids inhibit brain
ischemia-reperfusion-mediated leukocyte infiltration and pro-inflammatory gene
expression. J Biol Chem 278:43807–43817.
206. Soderberg M, Edlund C, Kristensson K et al. 1991. Fatty acid composition of brain
phospholipids in aging and in Alzheimer’s disease. Lipids 26:421–425.
207. Suzuki H, Park SJ, Tamura M et al. 1998. Effect of the long-term feeding of dietary
lipids on the learning ability, fatty acid composition of brain stem phospholipids and
synaptic membrane fluidity in adult mice: A comparison of sardine oil diet with palm oil
diet. Mech Ageing Dev 101:119–128.
208. Sugimoto Y, Taga C, Nishiga M et al. 2002. Effect of docosahexaenoic acid-fortified
Chlorella vulgaris strain CK22 on the radial maze performance in aged mice. Biol
Pharm Bull 25:1090–1092.
209. Moriguchi T, Salem N, Jr. 2003. Recovery of brain docosahexaenoate leads to recovery
of spatial task performance. J Neurochem 87:297–309.
210. Chung WL, Chen JJ, Su HM. 2008. Fish oil supplementation of control and (n-3) fatty
acid-deficient male rats enhances reference and working memory performance and
increases brain regional docosahexaenoic acid levels. J Nutr 138:1165–1171.
211. van Gelder BM, Tijhuis M, Kalmijn S et al. 2007. Fish consumption, n-3 fatty acids, and
subsequent 5-y cognitive decline in elderly men: The Zutphen Elderly Study. Am J Clin
Nutr 85:1142–1147.
212. Kalmijn S, Launer LJ, Ott A et al. 1997. Dietary fat intake and the risk of incident
dementia in the Rotterdam Study. Ann Neurol 42:776–782.
213. Morris MC, Evans DA, Tangney CC et al. 2005. Fish consumption and cognitive decline
with age in a large community study. Arch Neurol 62:1849–1853.
214. Kalmijn S, Feskens EJ, Launer LJ et al. 1997. Polyunsaturated fatty acids, antioxidants,
and cognitive function in very old men. Am J Epidemiol 145:33–41.
215. Barberger-Gateau P, Letenneur L, Deschamps V et al. 2002. Fish, meat, and risk of
dementia: Cohort study. BMJ 325:932–933.
216. Morris MC, Evans DA, Bienias JL et al. 2003. Consumption of fish and n-3 fatty acids
and risk of incident Alzheimer disease. Arch Neurol 60:940–946.
217. Kalmijn S, van Boxtel MP, Ocke M et al. 2004. Dietary intake of fatty acids and fish in
relation to cognitive performance at middle age. Neurology 62:275–280.
218. Nurk E, Drevon CA, Refsum H et al. 2007. Cognitive performance among the elderly
and dietary fish intake: The Hordaland Health Study. Am J Clin Nutr 86:1470–1478.
219. Dangour AD, Allen E, Elbourne D et al. 2009. Fish consumption and cognitive function
among older people in the UK: Baseline data from the OPAL study. J Nutr Health Aging
13:198–202.
220. Whalley LJ, Fox HC, Wahle KW et al. 2004. Cognitive aging, childhood intelligence,
and the use of food supplements: Possible involvement of n-3 fatty acids. Am J Clin Nutr
80:1650–1657.
221. Whalley LJ, Deary IJ, Starr JM et al. 2008. n-3 Fatty acid erythrocyte membrane con-
tent, APOE varepsilon4, and cognitive variation: An observational follow-up study in
late adulthood. Am J Clin Nutr 87:449–454.
222. Heude B, Ducimetiere P, Berr C. 2003. Cognitive decline and fatty acid composition of
erythrocyte membranes—The EVA Study. Am J Clin Nutr 77:803–808.
223. Schaefer EJ, Bongard V, Beiser AS et al. 2006. Plasma phosphatidylcholine docosa-
hexaenoic acid content and risk of dementia and Alzheimer disease: The Framingham
Heart Study. Arch Neurol 63:1545–1550.
224. Laurin D, Verreault R, Lindsay J et al. 2003. Omega-3 fatty acids and risk of cognitive
impairment and dementia. J Alzheimers Dis 5:315–322.
225. Chiu CC, Su KP, Cheng TC et al. 2008. The effects of omega-3 fatty acids monother-
apy in Alzheimer’s disease and mild cognitive impairment: A preliminary randomized
double-blind placebo-controlled study. Prog Neuropsychopharmacol Biol Psychiatry
32:1538–1544.
226. Quinn JF, Raman R, Thomas RG et al. 2010. Docosahexaenoic acid supplementation
and cognitive decline in Alzheimer disease: A randomized trial. JAMA 304:1903–1911.
227. Freund-Levi Y, Eriksdotter-Jonhagen M, Cederholm T et al. 2006. Omega-3 fatty acid
treatment in 174 patients with mild to moderate Alzheimer disease: OmegAD study:
A randomized double-blind trial. Arch Neurol 63:1402–1408.
228. Dangour AD, Allen E, Elbourne D et al. 2010. Effect of 2-y n-3 long-chain polyunsatu-
rated fatty acid supplementation on cognitive function in older people: A randomized,
double-blind, controlled trial. Am J Clin Nutr 91:1725–1732.
229. van de Rest O, Geleijnse JM, Kok FJ et al. 2008. Effect of fish oil on cognitive perfor-
mance in older subjects: A randomized, controlled trial. Neurology 71:430–438.
230. Yurko-Mauro K, McCarthy D, Rom D et al. 2010. Beneficial effects of docosahexaenoic
acid on cognition in age-related cognitive decline. Alzheimers Dement 6:456–464.
231. Schenk D, Barbour R, Dunn W et al. 1999. Immunization with amyloid-beta attenuates
Alzheimer-disease-like pathology in the PDAPP mouse. Nature 400:173–177.
232. Bayer AJ, Bullock R, Jones RW et al. 2005. Evaluation of the safety and immunogenicity
of synthetic Abeta42 (AN1792) in patients with AD. Neurology 64:94–101.
233. Orgogozo JM, Gilman S, Dartigues JF et al. 2003. Subacute meningoencephalitis in a
subset of patients with AD after Abeta42 immunization. Neurology 61:46–54.
234. Town T, Tan J, Flavell RA et al. 2005. T-cells in Alzheimer’s disease. Neuromolecular
Med 7:255–264.
235. Gilman S, Koller M, Black RS et al. 2005. Clinical effects of Abeta immunization
(AN1792) in patients with AD in an interrupted trial. Neurology 64:1553–1562.
236. Vellas B, Black R, Thal LJ et al. 2009. Long-term follow-up of patients immunized
with AN1792: Reduced functional decline in antibody responders. Curr Alzheimer Res
6:144–151.
237. Town T. 2009. Alternative Abeta immunotherapy approaches for Alzheimer’s disease.
CNS Neurol Disord Drug Targets 8:114–127.
238. Fu HJ, Liu B, Frost JL et al. 2010. Amyloid-beta immunotherapy for Alzheimer’s dis-
ease. CNS Neurol Disord Drug Targets 9:197–206.
239. Blennow K, Hampel H. 2003. CSF markers for incipient Alzheimer’s disease. Lancet
Neurol 2:605–613.
240. Bard F, Cannon C, Barbour R et al. 2000. Peripherally administered antibodies against
amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse
model of Alzheimer disease. Nat Med 6:916–919.
241. Schroeter S, Khan K, Barbour R et al. 2008. Immunotherapy reduces vascular amyloid-
beta in PDAPP mice. J Neurosci 28:6787–6793.
242. Wang A, Das P, Switzer RC, 3rd et al. 2011. Robust amyloid clearance in a mouse
model of Alzheimer’s disease provides novel insights into the mechanism of amyloid-
beta immunotherapy. J Neurosci 31:4124–4136.
243. Tucker SM, Borchelt DR, Troncoso JC. 2008. Limited clearance of pre-existing amyloid
plaques after intracerebral injection of Abeta antibodies in two mouse models of
Alzheimer disease. J Neuropathol Exp Neurol 67:30–40.
244. Goni F, Prelli F, Ji Y et al. 2010. Immunomodulation targeting abnormal protein con-
formation reduces pathology in a mouse model of Alzheimer’s disease. PloS One
5:e13391
245. Salloway S, Sperling R, Gilman S et al. 2009. A phase 2 multiple ascending dose trial of
bapineuzumab in mild to moderate Alzheimer disease. Neurology 73:2061–2070.
246. Blennow K, Zetterberg H, Rinne JO et al. 2012. Effect of immunotherapy with bap-
ineuzumab on cerebrospinal fluid biomarker levels in patients with mild to moderate
Alzheimer disease. Arch Neurol 69:1002–1010.
247. Rinne JO, Brooks DJ, Rossor MN et al. 2010. 11C-PiB PET assessment of change in
fibrillar amyloid-beta load in patients with Alzheimer’s disease treated with bapineu-
zumab: A phase 2, double-blind, placebo-controlled, ascending-dose study. Lancet
Neurol 9:363–372.
248. Racke MM, Boone LI, Hepburn DL et al. 2005. Exacerbation of cerebral amyloid
angiopathy-associated microhemorrhage in amyloid precursor protein transgenic mice
by immunotherapy is dependent on antibody recognition of deposited forms of amyloid
beta. J Neurosci 25:629–636.
249. Wilcock DM, Munireddy SK, Rosenthal A et al. 2004. Microglial activation facili-
tates Abeta plaque removal following intracranial anti-Abeta antibody administration.
Neurobiol Dis 15:11–20.
250. Sperling R, Salloway S, Brooks DJ et al. 2012. Amyloid-related imaging abnormalities
in patients with Alzheimer’s disease treated with bapineuzumab: A retrospective analy-
sis. Lancet Neurol 11:241–249.
251. Meyer-Luehmann M, Mora JR, Mielke M et al. 2011. T cell mediated cerebral hemor-
rhages and microhemorrhages during passive Abeta immunization in APPPS1 transgenic
mice. Mol Neurodegener 6:22.
252. Wilcock DM, Morgan D, Gordon MN et al. 2011. Activation of matrix metalloprotein-
ases following anti-Abeta immunotherapy; implications for microhemorrhage occur-
rence. J Neuroinflammation 8:115.
253. Wilcock DM, Alamed J, Gottschall PE et al. 2006. Deglycosylated anti-amyloid-beta
antibodies eliminate cognitive deficits and reduce parenchymal amyloid with minimal
vascular consequences in aged amyloid precursor protein transgenic mice. J Neurosci
26:5340–5346.
254. Wilcock DM, Jantzen PT, Li Q et al. 2007. Amyloid-beta vaccination, but not nitro-
nonsteroidal anti-inflammatory drug treatment, increases vascular amyloid and micro-
hemorrhage while both reduce parenchymal amyloid. Neuroscience 144:950–960.
255. Dovey HF, John V, Anderson JP et al. 2001. Functional gamma-secretase inhibitors
reduce beta-amyloid peptide levels in brain. J Neurochem 76:173–181.
256. Cirrito JR, May PC, O’Dell MA et al. 2003. In vivo assessment of brain interstitial fluid
with microdialysis reveals plaque-associated changes in amyloid-beta metabolism and
half-life. J Neurosci 23:8844–8853.
257. Best JD, Smith DW, Reilly MA et al. 2007. The novel gamma secretase inhibitor
N-[cis-4-[(4-chlorophenyl) sulfonyl]-4-(2,5-difluorophenyl) cyclohexyl]-1,1,1-trifl uoro-
methanesulfonamide (MRK-560) reduces amyloid plaque deposition without evidence
of notch-related pathology in the Tg2576 mouse. J Pharmacol Exp Ther 320:552–558.
258. Siemers ER, Quinn JF, Kaye J et al. 2006. Effects of a gamma-secretase inhibitor in a
randomized study of patients with Alzheimer disease. Neurology 66:602–604.
259. Siemers ER, Dean RA, Friedrich S et al. 2007. Safety, tolerability, and effects on
plasma and cerebrospinal fluid amyloid-beta after inhibition of gamma-secretase. Clin
Neuropharmacol 30:317–325.
260. Fleisher AS, Raman R, Siemers ER et al. 2008. Phase 2 safety trial targeting amyloid
beta production with a gamma-secretase inhibitor in Alzheimer disease. Arch Neurol
65:1031–1038.
261. Bateman RJ, Siemers ER, Mawuenyega KG et al. 2009. A gamma-secretase inhibitor
decreases amyloid-beta production in the central nervous system. Ann Neurol 66:48–54.
262. Green RC, Schneider LS, Amato DA et al. 2009. Effect of tarenflurbil on cognitive
decline and activities of daily living in patients with mild Alzheimer disease: A random-
ized controlled trial. JAMA 302:2557–2564.
263. May PC, Dean RA, Lowe SL et al. 2011. Robust central reduction of amyloid-beta
in humans with an orally available, non-peptidic beta-secretase inhibitor. J Neurosci
31:16507–16516.
264. Wright TM. 2006. Tramiprosate. Drugs Today (Barc) 42:291–298.
265. Aisen PS, Gauthier S, Vellas B et al. 2007. Alzhemed: A potential treatment for
Alzheimer’s disease. Curr Alzheimer Res 4:473–478.
266. Aisen PS, Gauthier S, Ferris SH et al. 2011. Tramiprosate in mild-to-moderate
Alzheimer’s disease—A randomized, double-blind, placebo-controlled, multi-centre
study (the Alphase Study). Arch Med Sci 7:102–111.
267. Saumier D, Duong A, Haine D et al. 2009. Domain-specific cognitive effects of tra-
miprosate in patients with mild to moderate Alzheimer’s disease: ADAS-cog subscale
results from the Alphase Study. J Nutr Health Aging 13:808–812.
268. Gauthier S, Aisen PS, Ferris SH et al. 2009. Effect of tramiprosate in patients with mild-
to-moderate Alzheimer’s disease: Exploratory analyses of the MRI sub-group of the
Alphase study. J Nutr Health Aging 13:550–557.
269. Tucker HM, Kihiko M, Caldwell JN et al. 2000. The plasmin system is induced by and
degrades amyloid-beta aggregates. J Neurosci 20:3937–3946.
270. Jacobsen JS, Comery TA, Martone RL et al. 2008. Enhanced clearance of Abeta in brain
by sustaining the plasmin proteolysis cascade. Proc Natl Acad Sci USA 105:8754–8759.
271. Schneider LS, Insel PS, Weiner MW. 2011. Treatment with cholinesterase inhibitors and
memantine of patients in the Alzheimer’s Disease Neuroimaging Initiative. Arch Neurol
68:58–66.
272. Golde TE, Schneider LS, Koo EH. 2011. Anti-abeta therapeutics in Alzheimer’s disease:
The need for a paradigm shift. Neuron 69:203–213.
273. Weiner MW, Veitch DP, Aisen PS et al. 2012. The Alzheimer’s Disease Neuroimaging
Initiative: A review of papers published since its inception. Alzheimers Dement
8:S1–S68.
274. Reines SA, Block GA, Morris JC et al. 2004. Rofecoxib: No effect on Alzheimer’s
disease in a 1-year, randomized, blinded, controlled study. Neurology 13:66–71.
275. Aisen PS, Schafer KA, Grundman M et al. 2003. Alzheimer’s disease cooperative study.
Effects of rofecoxib or naproxen vs placebo on Alzheimer disease progression: A ran-
domized controlled trial. JAMA 289:2819–2826.
276. Scharf S, Mander A, Ugoni A et al. 1999. A double-blind, placebo-controlled trial of
diclofenac/misoprostol in Alzheimer’s disease. Neurology 13:197–201.
277. de Jong D, Jansen R, Hoefnagels W et al. 2008. No effect of one-year treatment with
indomethacin on Alzheimer’s disease progression: A randomized controlled trial. PLoS
One 23:e1475.
278. Pasqualetti P, Bonomini C, Dal Forno G et al. 2009. A randomized controlled study on
effects of ibuprofen on cognitive progression of Alzheimer’s disease. Aging Clin Exp
Res 21:102–110.
279. Aisen PS, Schmeidler J, Pasinetti GM. 2002. Randomized pilot study of nimesulide
treatment in Alzheimer’s disease. Neurology 58:1050–1054.
13 Nutrition in
Autoimmunity
A Focus on Systemic
Lupus Erythematosus and
Rheumatoid Arthritis
Maureen McMahon
CONTENTS
Introduction............................................................................................................. 211
Caloric Restriction.................................................................................................. 212
Amino Acids........................................................................................................... 213
Taurine.................................................................................................................... 213
Isoflavones.............................................................................................................. 214
n-3 and n-6 Polyunsaturated Fatty Acids................................................................ 214
Furanocoumarins..................................................................................................... 216
Vitamin D................................................................................................................ 217
Vitamin E................................................................................................................ 218
Vitamins B6, B12, and Folate................................................................................. 219
Vitamin C................................................................................................................ 220
Curcumin................................................................................................................. 221
Conclusions............................................................................................................. 221
References............................................................................................................... 222
INTRODUCTION
A normal functioning immune system recognizes and eliminates foreign cells, bacte-
ria, viruses, and macromolecules while maintaining tolerance toward self. If mecha-
nisms of tolerance break down, the persistence of autoreactive T and B cells can
occur, leading to the formation of autoantibodies, the elaboration of inflammatory
cytokines, and ultimately to the development of autoimmune diseases such as sys-
temic lupus erythematosus (SLE) and rheumatoid arthritis (RA) [1]. The prevalence
and incidence of autoimmune diseases are increasing in the developing world, sug-
gesting that the Western diet and lifestyle may be contributing [2]. Multiple aspects
of nutrition, including dietary interventions and nutritional supplements, may have
211
implications for both the pathogenesis and the management of autoimmune diseases
such as SLE and RA [3]. In addition, patients with RA and SLE have been defined
as at-risk populations for cardiovascular disease (CVD) by the American Heart
Association (AHA) and may therefore benefit from dietary interventions aimed to
reduce heart disease risk as well [4]. Here, we will review the interactions between
nutrition and autoimmunity by focusing on two of the most common autoimmune
rheumatic diseases, RA and SLE.
CALORIC RESTRICTION
Excessive caloric intake by SLE patients has been linked to metabolic syndrome,
obesity, increased heart disease, and increased lupus disease activity [5]. Obesity
itself has been linked to immune impairment; for example, obese children
have impairment of cell-mediated immune reactions and reduced intracellular
bacterial killing by polymorphonuclear leukocytes [6]. Obesity can also lead to
resistance to the adipokine leptin, which can in turn lead to hyperleptinemia,
an increase in proinflammatory cytokines, and increased stimulation of antigen-
presenting cells [7]. Leptin also inhibits T-regulatory cells, which can further
impair self-tolerance [7].
Excessive calorie intake has been associated with increased arthritis and elevated
IL-17 and Th17 expression in a collagen-induced arthritis mouse model [8]. In lupus-
prone mice, a high-fat diet and exogenous leptin administration resulted in increased
atherosclerosis (ATH) and worsened renal disease [9]. Conversely, restriction of
calories by 30%–40% has been shown to prolong the life of lupus-prone MRL/lpr
mice [10]. Caloric restriction may contribute to this survival benefit through mul-
tiple immune-mediated phenomena; for example, fasted NZB/NZW lupus mice have
reduced secretion of IgG2A and platelet-derived growth factor, which in turn results
in reduced glomerular lesions [11]. Caloric restriction also attenuates the increased
levels of Th1 cytokines typically seen in these mice, including interleukin-2 (IL-2)
and interferon gamma (IFN-γ), and inhibits decreases in CD4+ and CD8+ lympho-
cytes [10,12].
Extreme protein intake may also have deleterious effects on SLE disease activity.
Mice fed moderate levels of protein had a delay in the development of autoimmunity
compared with those fed a normal (higher) protein diet [13].
In human studies, higher protein consumption also resulted in higher rates
of cortical bone loss in juvenile patients with SLE [14]. A protein-restricted diet
(<0.6 g/kg/day) also improved the mean glomerular filtration rate in a population
of predialytic chronic renal disease patients (not restricted to SLE) [15]. In one
study in lupus-prone NZB/W mice, however, mice fed a low-fat (4.5%), high-
protein (23%) diet had later onset of hemolytic anemia, decreased autoantibody
formation, and increased longevity compared with mice fed a higher-fat, lower-
protein diet [16]. In a retrospective analysis from the Nurse’s Health Study, there
was no association found between protein or meat consumption and incidence
of RA [17]. Currently, data are insufficient to recommend any low-protein diet
for patients with autoimmune diseases. The recommended daily allowances for
protein are 46 g for women and 56 g for men in the 19–70+ years age group [18].
Nutrition in Autoimmunity 213
Note that a low-protein diet is not currently recommended for lupus nephritis
patients to avoid a negative protein balance and/or malnutrition.
AMINO ACIDS
L-canavanine is a nonprotein amino acid found in grains such as soybean, alfalfa
sprouts, onions, and seeds. A natural homologue of l-arginine, it has antimetabolic
activity and can induce cell apoptosis [19]. l-canavanine also demonstrates a supe-
rior ability to induce T cells and immunoglobulin-secreting cells [20]. Alfalfa seed
may have beneficial cardiovascular effects, as ingestion has been shown to reduce
serum cholesterol levels and leads to both prevention and regression of atheroscle-
rotic plaques in rats, rabbits, and monkeys [19]. However, during a human trial of
alfalfa seeds, a previously healthy man developed an autoimmune syndrome with
splenomegaly, pancytopenia, Coombs positive autoimmune hemolytic anemia
(AIHA), antinuclear antibodies (ANA), and hypocomplementemia. This syndrome
resolved after the discontinuation of alfalfa [21].
Furthermore, when l-canavanine was fed to mice, DBA/2 mice were more
likely to develop anti-dsDNA and ANA antibodies, and NZB/W mice demonstrated
accelerated mortality, worsened renal damage, and an increased number of
immunoglobulin-producing cells [22]. Studies in cynomolgus monkeys have also
demonstrated that ingestion of alfalfa sprouts can induce a reversible lupus-like
autoimmune syndrome accompanied by positive ANA and double-stranded DNA
(dsDNA) antibodies and by hypocomplementemia [19].
The Baltimore Lupus Environmental Study also showed a significant association
between alfalfa sprout consumption and initiation of SLE disease [5]. Ingestion of
6–24 alfalfa tablets per day has also been found to flare lupus disease activity in
patients [19]. In general, these findings have led to the recommendation that lupus
patients avoid the ingestion of alfalfa sprouts or alfalfa derivatives. However, in one
study, supplementation with alfalfa-based ethyl acetate extract resulted in a signifi-
cant decrease in the levels of IFN-γ and inflammatory responses of self-reactive
lymphocytes, decreased the disease severity, and increased the survival and life span
of lupus-prone MRL/lpr mice [23]. Alfalfa sprouts do also have lipid-lowering prop-
erties, and some studies suggest that cooking can destroy the deleterious effects of
alfalfa without attenuating its lipid-lowering effects.
TAURINE
Taurine is a major intracellular B-amino acid found in foods such as eggs, meat,
oysters, and squid. Taurine is present in high concentrations in leukocytes and is
thought to regulate the immune system by protecting cells from oxidative stress,
primarily through the neutralization of hypochlorous acid [24,25]. Taurine can
also regulate the immune response by inhibiting the production of inflammatory
cytokines and prostaglandin E2 (PGE2) and by decreasing the activity of matrix
metalloproteinases [24].
There are some animal data to suggest that taurine supplementation could be
beneficial in rheumatic diseases. For example, administration of intraperitoneal
taurine chloride to rats with adjuvant-induced arthritis resulted in downregulation

of the inflammatory response (including histamine and oxygen radical species gen-
eration) [26]. Taurine chloride administration also attenuated symptoms of collagen-
induced arthritis in one study [27], but in another study, taurine did not alleviate
arthritis symptoms but did seem to prevent arthritis when administered early [28].
When a taurine-rich diet was fed to NZB/NZW mice on a high-cholesterol diet, signif-
icant reductions were seen in cardiac abnormalities [29] and apoptosis of hepatic cells
[30]. However, human studies of taurine supplementation in SLE and RA are lacking.
ISOFLAVONES
Soybean-based foods are high in isoflavones, which are structurally similar to
17B-estradiol. Soy isoflavones are commonly used to combat the symptoms of
menopause and are also known to have antioxidant [31] and immune-modulating
effects [32]. In a collagen-induced model of arthritis, rats treated with soy protein
and the isoflavones genistein and daidzein demonstrated decreased arthritis
symptoms and decreased serum concentrations of inflammatory cytokines and adi-
pokines, including tumor necrosis factor- alpha (TNF-α), IL-6, adiponectin, and
leptin [33]. Prevention of tissue damage and joint inflammation was also observed
following treatment [33]. In addition, arthritis-induced decreases in the levels of
protective antioxidant enzymes such as paraoxonase and arylesterase activity were
restored after treatment with soy protein and isoflavones [31]. However, there is some
concern that isoflavones could have negative effects on rheumatic diseases such as
SLE that are thought to have an estrogenic component. To date, data regarding the
effects of isoflavones in SLE are conflicting. In one study by Zhao et al., lupus-prone
MRL/lpr mice fed soybean-rich diets demonstrated increased serum creatinine,
decreased creatinine clearance, and increased the severity of glomerular disease
[34]. However, in another study, isoflavone supplements improved the survival of
SLE mice, decreased anti-dsDNA and anticardiolipin antibody production, and
decreased secretion of IFN-γ, compared to controls treated with tamoxifen [35].
Clinical studies of the effects of isoflavones on disease activity in patients with SLE
and RA are currently lacking.
n-3 AND n-6 POLYUNSATURATED FATTY ACIDS

The n-6 polyunsaturated fatty acid (PUFA) arachidonic acid (ARA) is a precursor
to prostaglandins (PGs) and cyclooxygenases (COXs), which are partially respon-
sible for inflammation in SLE and RA [36]. Conversely, n-3 fatty acids reduce the
arachidonic acid cascade by inhibiting COX and lipoxygenase (LOX) [37]. Fish
oil, one of the major sources of n-3 PUFA, such as eicosapentaenoic acid (EPA)
and docosahexaenoic acid (DHA), has multiple anti-inflammatory and antiautoim-
mune effects. Although EPA is a substrate for the COX and LOX enzymes that
produce eicosanoids, the mediators formed are different in structure and are often
less biologically active than ARA-derived mediators [36]. There does seem to be
a threshold dietary dose of EPA required to result in a clinical effect—between
1.35 and 2.7 g/day [38].
The fatty acid intake of the typical Western diet contains about 0.5%–1% of the
n-3 PUFA EPA and 1.5%–3% DHA compared to 10%–20% of the n-6 PUFA ARA
[36]. Modulation of the percentages of dietary fatty acid intake can result in altera-
tions in the fatty acid composition of blood leukocytes, including monocytes and
neutrophils [36].
n-3 fatty acid has been reported to result in downregulation of many inflamma-
tory mediators both in healthy patients and in patients with rheumatic diseases. For
instance, dietary fish oil supplementation leads to decreased leukocyte production
of PGE2 and leukotrienes in healthy volunteers [39], and decreased neutrophil [40]
and monocyte production of LTB4 [41] and PGE2 [42] in patients with RA. EPA and
DHA inhibit the production of inflammatory cytokines such as TNF-α, IL-6, and
IL-1 in both in vitro studies [43,44] and in some in vivo studies in healthy volun-
teers, although human studies have not been uniformly consistent [36]. DHA also
has significant inhibitory effects on NF-κB and TNF-α [37]. Cell culture and animal
studies indicate that both EPA and DHA inhibit T-cell proliferation and production
of IL-2 [45]. Although some clinical studies have supported these findings [46],
the results have been inconsistent [36]. n-3 fatty acids may also have antioxidant
effects: high doses of n-3 fatty acids (>3 g/day), but not lower doses, resulted in
decreased production of reactive oxygen species by neutrophils and monocytes in
the blood of healthy volunteers [47]. Supplementation with n-3 FA and a low n-6 FA
diet decreased the expression of serum sTNF-R p55 and C-reactive protein (CRP)
levels in patients with RA [48].
Multiple studies in animal models of RA have suggested a clinical benefit to sup-
plementation with fish oil [49]. In one study, animals with collagen-induced arthri-
tis that were fed fish oil had delayed onset of arthritis, reduced incidence, and less
severe disease compared to animals given a vegetable oil feed [50]. In another study,
animals treated with either fish oil or krill oil developed slower onset and less severe
disease than control animals [51].
n-3 fatty acids have had multiple favorable studies in SLE mouse models. Fish
oil supplementation decreased proteinuria and protected kidney tissues against
free radical–induced damage in multiple murine models of SLE [37], presumably
through reductions in PI3 lipid kinase [52], decreased apoptosis, and decreased
TGF-β expression in renal tissue [53]. DHA has also been demonstrated to reduce
IL-18, serum ds-DNA antibody levels, and IgG renal deposits in NZB/NZW mice
[52]. Flaxseed oil is also high in n-3 PUFA and has also been shown to decrease pro-
teinuria, preserve glomerular function, and decrease anticardiolipin and anti-dsDNA
antibodies in mouse models of SLE [37].
In humans, one large epidemiologic study from Sweden demonstrated that dietary
intake of oily fish was associated with a modestly decreased risk of developing RA
[54]. There have also been a number of clinical trials of fish oil in RA. These results
were examined in a meta-analysis by Goldberg et al. in 2007, which indicated that
fish oil in RA reduces patient-assessed joint pain, the number of painful and tender
joints, duration of morning joint stiffness, and nonsteroidal anti-inflammatory use.
The meta-analysis found no effect of fish oil on patient-assessed disease activity
or the Ritchie articular index [55]. These findings were contradictory to a previous
AHRQ-sponsored meta-analysis by MacLean et al., which concluded that fish oil
supplementation has no effect on patient-reported pain, swollen joint count, disease

activity, or patient global assessment; however, this meta-analysis differed from the
Goldberg analysis in that it included studies with flaxseed oil, one study with no
control arm, and one study using transdermal application [56]. Another recent sys-
tematic review also concludes that there appears to be a modest clinical efficacy for
fish oil in RA [36].
In a small clinical study of 12 SLE patients with lupus nephritis, treatment with
fish oil resulted in decreased ARA levels, decreased LTB4, and decreased plate-
let aggregation [57]. MacLean et al. concluded an AHRQ-sponsored systematic
review of fish oil in SLE. Three studies were identified, which was considered an
inadequate amount of data to perform a meta-analysis. However, it was concluded
that omega-3 fatty acids had variable effects on clinical activity in SLE. In one
study, improvement in disease activity was noted using an outcome measure that
was developed for this study (and that had not been previously validated) [58]. Two
other studies noted no improvement in disease activity after treatment with fish oil
[57,59]. No studies assessed the effect on end organ damage, patient perception of
disease, or requirements for other immunosuppressive drugs. One study showed no
effect of fish oil on corticosteroid requirements [57]. In one SLE study published
after the MacLean review, fish oil treatment resulted in significant improvements
in the disease activity and endothelial function compared to an olive oil control.
It was concluded that low-dose n-3 PUFA supplementation has a therapeutic effect
on disease activity and might also have cardiovascular benefits [60]. In fact, for all
women at risk for heart disease, the AHA recommends the consumption of omega-3
fatty acids in the form of fish or in capsule form (e.g., EPA 1800 mg/day) as a class
IIB recommendation for primary and secondary prevention, especially for women
with hypercholesterolemia and/or hypertriglyceridemia. Note that a class IIb recom-
mendation is considered when the usefulness/efficacy of an intervention is less well
established by evidence/opinion than for a IIa recommendation (weight of evidence/
opinion is in favor of usefulness/efficacy) or a class I recommendation (Intervention
is useful and effective) [4].
FURANOCOUMARINS
Photosensitivity is a hallmark of SLE disease activity [61]. Dietary furanocouma-
rins (FCs) are produced by a number of plants, including celery, parsley, grape-
fruit, and parsnips, and can cause photosensitization. FCs intercalate into DNA,
where they can be activated by UV light to form a bond with pyrimidine bases
(especially thymidine). FCs also induce the formation of reactive oxygen species
and free radicals [62].
The dietary levels of FCs required for the induction of photosensitivity in the
general population are fairly high; for example, a normal dietary portion of celery
root is 100–150 g, while portions five to seven times that size would normally be
required to reach the serum levels required to produce phototoxic reactions. However,
it is possible that the phototoxic threshold is lower in SLE because lupus patients are
known at baseline to have a significantly reduced minimum erythemal dose to UV
light compared to healthy subjects [62]. Renal insufficiency might also lower the
clearance of dietary FCs. Furthermore, photoadducts of the FC 8-methoxypsoralen

have been found to cross-react with anti-double-stranded DNA antibodies [63].
Although the potential for FC-rich diets to induce photosensitivity in SLE patients
has not been formally studied, it would be wise for lupus patients to avoid any fad
diets that include ingestion of large amounts of FCs, such as celery juice– or grape-
fruit juice–based diets. A food journal might also be helpful for SLE patients who
have experienced frequent or troublesome flares of photosensitive skin rashes; if a
high dietary intake of FC-rich foods is identified, dietary adjustments could be rec-
ommended as a relatively simple therapeutic approach.
VITAMIN D
The vitamin D receptor is expressed on the surface of many cells of the immune
system, including monocytes, macrophages, dendritic cells (DCs), and activated
T and B cells [64]. 1,25(OH)2D has several immune modulating effects, including
downregulating Th1 responses, decreasing proliferation of activated B cells, and
increasing regulatory T cells [65,66]. It also promotes the development of tolerogenic
DCs and inhibits the IFN-α signal that is typical of active SLE [67]. Vitamin D also
appears to play an important role in innate immunity, as monocyte and macrophage
Toll-like receptor responses to bacterial infections are heightened by 25(OH)D [68].
The vitamin D receptor is expressed on the surface of many cells of the immune sys-
tem, including monocytes, macrophages, DCs, and activated T and B cells [64]. SLE
patients with vitamin D deficiency are more likely to have higher mean IFN-α levels
than those without [64]. Vitamin D also can inhibit anti-dsDNA antibody production
in peripheral blood mononuclear cells from SLE patients [69].
SLE patients have been noted to have low serum vitamin 25(OH) D levels (mean
25.5 mmol/L) compared to the recommended levels of 50–80 mmol/L. SLE patients
have several unique clinical aspects, which may contribute to vitamin D deficiency.
Because of the photosensitive nature of SLE disease activity, photoprotection is
strongly recommended for all SLE patients; however, sunlight is the primary source
of vitamin D3 [66]. Chronic steroid use and higher serum creatinine levels are also
associated with decreased vitamin D levels [70]. Lupus disease activity itself may
result in lower vitamin D levels [71]. Although some studies have suggested that high
vitamin D intake (>37 mg/mL) is associated with decreased risks for type I diabetes,
inflammatory bowel disease, and multiple sclerosis, a large prospective study (the
Nurse’s Health Study) found no association between vitamin D intake and the inci-
dence of a new diagnosis of either SLE or RA [72]. There have been some studies
that have suggested an association between SLE disease activity and SLE; for exam-
ple, Petri et al. found that a 20 ng/mL increase in vitamin D was associated with a
modest decrease in the odds of having a high activity score and a 15% decrease in the
odds of having clinically important proteinuria [73]. A recent meta-analysis looked
at all the studies of the association between vitamin D and SLE disease activity.
They concluded that there is convincing evidence to support the association between
vitamin D and disease activity. Out of the 15 observational studies that looked at this
question, 10 studies (including the three largest) demonstrated a statistically signifi-
cant inverse relationship. There was no convincing evidence, however, to support an
association between damage and vitamin D levels [74]. In one randomized clinical
trial, SLE patients treated with vitamin D had improved markers of disease activity,
including lower anti-double-stranded DNA and anti-Smith antibodies, and higher
C4 levels [71]. There were also significant decreases in inflammatory cytokines and
markers, including IL-6, IL-18, IL-1, and fibrinogen [71]. In another study, vitamin
D supplementation in SLE induced increases of CD4+ T cells and regulatory T cells
and a decrease in effector Th1 and Th17 cells. Vitamin D also induced a decrease in
memory B cells and anti-DNA antibodies [75].
In RA, one observational study demonstrated that patients with RA were more
likely to have low vitamin D than matched controls and that vitamin D levels were
inversely related to disease activity measured by the visual analog scale, but not by
the Disease Activity Score (DAS) [76]; however, another cohort study found that
extremely low vitamin D levels (<15 nmol/L) were significantly associated with high
DAS and more severe RA disease [77]. In a meta-analysis, Song et al. concluded that
there is a significant inverse association between vitamin D supplement intake and
RA incidence [78]. They also found that most studies indicated an inverse associa-
tion between vitamin D intake and RA disease activity [78].
Vitamin D also has important implications for bone health in patients with rheu-
matic diseases. For instance, patients with SLE are at greater risk for both osteoporo-
sis and fractures. This increased risk has been attributed to corticosteroid use, renal
disease, and to disease activity itself, but vitamin D deficiency also appears to play
a role. Low levels of 25(OH) vitamin D results in decreased absorption of dietary
calcium and ultimately reduced bone mineral density [66]. Low vitamin D levels
have been associated with osteopenia in adolescent patients with SLE [79]. The
American College of Rheumatology recommends that all postmenopausal patients
with rheumatic diseases who are treated with glucocorticoids should be treated with
both vitamin D and calcium 1200–1500 mg/day [80].
Vitamin D has also been implicated in the risk for CVD. In one meta-analysis
of 28 studies from the general population, the highest levels of serum 25(OH)
vitamin D were associated with a 43% reduction in cardiometabolic disorders (OR
0.57, 95% [CI 0.48–0.68]) [81]. Studies in rheumatic disease populations also suggest
an association between vitamin D and cardiovascular risk. In one RA cohort study,
low levels of 25 (OH) vitamin D were associated with increased markers of endo-
thelial dysfunction, including E-selectin, ICAM, and low levels of HDL cholesterol,
even after controlling for disease activity [82]. In one study of SLE subjects, levels
of 25 (OH) vitamin D were inversely associated with carotid plaque [83]. Similarly,
Reynolds et al. found that vitamin D deficiency is associated with increased aortic
stiffness in SLE, independent of CVD risk factors and insulin [84].
VITAMIN E
Vitamin E is the most effective peroxyl radical scavenger and protects lipid mem-
branes against peroxidation [85]. In addition, vitamin E has multiple effects on the
immune system. Vitamin E deficiency impairs humoral and cellular immune func-
tions (2). Vitamin E also decreases production of PGE2 and decreases lymphocyte
proliferation [86].
The relationship of vitamin E to SLE has been conflicting. Some studies suggest
that MRL/lpr mice fed supplements of vitamin E had decreased inflammatory cyto-
kine production, decreased autoimmunity, and increased survival [2]. In one study in
MRL/lpr mice, low-dose vitamin E supplementation led to improved survival; how-
ever, high-dose supplementation had decreased survival compared to low dose [87].
Furthermore, high-dose vitamin E supplementation did not result in decreased levels
of tissue oxidation, but did result in increased production of anti-dsDNA and anticar-
diolipin antibodies. In the low-dose vitamin E group, IL-2 secretion was enhanced,
while the high dose inhibited IL-2 release [88]. In a separate study, NZB/W mice
on an oxidized oil diet had decreased oxidative stress, proinflammatory cytokine
production (IL-6 and IFN-γ), and decreased anti-dsDNA antibody production when
supplemented with vitamin E [89].
The data from clinical studies have also been conflicted. In one clinical cohort
study in RA, serum MDA levels were higher, and plasma concentration of vitamin E
was significantly lower than healthy controls [90]. In a large randomized controlled
trial, the Women’s Health study, vitamin E supplementation did not decrease the risk
of incident RA [91]. A meta-analysis of trials of vitamin E in RA concluded that the
clinical trials “have been methodologically weak and have produced contradictory
findings” [92].
The results of studies of vitamin E in SLE are also conflicted. In one study, vita-
min E intake was inversely associated with disease activity in SLE [93]; however,
this association was not seen in the Nurses Health Study [94]. In another study, a
combination of vitamin C and vitamin E intake resulted in decreased lipid peroxida-
tion (measured by malondialdehyde levels), but did not improve endothelial function
in SLE [95].
Thus, there is inconsistent evidence that suggests no clear benefit for vitamin E
supplementation in SLE and RA. In addition, in the AHA guidelines for the preven-
tion of CVD in women, supplementation with vitamin E was considered a class III
recommendation, meaning that the intervention was considered to be not useful and/
or harmful and therefore should not be used for the prevention of CVD [4].
VITAMINS B6, B12, AND FOLATE

Intake of vitamins B6, B12, and folate may influence serum levels of inflammatory
markers such as CRP [96]. These nutrients are also cofactors for the metabolism of
homocysteine [87]. Several groups have identified homocysteine as a predictor for
CAD and stroke in the general population [97] and for ATH in SLE, where high lev-
els predict increased coronary calcium [98], plaque progression [99], and IMT pro-
gression [100]. The importance of vitamins B6, B12, and folate in the metabolism of
homocysteine implies that deficiencies in any of these nutrients could lead to acceler-
ated heart disease in patients with rheumatic diseases [99]. Hyperhomocysteinemia
has also been linked to increased inflammation and disease activity in patients with
SLE and RA. For instance, levels of the proinflammatory cytokine MCP-1 were
strongly positively correlated with homocysteine concentrations in SLE [101].
Dietary fiber intake is also inversely associated with homocysteine levels, as well as
inflammatory cytokines and markers such as IL-6 and CRP [102].
Vitamin B6 might also directly modulate disease activity independent of homo-

cysteine; vitamins B6 and folate act as cofactors in the metabolism of antibodies
and cytokines [103]. Deficiency of vitamin B6 also impairs lymphocyte maturation
[103,104].
In one observational cohort study, RA patients with low vitamin B6 had an
increase in inflammatory markers such as IL-6 compared to those with adequate B6
levels; however, this difference was no longer significant after controlling for serum
albumin [105]. In one study in RA patients, supplementation with 50 mg/day vitamin
B6 corrected pyridoxine deficiency but did not result in any change in inflammatory
cytokines (106). However, in one small, single-blinded, placebo-controlled study,
high-dose vitamin B6 supplementation (100 mg/day) suppressed proinflammatory
cytokines (IL-6 and TNF-α) in patients with RA [107]. In one long-term prospective
Japanese cohort study, vitamin B6 intake was inversely associated with the risk of
active lupus, but not with cardiovascular events [96]. However, in the AHA guide-
lines for the prevention of CVD in women, supplementation with folic acid, with or
without B6 and B12 supplementation, was considered a class III recommendation
and therefore should not be used for primary or secondary prevention of CVD [4].
Thus, at this time, there is no strong evidence to support vitamins B6, B12, or folate
supplementation in women with SLE or RA.
VITAMIN C
Vitamin C is known to have antioxidant effects. Supplementation with monthly
vitamin C resulted in significant improvements in flow-mediated dilation in non-
autoimmune patients with coronary artery disease [3]. Oxidative stress is increased
in patients with SLE and RA, and thus vitamin C may exert a protective role in
these patients [108,109]. In mice, supplementation with vitamins C, E, and B reduces
lymphoproliferation, IgG, and anti-dsDNA antibody levels [110], while insuf-
ficiency resulted in increased oxidative stress and induction of inflammation [3].
In a collagen-induced model of arthritis, vitamin C supplementation resulted in
improved antioxidant status, with decreased plasma measures of lipid peroxidation,
ceruloplasmin, and PGE2 [111].
Vitamin C supplementation also resulted in small but significant reductions in
lipid peroxidation in SLE patients [3]. In one prospective longitudinal Japanese
cohort study, risk of active lupus disease was inversely associated with vitamin C
intake [93]. As noted earlier, a combination of vitamin C and E supplementation did
result in decreased measurements in some (but not all) markers of oxidative stress
in SLE patients, but it did not improve endothelial function [95]. In a small study
in RA patients, supplementation with quercetin + vitamin C (166 mg + 133 mg/
capsule) did not result in any improvement in disease severity or blood markers of
inflammation (TNF-α, IL-6, IL-1Β, or CRP) [112]. Overall, there are currently no
convincing data to recommend vitamin C supplementation for patients with RA or
SLE. In addition, dietary supplementation with vitamin C was considered a class
III recommendation (i.e., not recommended) for primary and secondary prevention
of CVD in women [4].
CURCUMIN
Curcumin (diferuloylmethane) is a yellow pigment found in the rhizome of turmeric.
Curcumin is an anti-inflammatory agent that is known to have antioxidant properties,
can inhibit the complement cascade, and may also play a role in the inhibition of auto-
immune diseases [113]. Curcumin downregulates inflammatory cytokines such as
IL-1β, IL-6, IL-12, TNF-α, and IFN-γ and also downregulates associated signaling
pathways in immune cells, such as the NF-κΒ, ERK1/2, and JAK/STAT pathways
[114,115]. Curcumin has also been reported to inhibit IFN-α-induced expression of
COX-2 and to downregulate PGE2 production [113]. Curcumin is also able to induce
apoptosis in the synovial fibroblasts of RA patients; [115] this antiproliferative activ-
ity may contribute to its ability to decrease the synovial hypertrophy that is charac-
teristic of RA [113].
In one collagen-induced mouse model, curcumin attenuated the progression and
severity of arthritis, accompanied with decreases of serum B-cell-activating factor
belonging to the TNF family (BAFF) levels and decreased serum IFN-γ and IL-6
[116]. When curcumin supplementation was given to lupus-prone NZB/W mice, IgG
immune complex deposition, renal inflammation, and anti-dsDNA antibody forma-
tion were all reduced; these protective effects seemed to be mediated by T-regulatory
cells [117]. However, when curcumin was used in an experimental mouse model of
central nervous system lupus, immune complex deposition and worsened neurologic
outcomes/cognitive performance (maze performance) were noted [118].
There has been one small randomized clinical trial on curcumin in RA, in which
curcumin-treated patients had significant improvements in Disease Activity Scores
(DAS) and a higher percentage achieved ACR 20, 50, and 70 scores compared to
diclofenac-treated patients [119]. In one small randomized placebo controlled trial
in lupus nephritis, curcumin supplementation decreased proteinuria, hematuria, and
systolic blood pressure compared to controls [120]. Further studies will need to be
done to establish the efficacy and safety of curcumin in the treatment of autoimmune
conditions.
CONCLUSIONS
Although nutrition can impact the development and progression of autoimmunity in
multiple ways, there is a paucity of strong clinical data regarding specific nutritional
interventions in patients with rheumatic diseases. To date, data for supplementation
with n-3 fatty acids and vitamin D are the most consistent. Until more adequate data
are obtained in large well-designed randomized controlled trials in patients with
autoimmune diseases, it is reasonable to base other specific dietary intake recom-
mendations for patients upon the AHA dietary recommendations for women at risk
for CVD [4].
According to these guidelines, “patients should be advised to consume a diet rich
in fruits and vegetables; to choose whole-grain, high-fiber foods; to consume fish,
especially oily fish, at least twice a week; to limit intake of saturated fat, cholesterol,
alcohol, sodium, and sugar; and to avoid trans-fatty acids. Note: Pregnant women
(and possibly those with future child-bearing potential) should avoid eating fish with
the highest level of mercury contamination (e.g., shark, swordfish, king mackerel,
or tile fish). Women should maintain or lose weight through an appropriate balance
of physical activity, caloric intake, and formal behavioral programs when indicated
to maintain or achieve an appropriate body weight (e.g., BMI < 25 kg/m2 in U.S.
women), waist size (e.g., <35 in), or other target metric of obesity” [4].
REFERENCES
1. Kim SJ, McMahon M. 2013. Diagnosis and treatment of systemic lupus erythematosus.
J Clin Outcomes Manage 20:85–95.
2. Hseih C-C, Lin B-F. 2011. Dietary factors regulate cytokines in murine models of
systemic lupus erythematosus. Autoimmun Rev 11:22–27.
3. Klack K, Bonfa E, Borba EF. 2012. Diet and nutritional aspects in systemic lupus
erythematosus. Rev Bras Reumatol 52:384–408.
4. Mosca L, Benjamin EJ, Berra K, Bezanson JL, Dolor RJ, Lloyd-Jones DM et al.
2011. Effectiveness-based guidelines for the prevention of cardiovascular disease in
women—2011 update: A guideline from the American Heart Association. Circulation
123:1243–1262.
5. Petri M. 2001. Diet and systemic lupus erythematosus: From mouse and monkey to
woman? Lupus 10:775–777.
6. Maki PA, Newberne PM. 1992. Dietary lipids and immune function. J Nutr 122:610–614.
7. Duntas LH, Biondi B. 2012. The Interconnections between obesity, thyroid function,
and autoimmunity: The multifold role of leptin. Thyroid 23:646–653.
8. Jhun J, Yoon B, Park M, Oh H, Byun J, Lee S et al. 2012. Obesity aggravates the
joint inflammation in a collagen-induced arthritis model through deviation to Th17
differentiation. Exp Mol Med 44:424–431.
9. Hahn BH, Lourencco EV, McMahon M, Skaggs B, Le E, Anderson M et al. 2010.
Pro-inflammatory high-density lipoproteins and atherosclerosis are induced in lupus-
prone mice by a high-fat diet and leptin. Lupus 19:913–917.
10. Muthukumar A, Jolly C, Zaman K, Fernandes G. 2000. Calorie restriction decreases
proinflammatory cytokines and polymeric Ig receptor expression in the submandibular
glands of autoimmune prone (NZB × NZW)F1 mice. J Clin Immunol 20:354–361.
11. Troyer D, Chandrasekar B, Barnes J, Fernandes G. 1997. Calorie restriction decreases
platelet-derived growth factor (PDGF)-A and thrombin receptor mRNA expression in
autoimmune murine lupus nephritis. Clin Exp Immunol 108:58–62.
12. Jolly C, Fernandez R, Muthukumar A, Fernandes G. 1999. Calorie restriction modulates
Th-1 and Th-2 cytokine-induced immunoglobulin secretion in young and old C57BL/6
cultured submandibular glands. Aging 11:383–389.
13. Brown AC. 2000. Lupus erythematosus and nutrition: A review of the literature. J Ren
Nutr 10:170–183.
14. Caetano M, Ortiz T, Terreri M, Sarni R, Silva S, Souza F et al. 2009. Inadequate dietary
intake of children and adolescents with juvenile idiopathic arthritis and systemic lupus
erythematosus. J Pediatr 85:509–515.
15. Milovanov I, Lysenko L, Milovanova L, Dobrosmyslov I. 2009. The role of balanced
low-protein diet in inhibition of predialysis chronic kidney disease progression in
patients with systemic diseases. Ter Arkh 81:52–57.
16. Fernandes G, Yunis E, Smith J, Good R. 1972. Dietary influence on breeding behavior,
hemolytic anemia, and longevity in NZB mice. Proc Soc Exp Biol Med 139:1189–1196.
17. Benito-Garcia E, Feskanich D, Hu F, Mandl L, Karlson E. 2007. Protein, iron, and meat
consumption and risk for rheumatoid arthritis: A prospective cohort study. Arthritis Res
Ther 9(1):R16.
18. Britten P, Cleveland LE, Koegel KL, Kuczynski KJ, Nickols-Richardson SM. 2012.
Updated US Department of Agriculture Food Patterns meet goals of the 2010 dietary
guidelines. J Acad Nutr Diet 112(10):1648–1655.
19. Akaogi J, Barker T, Kuroda Y, Nacionales DC, Yamasaki Y, Stevens BR et al. 2006. Role
of non-protein amino acid L-canavanine in autoimmunity. Autoimmun Rev 5:429–435.
20. Morimoto I, Shiozawa S, Tanaka Y, Fujita T. 1990. L-canavanine acts on suppressor-
inducer T cells to regulate antibody synthesis: Lymphocytes of systemic lupus erythema-
tosus patients are specifically unresponsive to L-canavanine. Clin Immunol Immunopathol
55:97–108.
21. Malinow MR, Bardana EJ, Jr., Goodnight SH, Jr. 1981. Pancytopenia during ingestion
of alfalfa seeds. Lancet 1(8220 Pt 1):615.
22. Prete PE. 1985. Effects of L-canavanine on immune function in normal and autoimmune
mice: Disordered B-cell function by a dietary amino acid in the immunoregulation of
autoimmune disease. Can J Physiol Pharmacol 63:843–854.
23. Hong YH, Huang CJ, Wang SC, Lin BF. 2009. The ethyl acetate extract of alfalfa
sprout ameliorates disease severity of autoimmune-prone MRL-lpr/lpr mice. Lupus
18:206–215.
24. Marcinkiewicz J, Kontny E. 2012. Taurine and inflammatory diseases. Amino Acids.
July, 2012 (epub ahead of print).
25. Hagar HH. 2004. The protective effect of taurine against cyclosporine A-induced oxida-
tive stress and hepatotoxicity in rats. Toxicol Lett 151:335–343.
26. Wojtecka-Lukasik E, Gujski M, Roguska K, Maslinska D, Maslinski S. 2005. Taurine
chloramine modifies adjuvant arthritis in rats. Inflamm Res 54(Suppl 1):S21–S22.
27. Wang Y, Cha YN, Kim KS, Kim C. 2011. Taurine chloramine inhibits osteoclastogenesis
and splenic lymphocyte proliferation in mice with collagen-induced arthritis. Eur
J Pharmacol 668:325–330.
28. Kwasny-Krochin B, Bobek M, Kontny E, Gluszko P, Biedron R, Chain BM et al. 2002.
Effect of taurine chloramine, the product of activated neutrophils, on the development
of collagen-induced arthritis in DBA 1/J mice. Amino Acids 23:419–426.
29. Huang CY, Hsu TC, Kuo WW, Wu SP, Lin YM, Yen CY et al. 2009. Beneficial effects
of taurine on cardiac abnormality in NZB/W F1 mice fed with a high-cholesterol diet.
J Agric Food Chem 57:8635–8642.
30. Hsu TC, Chiang SY, Wu JH, Tsai CC, Huang CY, Chen YC et al. 2008. Treatment with
taurine attenuates hepatic apoptosis in NZB/W F1 mice fed with a high-cholesterol diet.
J Agric Food Chem 56:9685–9691.
31. Mohammadshahi M, Haidari F, Saei AA, Rashidi B, Mahboob S, Rashidi MR. 2013.
Soy protein, genistein, and daidzein improve serum paraoxonase activity and lipid pro-
files in rheumatoid arthritis in rats. J Med Food 16:147–154.
32. Wang J, Zhang Q, Jin S, He D, Zhao S, Liu S. 2008. Genistein modulate immune
responses in collagen-induced rheumatoid arthritis model. Maturitas 59:405–412.
33. Mohammad Shahi M, Rashidi MR, Mahboob S, Haidari F, Rashidi B, Hanaee J. 2011.
Protective effect of soy protein on collagen-induced arthritis in rat. Rheumatol Int
32:2407–2414.
34. Zhao JH, Sun SJ, Horiguchi H, Arao Y, Kanamori N, Kikuchi A et al. 2005. A soy diet
accelerates renal damage in autoimmune MRL/Mp-lpr/lpr mice. Int Immunopharmacol
5:1601–1610.
35. Hong Y, Wang T, Huang C, Cheng W, Lin B. 2008. Soy isoflavones supplementa-
tion alleviates disease severity in autoimmune-prone MRL-lpr/lpr mice. Lupus
17:814–821.
36. Miles EA, Calder PC. 2012. Influence of marine n-3 polyunsaturated fatty acids on
immune function and a systematic review of their effects on clinical outcomes in
rheumatoid arthritis. Br J Nutr 107(Suppl 2):S171–S184.
37. Pestka JJ. 2010. n-3 Polyunsaturated fatty acids and autoimmune-mediated glomerulo-
nephritis. Prostaglandins Leukot Essent Fatty Acids 82:251–258.
38. Rees D, Miles EA, Banerjee T, Wells SJ, Roynette CE, Wahle KW et al. 2006. Dose-
related effects of eicosapentaenoic acid on innate immune function in healthy humans:
A comparison of young and older men. Am J Clin Nutr 83:331–342.
39. von Schacky C, Kiefl R, Marcus AJ, Broekman MJ, Kaminski WE. 1993. Dietary n-3
fatty acids accelerate catabolism of leukotriene B4 in human granulocytes. Biochim
Biophys Acta 1166:20–24.
40. Kremer JM, Bigauoette J, Michalek AV, Timchalk MA, Lininger L, Rynes RI et al.
1985. Effects of manipulation of dietary fatty acids on clinical manifestations of rheu-
matoid arthritis. Lancet 1:184–187.
41. Cleland LG, French JK, Betts WH, Murphy GA, Elliott MJ. 1988. Clinical and bio-
chemical effects of dietary fish oil supplements in rheumatoid arthritis. J Rheumatol
15:1471–1475.
42. Cleland LG, Caughey GE, James MJ, Proudman SM. 2006. Reduction of cardiovascular
risk factors with long term fish oil treatment in early rheumatoid arthritis. J Rheumatol
33:1973–1979.
43. De Caterina R, Cybulsky MI, Clinton SK, Gimbrone MA, Jr., Libby P. 1994. The
omega-3 fatty acid docosahexaenoate reduces cytokine-induced expression of proath-
erogenic and proinflammatory proteins in human endothelial cells. Arterioscler Thromb
14:1829–1836.
44. Novak TE, Babcock TA, Jho DH, Helton WS, Espat NJ. 2003. NF-kappa B inhibition
by omega-3 fatty acids modulates LPS-stimulated macrophage TNF-alpha transcription.
Am J Physiol Lung Cell Mol Physiol 284:L84–L89.
45. Calder PC, Newsholme EA. 1992. Polyunsaturated fatty acids suppress human periph-
eral blood lymphocyte proliferation and interleukin-2 production. Clin Sci (Lond)
82:695–700.
46. Meydani SN, Endres S, Woods MM, Goldin BR, Soo C, Morrill-Labrode A et al. 1991.
Oral (n-3) fatty acid supplementation suppresses cytokine production and lymphocyte
proliferation: Comparison between young and older women. J Nutr 121:547–555.
47. Varming K, Schmidt EB, Svaneborg N, Moller JM, Lervang HH, Grunnet N et al. 1995.
The effect of n-3 fatty acids on neutrophil chemiluminescence. Scand J Clin Lab Invest
55:47–52.
48. Sundrarjun T, Komindr S, Archararit N, Dahlan W, Puchaiwatananon O, Angthararak S
et al. 2004. Effects of n-3 fatty acids on serum interleukin-6, tumour necrosis factor-
alpha and soluble tumour necrosis factor receptor p55 in active rheumatoid arthritis.
J Int Med Res 32:443–454.
49. Fisher M, Levine PH. 1991. Effects of dietary omega 3 fatty acid supplementation on
leukocyte free radical production. World Rev Nutr Diet 66:245–249.
50. Leslie CA, Gonnerman WA, Ullman MD, Hayes KC, Franzblau C, Cathcart ES. 1985.
Dietary fish oil modulates macrophage fatty acids and decreases arthritis susceptibility
in mice. J Exp Med 162:1336–1349.
51. Ierna M, Kerr A, Scales H, Berge K, Griinari M. 2010. Supplementation of diet with
krill oil protects against experimental rheumatoid arthritis. BMC Musculoskelet Disord
11:136.
52. Halade GV, Rahman MM, Bhattacharya A, Barnes JL, Chandrasekar B,
Fernandes G. 2010. Docosahexaenoic acid-enriched fish oil attenuates kidney disease
and prolongs median and maximal life span of autoimmune lupus-prone mice. J
Immunol 184:5280–5286.
53. Chandrasekar B, Troyer DA, Venkatraman JT, Fernandes G. 1995. Dietary omega-3
lipids delay the onset and progression of autoimmune lupus nephritis by inhibiting
transforming growth factor beta mRNA and protein expression. J Autoimmun 8:381–393.
54. Rosell M, Wesley A, Rydin K, Klareskog L, Alfredsson L. 2009. Dietary fish and fish oil
and the risk of rheumatoid arthritis. Epidemiology 20:896–901.
55. Goldberg RJ, Katz J. 2007. A meta-analysis of the analgesic effects of omega-3
polyunsaturated fatty acid supplementation for inflammatory joint pain. Pain
129:210–223.
56. MacLean CH, Mojica WA, Morton SC, Pencharz J, Hasenfeld Garland R, Tu W et al.
2004. Effects of omega-3 fatty acids on lipids and glycemic control in type II diabetes
and the metabolic syndrome and on inflammatory bowel disease, rheumatoid arthritis,
renal disease, systemic lupus erythematosus, and osteoporosis. Evid Rep Technol Assess
(Summ) 89:1–4.
57. Clark W, Parbtani A, Naylor C, Levinton C, Muirhead N, Spanner E et al. 1993. Fish
oil in lupus nephritis: Clinical findings and methodological implications. Kidney Int
44:75–86.
58. Walton A, Snaith M, Locniskar M, Cumberland A, Morrow W, Isenberg D. 1991. Dietary
fish oil and the severity of symptoms in patients with systemic lupus erythematosus. Ann
Rheum Dis 50:463–466.
59. Westberg G, Tarkowski A. 1990. Effect of MaxEPA in patients with SLE. A double-
blind, crossover study. Scand J Rheumatol 19:137–143.
60. Wright SA, O’Prey FM, McHenry MT, Leahey WJ, Devine AB, Duffy EM et al. 2008.
A randomised interventional trial of omega-3-polyunsaturated fatty acids on endothe-
lial function and disease activity in systemic lupus erythematosus. Ann Rheum Dis
67:841–848.
61. Kim A, Chong BF. 2013. Photosensitivity in cutaneous lupus erythematosus.
Photodermatol Photoimmunol Photomed 29:4–11.
62. Rastmanesh R, Baer A. 2011. Possible augmentation of photosensitivity by dietary fura-
nocoumarins in patients with systemic lupus erythematosus. Lupus 20:1005–1009.
63. Arjumand S, Ali A. 1994. Cross-reactions of human lupus autoantibodies with
8-methoxypsoralen photomodified DNA fragments. Microbiol Immunol 38:239–243.
64. Ritterhouse LL, Crowe SR, Niewold TB, Kamen DL, Macwana SR, Roberts VC et al.
2011. Vitamin D deficiency is associated with an increased autoimmune response in
healthy individuals and in patients with systemic lupus erythematosus. Ann Rheum Dis
70:1569–1574.
65. Chen S, Sims GP, Chen XX, Gu YY, Lipsky PE. 2007. Modulatory effects of
1,25-dihydroxyvitamin D3 on human B cell differentiation. J Immunol 179:1634–1647.
66. Singh A, Kamen DL. 2012. Potential benefits of vitamin D for patients with systemic
lupus erythematosus. Dermatoendocrinology 4:146–151.
67. Ben-Zvi I, Aranow C, Mackay M, Stanevsky A, Kamen DL, Marinescu LM et al. 2010.
The impact of vitamin D on dendritic cell function in patients with systemic lupus ery-
thematosus. PLoS One 5(2):e9193.
68. Kamen DL, Tangpricha V. 2010. Vitamin D and molecular actions on the immune sys-
tem: Modulation of innate and autoimmunity. J Mol Med (Berl) 88:441–450.
69. Linker-Israeli M, Elstner E, Klinenberg JR, Wallace DJ, Koeffler HP. 2001. Vitamin
D(3) and its synthetic analogs inhibit the spontaneous in vitro immunoglobulin produc-
tion by SLE-derived PBMC. Clin Immunol 99:82–93.
70. Chaiamnuay S, Chailurkit LO, Narongroeknawin P, Asavatanabodee P,
Laohajaroensombat S, Chaiamnuay P. 2013. Current daily glucocorticoid use and serum
creatinine levels are associated with lower 25(OH) vitamin D levels in Thai patients with
systemic lupus erythematosus. J Clin Rheumatol 19:121–125.
71. Abou-Raya A, Abou-Raya S, Helmii M. 2012. The effect of vitamin D supplementa-
tion on inflammatory and hemostatic markers and disease activity in patients with
systemic lupus erythematosus: A randomized placebo-controlled trial. J Rheumatol
40:265–272.
72. Costenbader KH, Feskanich D, Holmes M, Karlson EW, Benito-Garcia E. 2008.

Vitamin D intake and risks of systemic lupus erythematosus and rheumatoid arthritis
in women. Ann Rheum Dis 67:530–535.
73. Petri M, Bello KJ, Fang H, Magder LS. 2013. Vitamin D in SLE: Modest association
with disease activity and urine protein/creatinine ratio. Arthritis Rheum 65:1865–1871.
74. Sakthiswary R, Raymond AA. 2013. The clinical significance of vitamin D in systemic
lupus erythematosus: A systematic review. PLoS One 8(1):e55275.
75. Terrier B, Derian N, Schoindre Y, Chaara W, Geri G, Zahr N et al. 2012. Restoration of
regulatory and effector T cell balance and B cell homeostasis in systemic lupus erythe-
matosus patients through vitamin D supplementation. Arthritis Res Ther 14:R221.
76. Higgins MJ, Mackie SL, Thalayasingam N, Bingham SJ, Hamilton J, Kelly CA. 2013.
The effect of vitamin D levels on the assessment of disease activity in rheumatoid arthri-
tis. Clin Rheumatol 32:863–867.
77. Haga HJ, Schmedes A, Naderi Y, Moreno AM, Peen E. 2013. Severe deficiency of
25-hydroxyvitamin D(3) (25-OH-D (3)) is associated with high disease activity of rheu-
matoid arthritis. Clin Rheumatol 32:629–633.
78. Song GG, Bae SC, Lee YH. 2012. Association between vitamin D intake and the risk of
rheumatoid arthritis: A meta-analysis. Clin Rheumatol 31:1733–1739.
79. O’Regan S, Chesney RW, Hamstra A, Eisman JA, O’Gorman AM, Deluca HF. 1979.
Reduced serum 1,25-(OH)2 vitamin D3 levels in prednisone-treated adolescents with
systemic lupus erythematosus. Acta Paediatr Scand 68:109–111.
80. Grossman JM, Gordon R, Ranganath VK, Deal C, Caplan L, Chen W et al. 2010.
American College of Rheumatology 2010 recommendations for the prevention and
treatment of glucocorticoid-induced osteoporosis. Arthritis Care Res (Hoboken)
62:1515–1526.
81. Parker J, Hashmi O, Dutton D, Mavrodaris A, Stranges S, Kandala NB et al. 2009.
Levels of vitamin D and cardiometabolic disorders: Systematic review and meta-
analysis. Maturitas 65:225–236.
82. Haque UJ, Bathon JM, Giles JT. 2012. Association of vitamin D with cardiometabolic
risk factors in rheumatoid arthritis. Arthritis Care Res (Hoboken) 64:1497–1504.
83. Ravenell RL, Kamen DL, Spence JD, Hollis BW, Fleury TJ, Janech MG et al. 2012.
Premature atherosclerosis is associated with hypovitaminosis D and angiotensin-
converting enzyme inhibitor non-use in lupus patients. Am J Med Sci 344(4):268–273.
84. Reynolds JA, Haque S, Berry JL, Pemberton P, Teh LS, Ho P et al. 2011.
25-Hydroxyvitamin D deficiency is associated with increased aortic stiffness in patients
with systemic lupus erythematosus. Rheumatology (Oxford) 51:544–551.
85. Rock CL, Jacob RA, Bowen PE. 1996. Update on the biological characteristics of the
antioxidant micronutrients: Vitamin C, vitamin E, and the carotenoids. J Am Diet Assoc
96:693–702.
86. Hsieh CC, Lin BF. 2011. Dietary factors regulate cytokines in murine models of sys-
temic lupus erythematosus. Autoimmun Rev 11:22–27.
87. McKinley M. 2000. Nutritional aspects and possible pathological mechanisms of hyper-
homocysteinaemia: An independent risk factor for vascular disease. Proc Nutr Soc
59:37.
88. Hseih C-C, Lin B-F. 2005. Opposite effects of low and high dose supplementation of
vitamin E on survival of MRL/lpr mice. Nutrition 21:940–948.
89. Hsieh C, Lin B. 2005. The effects of vitamin E supplementation on autoimmune-prone
New Zealand black × New Zealand white F1 mice fed an oxidised oil diet. Br J Nutr
93:655–662.
90. Aryaeian N, Djalali M, Shahram F, Jazayeri S, Chamari M, Nazari S. 2011. Beta-
carotene, vitamin E, MDA, glutathione reductase and arylesterase activity levels in
patients with active rheumatoid arthritis. Iran J Public Health 40:102–109.
91. Karlson E, Shadick N, Cook N, Buring J, Lee I. 2008. Vitamin E in the primary preven-
tion of rheumatoid arthritis: The Women’s Health Study. Arthritis Rheum 59:1589–1595.
92. Canter PH, Wider B, Ernst E. 2007. The antioxidant vitamins A, C, E and selenium in the
treatment of arthritis: A systematic review of randomized clinical trials. Rheumatology
(Oxford) 46:1223–1233.
93. Minami Y, Sasaki T, Arai Y, Kurisu Y, Hisamichi S. 2003. Diet and systemic lupus
erythematosus: A 4 year prospective study of Japanese patients. J Rheumatol
30:747–754.
94. Costenbader K, Kang J, Karlson E. 2010. Antioxidant intake and risks of rheumatoid
arthritis and systemic lupus erythematosus in women. Am J Epidemiol 172:205–216.
95. Tam L, Li E, Leung V, Griffith J, Benzie I, Lim P et al. 2005. Effects of vitamins C and E
on oxidative stress markers and endothelial function in patients with systemic lupus ery-
thematosus: A double blind, placebo controlled pilot study. J Rheumatol 32:275–282.
96. Minami YH, Nagata, C, Ishii T, Harigae H, Sasaki, T. 2011. Intakes of vitamin B6 and
dietary fiber and clinical course of systemic lupus erythematosus: A prospective study
of Japanese female patients. J Epidemiol 21:246–254.
97. Collaboration HS. 2002. Homocysteine and risk of ischemic heart disease and stroke:
A meta-analysis. JAMA 288:2015–2022.
98. Von Feldt JM, Scalzi LV, Cucchiara AJ, Morthala S, Kealey C, Flagg SD et al. 2006.
Homocysteine levels and disease duration independently correlate with coronary
artery calcification in patients with systemic lupus erythematosus. Arthritis Rheum
54:2220–2227.
99. Roman MJ, Crow MK, Lockshin MD, Devereux RB, Paget SA, Sammaritano L et al.
2007. Rate and determinants of progression of atherosclerosis in systemic lupus erythe-
matosus. Arthritis Rheum 56:3412–3419.
100. Rua-Figueroa I, Arencibia-Mireles O, Elvira M, Erausquin C, Ojeda S, Francisco F
et al. 2009. The factors involved in the progress of preclinical atherosclerosis associ-
ated with systemic lupus erythematosus: A two year longitudinal study. Ann Rheum Dis
69:1136–1139.
101. Brown KS, Nackos E, Morthala S, Jensen LE, Whitehead AS, Von Feldt JM. 2007.
Monocyte chemoattractant protein-1: Plasma concentrations and A(-2518)G promoter
polymorphism of its gene in systemic lupus erythematosus. J Rheumatol 34:740–746.
102. Ma Y, Hebert J, Li W, Bertone-Johnson E, Olendzki B, Pagoto S. 2008. Association
between dietary fiber and markers of systemic inflammation in the Women’s Health
Initiative Observational Study. Nutrition 12:63.
103. Maggini S, Wintergerst E, Beveridge S, Hornig D. 2007. Selected vitamins and trace
elements support immune function by strengthening epithelial barriers and cellular and
humoral immune responses. Br J Nutr 98:S29–S35.
104. Wintergerst E, Maggini S, Hornig D. 2007. Contribution of selected vitamins and trace
elements to immune function. Ann Nutr Metab 51:301–323.
105. Huang SC, Wei JC, Lin PT, Wu DJ, Huang YC. 2012. Plasma pyridoxal 5′-phosphate
is not associated with inflammatory and immune responses after adjusting for serum
albumin in patients with rheumatoid arthritis: A preliminary study. Ann Nutr Metab
60:83–89.
106. Chiang EP, Selhub J, Bagley PJ, Dallal G, Roubenoff R. 2005. Pyridoxine supplementa-
tion corrects vitamin B6 deficiency but does not improve inflammation in patients with
rheumatoid arthritis. Arthritis Res Ther 7:R1404–R1411.
107. Huang SC, Wei JC, Wu DJ, Huang YC. 2010. Vitamin B(6) supplementation improves
pro-inflammatory responses in patients with rheumatoid arthritis. Eur J Clin Nutr
64:1007–1013.
108. Alves JD, Grima B. 2003. Oxidative stress in systemic lupus erythematosus and antiphos-
pholipid syndrome: A gateway to atherosclerosis. Curr Rheumatol Rep 5:383–390.
109. Shah D, Kiran R, Wanchu A, Bhatnagar A. 2010. Oxidative stress in systemic lupus ery-
thematosus: Relationship to Th1 cytokine and disease activity. Immunol Lett 129:7–12.
110. Weimann B, Weiser H. 1992. Effects of antioxidant vitamins C, E, and B-Carotene on
immune functions in MRL/lpr mice and rats. Ann NY Acad Sci 669:390–392.
111. Meki A, Hamed E, Ezam K. 2009. Effect of green tea extract and vitamin C on oxidant or
antioxidant status of rheumatoid arthritis rat model. Indian J Clin Biochem 24:280–287.
112. Bae SC, Jung WJ, Lee EJ, Yu R, Sung MK. 2009. Effects of antioxidant supplements
intervention on the level of plasma inflammatory molecules and disease severity of rheu-
matoid arthritis patients. J Am Coll Nutr 28:56–62.
113. Park C, Moon D, Choi I, Choi B, Nam T, Rhu C et al. 2007. Curcumin induces apoptosis
and inhibits prostaglandin E2 production in synovial fibroblasts of patients with rheu-
matoid arthritis. Int J Mol Med 20:365–372.
114. Funk J, Oyarzo J, Frye J, Chen G, Lantz R, Jolad S et al. 2006. Turmeric extracts contain-
ing curcuminoids prevent experimental rheumatoid arthritis. J Nat Prod 69:351–355.
115. Kloesch B, Becker T, Dietersdorfer E, Kiener H, Steiner G. 2013. Anti-inflammatory
and apoptotic effects of the polyphenol curcumin on human fibroblast-like synovio-
cytes. Int Immunopharmacol 15:400–405.
116. Huang G, Xu Z, Huang Y, Duan X, Gong W, Zhang Y et al. 2012. Curcumin protects
against collagen-induced arthritis via suppression of BAFF production. J Clin Immunol
33:550–557.
117. Lee H, Kim H, Lee G, Chung HS, Bae H. 2012. Curcumin attenuates lupus nephritis
upon interaction with regulatory T cells in New Zealand Black/White mice. Br J Nutr
110:1–8.
118. Foxley S, Zamora M, Hack B, Alexander R, Roman B, Quigg R et al. 2013. Curcumin
aggravates CNS pathology in experimental systemic lupus erythematosus. Brain Res
1504:85–96.
119. Chandran B, Goel A. 2012. A randomized, pilot study to assess the efficacy and safety
of curcumin in patients with active rheumatoid arthritis. Phytother Res 26:1719–1725.
120. Khajehdehi P, Zanjaninejad B, Aflaki E, Nazarinia M, Azad F, Malekmakan L et al.
2012. Oral supplementation of turmeric decreases proteinuria, hematuria, and systolic
blood pressure in patients suffering from relapsing or refractory lupus nephritis: A ran-
domized and placebo-controlled study. J Ren Nutr 22:50–57.
14 Asthma and
Inflammation
Andre Nel and David Heber
CONTENTS
Introduction............................................................................................................. 229
Asthma: A Complex Disorder................................................................................. 230
Air Pollution........................................................................................................... 230
Oxidant Stress and Asthma..................................................................................... 231
Nrf2 and Oxidant Stress.......................................................................................... 232
Cellular Mechanisms of Inflammation in Allergic Asthma.................................... 232
Effects of Particulate Matter in Allergic Asthma.................................................... 233
Asthma and Obesity................................................................................................ 235
Conclusion.............................................................................................................. 237
References............................................................................................................... 237
INTRODUCTION
Allergic diseases affect approximately one-third of the general population, and
asthma is a heterogeneous disorder, characterized by reversible airway obstruction
and bronchial hyperresponsiveness, which is commonly associated with atopy [1,2].
Among the multiple factors that contribute to asthma are genetic predisposition,
immunological aberration, and the possible involvement of noxious environmental
factors [3]. Epidemiological studies, in particular, have suggested that worldwide
increases in allergic and respiratory disease may be associated with environmen-
tal pollutants such as air pollution [4,5]. The major component of airborne particu-
late matter (PM) is diesel exhaust particles (DEPs), which can induce and enhance
allergic responses [6] by entering cells as nanoparticles and directly and indirectly
generating reactive oxygen species (ROS) [7,8]. The defensive response of the lung
alveolar cells to ROS is mediated through nuclear factor (erythroid-derived 2)-like 2
(Nrf2), and one of the primary cellular defenses to this reaction is mediated through
glutathione S-transferases such as GSTM1 [9–11]. The GSTM1 null mutation is
present in up to 50% of normal individuals and predisposes these individuals to a
higher risk of acquiring environmentally related diseases, including some forms of
cancer [11]. It has also been reported that individuals with the null mutation have a
higher induction of IgE in response to DEP plus secondhand smoke exposure [12].
DEP increases inflammation and stimulates oxidant stress pathways in the normal
bronchial epithelium, but asthmatics are more sensitive to the effects of DEPs, and
229
individuals expressing GSTP1 ile/ile105 have higher histamine inductions when

exposed to DEP and secondhand smoke [13,14]. In this chapter, the role of airborne
PM predominated by diesel particles will be examined in light of their ability to
increase oxidative stress and inflammation, which may be affected in many individu-
als by obesity, allergy, dietary antioxidants, and other bioactive substances.
ASTHMA: A COMPLEX DISORDER

The incidence of asthma has been increasing over the last 30 years in parallel with
the epidemic of obesity and lifestyle-associated chronic diseases where inflamma-
tion plays a significant etiological role. The etiology and pathogenesis of asthma
remain poorly understood, but the role of inflammation has influenced the approach
to therapy, which has changed in the last 30 years from simply addressing the hyper-
reactivity of airways with pharmacology to addressing the immune system underly-
ing the hyperreactivity of airways. Asthma can be defined as a respiratory disease
with three primary features: (a) airway inflammation associated with eosinophilic
infiltration and altered T-cell lymphocytic function; (b) thickening of the base-
ment membrane, mucin hypersecretion, loss or altered ciliary structure, and altered
cytokine and other inflammatory mediator production in lung epithelial cells; and
(c) clinical presentation with recurrent airflow obstruction presented in dual phases as
decreased forced expiratory volume, bronchospasm, and/or airway hyperreactivity.
While atopic individuals have a much higher incidence of asthma, there are many
individuals with asthma due to environmental exposures where allergy does not
appear to play a primary role [15,16]. This observation suggests that diet and lifestyle
play a major role. In particular, the childhood epidemic of obesity and diabetes has
been associated with a significant increase in asthma among obese children.
These individuals respond with airway hyperreactivity to many agents, including
DEPs, dry air, aerosols, and sulfur dioxide. Consequently, this condition is called
nonspecific airway hyperreactivity, which many clinical investigators consider the
hallmark of asthma [17,18]. Oxidative stress and inflammation may both play a sig-
nificant role in this aspect of asthma and provide an avenue for improved preven-
tion and therapy based on antioxidants from the diet and extracts rich in bioactive
substances.
AIR POLLUTION
Epidemiological studies have demonstrated an association between air pollution
and the prevalence of respiratory symptoms characteristic of asthma throughout
the world. When the association was examined for atopic and nonatopic individu-
als separately, the association of air pollution with asthma was unaffected [19–21].
Besides increases in symptoms, air pollution has been associated with decreases in
pulmonary function, including depressed forced expiratory volume-1 second (FEVy)
or peak expiratory flow rates [22,23].
Environmental factors, including weather, pollen, and tobacco smoke, are impor-
tant risk factors in asthma, but each has been found to act independently of air pol-
lution and these factors do not explain the association between air pollution and
Asthma and Inflammation 231
asthma [24–29]. Moreover, stationary sources of air pollution have been associated
with increased risks of asthma [19,30–38].
The major component of airborne PM is DEPs, which can induce and enhance
allergic responses [6] by entering cells as nanoparticles and directly and indirectly
generating ROS [7,8].
OXIDANT STRESS AND ASTHMA

Oxidative stress is widely recognized as an important component of airway inflam-
mation in asthma [39–41]. While under normal physiological conditions a small
amount of ROS are produced in mitochondria, the production of superoxide
anion, hydrogen peroxide, and hydroxyl radicals can be considerably increased by
xenobiotic metabolism, redox cycling chemicals, arachidonic acid metabolism, and
peroxidase activity in immune cells in bronchial tissue. Excessive oxidant stress can
lead to damage to cells and organelles through the oxidation of proteins, lipids, and
nucleic acids. In addition, excessive oxidant stress can deplete antioxidant networks,
including glutathione, with an accumulation of oxidized forms. Oxidant stress can
activate additional cellular responses that are either defensive or injurious. The out-
come of oxidative stress is determined by a dynamic equilibrium between protective
and injurious oxidative stress response pathways. In order to better visualize this
equilibrium, a hierarchical oxidative stress model can be employed to explain the
relationship between the level of oxidative stress and the outcome of the cellular
response (Figure 14.1) [40]. At low levels of oxidative stress (tier 1), Nrf2 translocates
High Low
GSH/GSSG GSH/GSSG
ratio ratio
oxidative stress
Level of
Tier 1 Tier 2 Tier 3
Cell response Normal Antioxidant Inflammation Toxicity

pathway: defense
Signaling pathway: Nrf-2 MAPK Mitochondrial

NF-κB perturbation
Genetic response: ARE AP-1 PT pore
NF-κB
FIGURE 14.1 Hierarchical oxidative stress responses. At a low level of oxidative stress
(tier 1), antioxidant enzymes are induced to restore cellular redox homeostasis. At an interme-
diate level of oxidative stress (tier 2), activation of MAPK and NF-κB cascades induces pro-
inflammatory responses, for example, cytokines and chemokines. At a high level of oxidative
stress (tier 3), perturbation of the mitochondrial permeability transition pore and disruption
of electron transfer result in cellular apoptosis or necrosis.
to the nucleus where it is responsible for the transcriptional activation of more than
200 genes that express proteins to defend against oxidative stress.
NRF2 AND OXIDANT STRESS

Nuclear factor-erythroid-2-related factor 2 (Nrf2) plays a crucial role in the coordi-
nated induction of genes encoding many oxidant stress–responsive and cytoprotective
enzymes and related proteins. These include NAD(P)H:-quinone oxidoreductase-1,
heme oxygenase-1,glutamate cysteine ligase, glutathione S-transferase, glutathione
peroxidase, and thioredoxin [42–46]. In resting cells, Nrf2 is sequestered in the cyto-
plasm as an inactive complex with the repressor Kelch-like ECH-associated protein
1 (Keap1). The release of Nrf2 from its repressor is secondary to alterations in the
structure of Keap1 via the oxidation of several reactive cysteine residues that func-
tion as sensors of cellular redox changes. Oxidation or covalent modification of some
of these critical cysteine thiols stabilize Nrf2, thereby facilitating nuclear accumula-
tion of Nrf2. After translocation into the nucleus, Nrf2 forms a heterodimer with
other transcription factors, such as small Maf, which in turn binds to the 5′-upstream
cis-acting regulatory sequence, termed antioxidant response elements or electrophile
response elements (EpRE), located in the promoter region of genes encoding vari-
ous antioxidant and phase 2 detoxifying enzymes. Certain dietary chemopreventive
agents such as sulforaphane target Keap1 by oxidizing or chemically modifying one
or more of its specific cysteine thiols, thereby stabilizing Nrf2. In addition, phos-
phorylation of specific serine or threonine residues present in Nrf2 by upstream
kinases may also facilitate the nuclear localization of Nrf2. Multiple mechanisms
of Nrf2 activation by signals mediated by one or more of the upstream kinases, such
as mitogen-activated protein kinases, phosphatidylinositol-3-kinase/Akt, protein
kinase C, and casein kinase-2, have recently been proposed. Transgenic mice defi-
cient in Nrf2 do not induce genes responsible for protection against oxidative stress
or detoxification of carcinogens [47,48]. When Nrf2-deficient mice are exposed to
the carcinogen benzo[a]pyrene (B[a]P), more gastric neoplasia was observed. The
naturally occurring chemopreventive agent, sulforaphane, reduced B[a]P-induced
forestomach tumorigenesis in mice, most likely via the induction of phase 2 detoxifi-
cation/antioxidant enzymes, as this protective effect did not occur in Nrf2-null mice
[49]. Besides its role in detoxification and cellular antioxidative defense, Nrf2 also
has anti-inflammatory functions [50,51].
CELLULAR MECHANISMS OF INFLAMMATION

IN ALLERGIC ASTHMA
Airway hyperreactivity to bronchoconstrictor mediators is a main characteristic in
the majority of asthmatic patients and correlates well with the severity of the disease.
At one extreme, anaphylaxis and asthma can be life threatening, and every year
deaths often occur in young people that are avoidable. Fortunately, most allergic
disorders are not life threatening. Allergic rhinitis, asthma, and eczema all inter-
fere with sleep, intellectual functioning, and activities of daily living. The airways
of asthmatic patients are characterized by an inflammatory state resulting in the
activation of lung tissue cells and attraction and infiltration of leukocytes from the
blood. The accumulation of eosinophilic leukocytes is a prominent feature of inflam-
matory reactions that occurs in allergic asthma [52].
A wide range of cellular responses underlies chronic allergic disease, includ-
ing the production of inflammatory substances that mediate the reactions. Both
histamine and cysteinyl leukotrienes account for most of the early- and late-phase
responses. They are typically released from mast cells in the early phase and eosin-
ophils, basophils, and macrophages in the late phase. However, the magnitude of
both acute and chronic asthmatic reactions correlates with the number of eosinophils
present in the lung [53], and the suppression of this eosinophil accumulation impairs
the development and progression of these processes [54]. The increase in the number
of eosinophils is important since it correlates in time with an increase in bronchial
hyperresponsiveness [55,56]. From biopsy studies, it is known that infiltrating eosin-
ophils mainly show degranulation at subepithelial sites but also at sites deeper in the
interstitium [57]. The causal relationship between an increase in infiltrating eosino-
phils and bronchial hyperresponsiveness is not finally settled. It is generally assumed
that the deposition of toxic products of eosinophils—for example, eosinophilic cat-
ionic protein and major basic protein—is able to induce the shedding of epithelial
cells as seen in human bronchial tissues from asthmatics. Eosinophilic cationic pro-
tein is present in the bronchial submucosa of adults with asthma, especially in areas
where sloughing of epithelium has occurred [58]. In addition, this protein has also
been implicated in the inflammatory response accompanying allergic rhinitis [59].
Guinea pigs sensitized to ovalbumin (OVA), an animal model for allergic asthma,
have been shown to develop airway hyperresponsiveness (AHR) after antigen chal-
lenge. This hyperresponsiveness coincided with infiltration of eosinophils in airways
[60–62]. Moreover, a significant increase in the levels of eosinophil peroxidase and
major basic protein was observed in bronchoalveolar lavage fluid of these sensitized
and challenged animals [59,62,63].
EFFECTS OF PARTICULATE MATTER IN ALLERGIC ASTHMA

The adjuvant effects of ambient PM with its predominant DEPs have been
demonstrated in a number of human and animal studies [64–68]. Combined DEP
and ragweed nasal challenge significantly enhances ragweed-specific IgE and IL-4
production in humans [66]. In addition, intranasal instillation of DEP also increased
the expression of several CC chemokines, including RANTES, MIP-1α, and MCP-1,
in the human nose [65]. Gilliland et al. reported that individuals with GST M1 null
genotype exhibit increased nasal allergic and allergen-specific IgE response to nasal
DEP challenge, thereby demonstrating the possible linkage of these responses to an
oxidative stress mechanism [12]. This finding suggests that the antioxidant and anti-
inflammatory effects of phase II enzymes could play an important role in protect-
ing against the proinflammatory and proallergic effects of PM [69–71]. In animal
studies, DEP has been shown to enhance OVA-induced eosinophilic airway inflam-
mation, OVA-specific IgG1 and IgE production, goblet cell proliferation, and local
expression of several Th2 cytokines and chemokines [72]. Similar results have also
been reported in animals receiving intratracheal instillation of the dust mite allergen,
Der f, in the presence of DEP [73,74]. Furthermore, when Balb/c mice were exposed
to an aerosolized leachate of residual oil fly ash, their offspring demonstrated
a significant increase in AHR, eosinophilic inflammation, and IgE production in
response to sensitization with a suboptimal dose of OVA [75]. Cultured splenocytes
from these offspring demonstrated an increased IL-4/IFNγ ratio, suggesting a skew-
ing toward Th2 immunity [75]. The immunological basis for the adjuvant effects of
PM is still improperly understood.
Several cell types are involved in allergen sensitization and asthma pathogenesis,
including antigen-presenting cells (APCs), T-helper 2 (Th2) lymphocytes, IgE-
secreting plasma cells, mast cells, eosinophils, neutrophils, mucus-secreting goblet
cells, smooth muscle cells, and endothelial cells. DEP can directly impact a number of
cells that play a role in the afferent or efferent immune response [76–83]. Traditional
adjuvants exert their effects on the afferent or early phase of the immune response,
which implies possible effects on APCs [84,85]. Consequently, a lot of attention is
currently being directed at the possible contribution of dendritic cells (DCs). DCs
play a crucial role in initiating T-cell activation and are the main APCs that are
responsible for allergen processing and presentation in asthma. Airway DCs continu-
ously sample their environment for antigens and allergens [86–88]. After allergen
capture and receipt of a danger signal, DCs upregulate CCR7 expression, enter the
afferent lymphatic vessels, and carry the allergen to the draining lymph nodes, where
it is presented by MHC in the presence of costimulatory molecules. Allergen-specific
T-cells are selected for antigen specificity and induced to proliferate. Depending on
the cytokine milieu and other variables, DCs could initiate a primary Th2 response in
regional lymph nodes [86–91]. Following immune excitation, memory/effector CD4+
Th2 cells then leave the draining lymph nodes and extravasate at sites of inflammation
during the challenge phase. Once in the tissues, Th2 cells interact with IgE-bearing
local DCs to increase IL-4, IL-5, IL-9, and IL-13 production [86–88,92–98]. These
cytokines are important for inducing tissue eosinophilia, airway hyperreactivity, and
the production of chemokines that attract further inflammatory cells.
In addition to adjuvant effects, PM exposure induces acute asthma exacerba-
tions independent of their effects on allergic sensitization [99]. For instance, it is
capable of inducing AHR in naive mice in the absence of allergen [100,101]. It has
also been demonstrated that DEP alone can induce increased AHR in asthmatic
individuals [102]. While these effects may be related to PM effects on the immune
system, the particles and their components may directly contribute to increased
AHR during asthma attack [103–105]. One possible mechanism is nitric oxide gen-
eration, as evidenced by the ability of nitric oxide synthase inhibitors to interfere
with DEP-induced AHR in mice [106]. Shedding of airway epithelial cells is another
possibility, based on the ability of DEP to induce acute epithelial damage in vivo and
in vitro [107–110]. Two recent reviews have summarized the potential mechanisms
of PM–lung interaction and particle translocation to other tissues with a focus on the
ultrafine particle (UFP) [111,112]. It has been suggested that the unique physical and
chemical properties of UFP play important roles in particle deposition in the lung
and translocation to the extralung tissues. When inhaled UFPs deposit on the epithe-
lial surface of the peripheral lungs, their contact with surfactant layer and epithelial
lining fluid (ELF) leads to their interactions with proteins and other biomolecules in
the ELF. The large number concentration of UFP, compared with that of micrometer-
sized PM, allows them to deposit over a large surface area of alveoli. This may result
in a scattered chemotractant signal that leads to less recognition and phagocytosis of
UFP by alveolar macrophages. In addition, PM may form complexes with proteins
in the ELF. While proteins on the surface of micrometer-sized PM are immobi-
lized and therefore allow rapid phagocytosis by alveolar macrophages, the extremely
small size of UFP may make UFP–protein complexes that are protein specific and
less accessible to the cells of defense system such as macrophages in the lung epi-
thelium. Modifications of UFP may also allow DCs to process these particles, take
up antigenic material, and carry it to the immune system, where it elicits an immune
response (Figure 14.2) [111,112].
ASTHMA AND OBESITY

Epidemiological studies, including cross-sectional [113–117], case–control [118],
and prospective cohort [119,120], have demonstrated an association of asthma and
obesity [121–125]. Obesity as measured by body mass index has been associated
with an increased incidence and prevalence of asthma, as well as greater severity of
asthmatic symptoms with poor responses to medications [126–128], suggesting some
specific effects of excess adiposity on the pathogenesis or progression of asthmatic
symptoms [129,130].
The mechanisms by which obesity enhances the clinical expression of asthma-
related physiological changes have not been fully elucidated. In murine models
of diet-induced obesity and allergic diseases, OVA challenge promotes hyperre-
sponsiveness and eosinophilic inflammation associated with increased lung Th2
cytokines, serum IgE, and lung parenchyma remodeling [131–133]. Recent studies
reported that the number of eosinophils in sputum or serum does not significantly
differ between obese or nonobese asthmatics [134,135]. Obesity associated with
increased serum leptin and TNF-α levels enhances eosinophil chemotaxis and
adhesion in asthmatic children and adolescents. Despite effective asthma control
with the regular use of inhaled corticosteroids, there is increased proinflammatory
activity in circulating eosinophils from obese asthmatics. Thus, there is a pressing
need to improve our understanding of the mechanisms underlying the relationship
between asthma and obesity since this would have significant medical and public
health implications.
A recent trial compared the effects of weight loss achieved by dietary restriction,
exercise, or combined dietary restriction and exercise on airway inflammation and
clinical outcomes in overweight and obese adults with asthma [136]. A 5%–10%
weight loss resulted in clinically important improvements in asthma control in 58%
of 43 patients while quality of life improved in 83% of the subjects. Gynoid adipose
tissue reduction was associated with reduced neutrophilic airway inflammation in
women (p = 0.047), whereas a reduction in dietary saturated fat was associated with
reduced neutrophilic airway inflammation in males (p = 0.041). The exercise inter-
vention resulted in a significant reduction to sputum eosinophils (p = 0.028). This
study suggests that the obese-asthma phenotype may involve both innate and allergic
inflammatory pathways. Further studies are needed to determine whether a modest
UFP
Redox NP
chemistry
Mito Endosome
NADPH
oxidase
Fe2+
Lysosome
PAHs Fenton ROS
Quinones reaction Mito
Ca2+
ROS ROS Ca2+
ROS
ATP ?
Cyt C
Nrf2 Cyt C Nrf2 Ca2+
JNK, NF-κB ATP
Caspases
Caspases
HO-1, Phase II
enzymes
Cytokines
Tier 1: Cell defense

Tier 2: Proinflammation
Tier 3: Apoptosis/necrosis
FIGURE 14.2 (See color insert.) Comparison of the mechanisms of ROS generation
induced by UFP and NM outside or inside of cells. Ambient UFP usually contains large
amount of organic chemical such as polycyclic aromatic hydrocarbons (PAHs) and quinines
and transition metals such as Fe and Cu, which can generate ROS through redox chemistry
both outside and inside of cells. UFPs have also been found to lodge in mitochondria, causing
damage to mitochondrial function and structure, which can also produce more ROS. Cells
under oxidative stress will have tiered responses, including cell defense (tier 1), proinflamma-
tion (tier 2), and mitochondria-mediated cell death (tier 3). Nanomaterial (NM) are uniform in
size and can also generate ROS via crystal structural defects or under UV conditions. NM are
taken up into cells via endocytosis, which includes phagocytosis, clathrin-dependent endocy-
tosis, caveolae-mediated endocytosis, or macropinocytosis depending on specific cell types.
After cells take up NM, endosomes are formed, and ROS can be produced via the formation
of NADPH oxidase. After a series of fusion and fission processes, endosomes will fuse with
lysosomes. NM can break loose from lysosomes and interact with other organelles such as
mitochondria, which can produce more ROS. The cells under oxidative stress will go through
tiered oxidative stress responses as described previously. (From Li, N. et al., Free Radic. Biol.
Med., 44(9), 1689, 2008.)
weight loss goal of 5%–10% could assist in the clinical management of overweight
and obese adults with asthma.
CONCLUSION
As reviewed in this chapter, asthma is a complex disease influenced by changes in
environmental exposures to air pollution and nutritional factors including a proin-
flammatory obesogenic diet and sedentary lifestyles. The role of PM in air pollution
has been shown to lead to oxidative stress in lung tissue. Phytochemicals that can
impact Nrf2 such as sulforaphane hold promise for reducing inflammation due to
oxidant stress in the lung. Oxidant stress, in turn, stimulates both inflammation and
atopy so that asthmatic symptoms reflect a complex mixture of influences. Innate
immunity due to chronic low-grade inflammation associated with obesity may play
a role. Therefore, an integrative approach to nutritional intervention that includes
caloric restriction, increased dietary antioxidant intake, and increased physical
activity holds promise for the treatment of the obese phenotype of asthma in both
children and adults.
REFERENCES
1. Hwang CY, Chen YJ, Lin MW, Chen TJ, Chu SY, Chen CC et al. 2010. Prevalence of
atopic dermatitis, allergic rhinitis and asthma in Taiwan: A national study 2000 to 2007.
Acta Derm Venereol 90:589–594.
2. Nadif R, Siroux V, Oryszczyn MP, Ravault C, Pison C, Pin I et al. 2009. Heterogeneity
of asthma according to blood inflammatory patterns. Thorax 64:374–380.
3. Gibson PG. 2009. Inflammatory phenotypes in adult asthma: Clinical applications. Clin
Respir J 3:198–206.
4. Lewtas J. 2007. Air pollution combustion emissions: Characterization of causative
agents and mechanisms associated with cancer, reproductive, and cardiovascular effects.
Mutat Res 636:95–133.
5. Dockery DW, Schwartz J, Spengler JD. 1992. Air pollution and daily mortality:
Associations with particulates and acid aerosols. Environ Res 59:362–373.
6. Polosa R, Salvi S, Di Maria GU. 2002. Allergic susceptibility associated with diesel
exhaust particle exposure: Clear as mud. Arch Environ Health 57:188–193.
7. Muller L, Riediker M, Wick P, Mohr M, Gehr P, Rothen-Rutishauser B. 2010. Oxidative
stress and inflammation response after nanoparticle exposure: Differences between
human lung cell monocultures and an advanced three-dimensional model of the human
epithelial airways. J R Soc Interface 7(Suppl 1):S27–S40.
8. Auger F, Gendron MC, Chamot C, Marano F, Dazy AC. 2006. Responses of well-
differentiated nasal epithelial cells exposed to particles: Role of the epithelium in airway
inflammation. Toxicol Appl Pharmacol 215:285–294.
9. Li N, Alam J, Venkatesan MI, Eiguren-Fernandez A, Schmitz D, Di Stefano E et al.
2004. Nrf2 is a key transcription factor that regulates antioxidant defense in macro-
phages and epithelial cells: Protecting against the proinflammatory and oxidizing effects
of diesel exhaust chemicals. J Immunol 173:3467–3481.
10. Baulig A, Sourdeval M, Meyer M, Marano F, Baeza-Squiban A. 2003. Biological effects
of atmospheric particles on human bronchial epithelial cells. Comparison with diesel
exhaust particles. Toxicol In Vitro 17:567–573.
11. Brockmoller J, Cascorbi I, Kerb R, Roots I. 1996. Combined analysis of inherited poly-
morphisms in arylamine N-acetyltransferase 2, glutathione S-transferases M1 and T1,
microsomal epoxide hydrolase, and cytochrome P450 enzymes as modulators of blad-
der cancer risk. Cancer Res 56:3915–3925.
12. Gilliland FD, Li YF, Gong H Jr, Diaz-Sanchez D. 2006. Glutathione s-transferases M1
and P1 prevent aggravation of allergic responses by secondhand smoke. Am J Respir
Crit Care Med 174:1335–1341.
13. Takizawa H. 2004. Diesel exhaust particles and their effect on induced cytokine expres-
sion in human bronchial epithelial cells. Curr Opin Allergy Clin Immunol 4:355–359.
14. Bayram H, Devalia JL, Khair OA, Abdelaziz MM, Sapsford RJ, Sagai M et al. 1998.
Comparison of ciliary activity and inflammatory mediator release from bronchial
epithelial cells of nonatopic nonasthmatic subjects and atopic asthmatic patients and the
effect of diesel exhaust particles in vitro. J Allergy Clin Immunol 102:771–782.
15. Burrows B, Martinez FD, Halonen M, Barbee RA, Cline MG. 1989. Association
of asthma with serum IgE levels and skin test reactivity to allergens. N Engl J Med
320:271–277.
16. Bernstein IL, Chan-Yeung M, Malo JL, Berstein DI eds. 1993. Asthma in the Workplace.
New York: Marcel Dekker.
17. Barnes PJ, Rodgers IW, Thomson NC. 1992. Asthma: Basic Mechanisms and Clinical
Management, 2nd edn. London, U.K.: Academic Press.
18. Boushey HA, Holtzman MJ, Sheller JR, Nadel JA. 1980. Bronchial hyperresponsive-
ness. Am Rev Respir Dis 12:389–414.
19. Henry RL, Abramson R, Adler JA, Wiodarcyzk J, Hensley MJ. 1991. Asthma in the
vicinity of power stations: I. A prevalence study. Pediatr Pulmonol 11:127–133.
20. Corbo GM, Forastiere F, Dell’Orco V, Pistelli R, Agabiti N, De Stefanis B et al. 1993.
Effects of environment on atopic status and respiratory disorders in children. J Allergy
Clin Immunol 92:616–623.
21. Rutishauser M, Ackermann U, Braun C, Gnehm HP, Wanner HU. 1990. Significant
association between outdoor NO2 and respiratory symptoms in preschool children. Lung
168:347–352.
22. Pope CA III, Dockery DW, Spengler JD, Raizenne ME. 1991. Respiratory health and
PM10 pollution. Am Rev Respir Dis 144:668–674.
23. Koenig JQ, Larson TV, Hanley QS, Rebolledo V, Dumler K, Checkoway H et al. 1993.
Pulmonary function changes in children associated with fine particulate matter. Environ
Res 63:26–38.
24. Forsberg B, Stjernberg N, Falk M, Lundback B, Wall S. 1993. Air pollution levels,
meteorological conditions and asthma symptoms. Eur Respir J 6:1109–1115.
25. Bates DV, Baker-Anderson M, Sizto R. 1990. Asthma attack periodicity: A study of
hospital emergency visits in Vancouver. Environ Res 51:51–70.
26. Rossi OVJ, Kinnula VL, Tienari J, Huhti E. 1993. Association of severe asthma attacks
with weather, pollen, and air pollutants. Thorax 48:244–248.
27. Euler GL, Abbey DE, Magie AR, Hodgkin JE. 1987. Chronic obstructive pulmonary
disease symptom effects of long-term cumulative exposure to ambient levels of total
suspended particulates and sulfur dioxide in California Seventh-Day Adventist resi-
dents. Arch Environ Health 42:213–222.
28. O’Hollaren MT, Yunginger JW, Offord KP, Somer MJ, O’Connell EJ, Ballard DJ et al.
1991. Exposure to an aeroallergen as a possible precipitating factor in respiratory arrest
in young patients with asthma. N Engl J Med 324:359–363.
29. Dockery DW, Pope CA III, Xu X, Spengler JD, Ware JH, Fay ME et al. 1993. An associa-
tion between air pollution and mortality in six U.S. cities. N Engl J Med 329:1753–1759.
30. Dockery DW, Speizer FE, Strain DO, Ware JH, Spengler JD, Ferris BG Jr. 1989. Effects
of inhalable particles on respiratory health of children. Am Rev Respir Dis 139:587–594.
31. Schwartz J, Slater D, Larson TV, Pierson WE, Koenig JQ. 1993. Particulate air pol-
lution and hospital emergency room visits for asthma in Seattle. Am Rev Respir Dis
147:826–831.
32. Dodge R. 1980. The respiratory health of school children in smelter communities. Am J
Indust Med 1:359–364.
33. Dockery DW, Ware JH, Ferris BG Jr, Speizer FE, Cook NR. 1982. Change in pulmonary
function in children associated with air pollution episodes. J Air Pollut Control Assoc
32:937–942.
34. Schwartz J. 1991. Particulate air pollution and daily mortality in Detroit. Environ Res
56:204–213.
35. Pope CA III, Schwartz J, Ransom MR. 1992. Daily mortality and PM10 pollution in
Utah Valley. Arch Environ Health 47:211–217.
36. Ransom MR, Pope CA III. 1992. Elementary school absences and PM pollution in Utah
Valley. Environ Res 58:204–219.
37. Schwartz J, Dockery DW. 1992. Increased mortality in Philadelphia associated with
daily air pollution concentrations. Am Rev Respir Dis 145:600–604.
38. Schwartz J, Dockery DW. 1992. Particulate air pollution and daily mortality in
Steubenville, Ohio. Am J Epidemiol 135:12–19.
39. Bowler RP, Crapo JD. 2002. Oxidative stress in allergic respiratory disease. J Allergy
Clin Immunol 110:349356.
40. Li N, Hao M, Phalen RF, Hinds WC, Nel AE 2003. Particulate air pollutants and asthma:
A paradigm for the role of oxidative stress in PM-induced adverse health effects. Clin
Immunol 109:250–265.
41. Nel A. 2005. Atmosphere. Air pollution-related illness: Biomolecular effects of par-
ticles. Science 308:804.
42. Chen C, Kong AN. 2004. Dietary chemopreventive compounds and ARE/EpRE
signaling. Free Radic Biol Med 36:1505–1516.
43. Lee JS, Surh YJ. 2005. Nrf2 as a novel molecular target for chemoprevention. Cancer
Lett 224:171–184.
44. Dinkova-Kostova AT, Talalay P. 2008. Direct and indirect antioxidant properties of
inducers of cytoprotective proteins. Mol Nutr Food Res 52:128–138.
45. Eggler AL, Gay KA, Mesecar AD. 2008. Molecular mechanisms of natural products
in chemoprevention: Induction of cytoprotective enzymes by Nrf2. Mol Nutr Food Res
52:S84–S94.
46. Motohashi H, Yamamoto M. 2004. Nrf2-Keap1 defines a physiologically important
stress response mechanism. Trends Mol Med 10:549–557.
47. Ramos-Gomez M, Kwak MK, Dolan PM, Itoh K, Yamamoto M, Talalay P et al. 2001.
Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme
inducers is lost in nrf2 transcription factor-deficient mice. Proc Natl Acad Sci USA
98:3410–3415.
48. Chan K, Kan YW. 1999. Nrf2 is essential for protection against acute pulmonary injury
in mice. Proc Natl Acad Sci USA 96:12731–12736.
49. Fahey JW, Haristoy X, Dolan PM, Kensler TW, Scholtus I, Stephenson KK et al. 2002.
Sulforaphane inhibits extracellular, intracellular, and antibiotic resistant strains of
Helicobacter pylori and prevents benzo[a]pyrene induced stomach tumors. Proc Natl
Acad Sci USA 99:7610–7615.
50. Rangasamy T, Guo J, Mitzner WA, Roman J, Singh A, Fryer AD et al. 2005. Disruption
of Nrf2 enhances susceptibility to severe airway inflammation and asthma in mice.
J Exp Med 202:47–59.
51. Thimmulappa RK, Scollick C, Traore K, Yates M, Trush MA, Liby KT et al. 2006. Nrf2-
dependent protection from LPS induced inflammatory response and mortality by CDDO
imidazolide. Biochem Biophys Res Commun 351:883–889.
52. Kraneveld AD, Folkerts G, Van Oosterhout AJ, Nijkamp FP. 1997. Airway hyper-
responsiveness: First eosinophils and then neuropeptides. Int J Immunopharmacol
19:517–527.
53. Bradley BL, Azzawi M, Jacobson M, Assoufi B, Collins JV, lrani A-M et al. 1991.
Eosinophils, T-lymphocytes. mast cells, neutrophils and macrophages in bronchial
biopsy specimens from atopic subjects with asthma: Comparison with biopsy speci-
mens from atopic subjects without asthma and normal control subjects and relationship
to bronchial hyperresponsiveness. J Allergy Clin lmmunol 88:661–670.
54. Wegner CD, Gundel RH, Reilly P, Haynes N, Letts LG, Rothlein R. 1990. Intercellular
adhesion molecule-1 (ICAM-1) in the pathogenesis of asthma. Science 247:445–459.
55. Cartier A, Thomson NC, Frith PA, Roberts R, Hargcave FE. 1982. Allergen-induced
increase in bronchial responsiveness to histamine: Relationship to the late asthmatic
response and change in airway caliber. J Allergy Clin Immunol 70:170–177.
56. Cockroft DW, Murdock KY. 1987. Comparative effects of inhaled salbutamol, sodium
cromoglycate, and beclomethasone dipropionate on allergen-induced early asthmatic
responses, late asthmatic responses, and increased bronchial responsiveness to hista-
mine. J Allergy Clin Immunol 79:734–740.
57. Aalbers R, De Monchy JGR, Kauffman HF, Smith M, Hoekstra Y, Vrugt B et al. 1993.
Dynamics of eosinophil infiltration in the bronchial mucosa before and after the late
asthmatic reaction. Eur Respir J 6:840–847.
58. Bousquet J, Chanez P, Lacostc JY. 1990. Eosinophilic inflammation in asthma. New
Engl J Med 323:1033–1039.
59. Linder A, Venge P, Deuschl H. 1987. Eosinophil cationic protein and myeloperoxidase
in nasal secretion as markers of inflammation in allergic rhinitis. Allergy 42:583–591.
60. Van Oosterhout AJM, Ladenius ARC, Savelkoul HFJ, Van Ark I, Delsman KC, Nijkamp
FP. 1993. Effect of anti-IL5 and IL-5 on airway hyperreactivity and eosinophils in
guinea pigs. Am Rev Respir Dis 147:548–552.
61. Mauser PJ, Pitman A, Witt A, Fernandez X, Zurcher J, Kung T et al. 1993. Inhibitory
effect of the TRFK-5 anti-I L-5 antibody in a guinea pig model of asthma. Am Rev
Respir Dis 148:1623–1627.
62. Pretolani M, Ruffle C, Joseph D, Campos MG, Church MK, Lefort J et al. 1994. Role of
eosinophil activation in the bronchial reactivity of allergic guinea pigs. Am J Respir Crit
Care Med 149:1167–1174.
63. Van Oosterhout AJM, Van Ark I, Hofman G, Van der Linde HJ, Fattah D, Nijkamp FP.
1996. Role of interleukin 5 and substance P in development of airway hyperreactivity to
histamine in guinea pigs. Eur Respir J 9:493–499.
64. de Haar C, Hassing I, Bol M, Bleumink R, Pieters R. 2006. Ultrafine but not fine particu-
late matter causes airway inflammation and allergic airway sensitization to co-adminis-
tered antigen in mice. Clin Exp Allergy 36:1469–1479.
65. Diaz-Sanchez D, Garcia MP, Wang M, Jyrala M, Saxon A. 1999. Nasal challenge with
diesel exhaust particles can induce sensitization to a neoallergen in the human mucosa.
J Allergy Clin Immunol 104:1183–1188.
66. Diaz-Sanchez D, Tsien A, Fleming J, Saxon A. 1997. Combined diesel exhaust particu-
late and ragweed allergen challenge markedly enhances human in vivo nasal ragweed-
specific IgE and skews cytokine production to a T helper cell 2-type pattern. J Immunol
158:2406–2413.
67. Kleinman MT, Sioutas C, Froines JR, Fanning E, Hamade A, Mendez L et al. 2007.
Inhalation of concentrated ambient particulate matter near a heavily trafficked road stim-
ulates antigen-induced airway responses in mice. Inhal Toxicol 19(Suppl 1):117–126.
68. Muranaka M, Suzuki S, Koizumi K, Takafuji S, Miyamoto T, Ikemori R et al. 1986.
Adjuvant activity of diesel-exhaust particulates for the production of IgE antibody in
mice. J Allergy Clin Immunol 77:616–623.
69. Ritz SA, Wan J, Diaz-Sanchez D. 2007. Sulforaphane-stimulated phase II enzyme

induction inhibits cytokine production by airway epithelial cells stimulated with diesel
extract. Am J Physiol Lung Cell Mol Physiol 292:L33–L39.
70. Wan J, Diaz-Sanchez D. 2006. Phase II enzymes induction blocks the enhanced IgE
production in B cells by diesel exhaust particles. J Immunol 177:3477–3483.
71. Wan J, Diaz-Sanchez D. 2007. Antioxidant enzyme induction: A new protective
approach against the adverse effects of diesel exhaust particles. Inhal Toxicol 19
(Suppl 1):177–182.
72. Ichinose T, Takano H, Miyabara Y, Sadakaneo K, Sagai M, Shibamoto T. 2002.
Enhancement of antigen-induced eosinophilic inflammation in the airways of mast-cell
deficient mice by diesel exhaust particles. Toxicology 180(3):293–301.
73. Yanagisawa R, Takano H, Inoue KI, Ichinose T, Sadakane K, Yoshino S et al. 2006.
Components of diesel exhaust particles differentially affect Th1/Th2 response in a
murine model of allergic airway inflammation. Clin Exp Allergy 36:386–395.
74. Ichinose T, Takano H, Sadakane K, Yanagisawa R, Kawazato H, Sagai M et al. 2003.
Differences in airway-inflammation development by house dust mite and diesel exhaust
inhalation among mouse strains. Toxicol Appl Pharmacol 187:29–37.
75. Sadakane K, Ichinose T, Takano H, Yanagisawa R, Sagai M, Yoshikawa T et al. 2002.
Murine strain differences in airway inflammation induced by diesel exhaust particles
and house dust mite allergen. Int Arch Allergy Immunol 128:220–228.
76. Nel AE, Diaz-Sanchez D, Ng D, Hiura T, Saxon A. 1998. Enhancement of allergic
inflammation by the interaction between diesel exhaust particles and the immune sys-
tem. J Allergy Clin Immunol 102:539–554.
77. Becker S, Soukup JM, Gilmour MI, Devlin RB. 1996. Stimulation of human and rat
alveolar macrophages by urban air particulates: Effects on oxidant radical generation
and cytokine production. Toxicol Appl Pharmacol 141:637–648.
78. Boland S, Baeza-Squiban A, Fournier T, Houcine O, Gendron MC, Chevrier M et al.
1999. Diesel exhaust particles are taken up by human airway epithelial cells in vitro and
alter cytokine production. Am J Physiol 276:L604–L613.
79. Goldsmith CA, Frevert C, Imrich A, Sioutas C, Kobzik L. 1997. Alveolar
macrophage interaction with air pollution particulates. Environ Health Perspect 105
(Suppl 5):1191–1195.
80. Li XY, Gilmour PS, Donaldson K, MacNee W. 1996. Free radical activity and pro-
inflammatory effects of particulate air pollution (PM10) in vivo and in vitro. Thorax
51:1216–1222.
81. Martin LD, Krunkosky TM, Dye JA, Fischer BM, Jiang NF, Rochelle LG et al. 1997.
The role of reactive oxygen and nitrogen species in the response of airway epithelium to
particulates. Environ Health Perspect 105(Suppl 5):1301–1307.
82. Ohtoshi T, Takizawa H, Okazaki H, Kawasaki S, Takeuchi N, Ohta K et al. 1998. Diesel
exhaust particles stimulate human airway epithelial cells to produce cytokines relevant
to airway inflammation in vitro. J Allergy Clin Immunol 101:778–785.
83. Yang HM, Ma JY, Castranova V, Ma JK. 1997. Effects of diesel exhaust particles on the
release of interleukin-1 and tumor necrosis factor-alpha from rat alveolar macrophages.
Exp Lung Res 23:269–284.
84. Gamvrellis A, Leong D, Hanley JC, Xiang SD, Mottram P, Plebanski M. 2004. Vaccines
that facilitate antigen entry into dendritic cells. Immunol Cell Biol 82:506–516.
85. Schijns VE. 2001. Induction and direction of immune responses by vaccine adjuvants.
Crit Rev Immunol 21:75–85.
86. Lambrecht BN. 2001. Allergen uptake and presentation by dendritic cells. Curr Opin
Allergy Clin Immunol 1:51–59.
87. Lambrecht BN. 2005. Dendritic cells and the regulation of the allergic immune response.
Allergy 60:271–282.
88. Lambrecht BN, Hammad H. 2003. Taking our breath away: Dendritic cells in the patho-
genesis of asthma. Nat Rev Immunol 3:994–1003.
89. Eisenbarth SC, Piggott DA, Bottomly K. 2003. The master regulators of allergic inflam-
mation: Dendritic cells in Th2 sensitization. Curr Opin Immunol 15:620–626.
90. Moser M, Murphy KM. 2000. Dendritic cell regulation of TH1–TH2 development. Nat
Immunol 1:199–205.
91. O’Garra A, Arai N. 2000. The molecular basis of T helper 1 and T helper 2 cell differen-
tiation. Trends Cell Biol 10:542–550.
92. Edwan JH, Perry G, Talmadge JE, Agrawal DK. 2004. Flt-3 ligand reverses late allergic
response and airway hyper-responsiveness in a mouse model of allergic inflammation.
J Immunol 172:5016–5023.
93. Julia V, Hessel EM, Malherbe L, Glaichenhaus N, O’Garra A, Coffman RL. 2002.
A restricted subset of dendritic cells captures airborne antigens and remains able to
activate specific T cells long after antigen exposure. Immunity 16:271–283.
94. Lambrecht BN, De Veerman M, Coyle AJ, Gutierrez-Ramos JC, Thielemans K, Pauwels
RA. 2000. Myeloid dendritic cells induce Th2 responses to inhaled antigen, leading to
eosinophilic airway inflammation. J Clin Invest 106:551–559.
95. Lambrecht BN, Salomon B, Klatzmann D, Pauwels RA. 1998. Dendritic cells are
required for the development of chronic eosinophilic airway inflammation in response
to inhaled antigen in sensitized mice. J Immunol 160:4090–4097.
96. Thepen T, McMenamin C, Girn B, Kraal G, Holt PG. 1992. Regulation of IgE produc-
tion in pre-sensitized animals: In vivo elimination of alveolar macrophages preferen-
tially increases IgE responses to inhaled allergen. Clin Exp Allergy 22:1107–1114.
97. van Rijt LS, Jung S, Kleinjan A, Vos N, Willart M, Duez C, Hoogsteden HC, Lambrecht
BN. 2005. In vivo depletion of lung CD11c+ dendritic cells during allergen challenge
abrogates the characteristic features of asthma. J Exp Med 201:981–991.
98. van Rijt LS, Prins JB, Leenen PJ, Thielemans K, de Vries VC, Hoogsteden HC et al.
2002. Allergen-induced accumulation of airway dendritic cells is supported by an
increase in CD31(hi) Ly-6C(neg) bone marrow precursors in a mouse model of asthma.
Blood 100:3663–3671.
99. Peden DB. 2002. Pollutants and asthma: Role of air toxics. Environ Health Perspect
110(Suppl 4):565–568.
100. Sagai M, Furuyama A, Ichinose T. 1996. Biological effects of diesel exhaust parti-
cles (DEP). III. Pathogenesis of asthma like symptoms in mice. Free Radic Biol Med
21:199–209.
101. Takenaka H, Zhang K, Diaz-Sanchez D, Tsien A, Saxon A. 1995. Enhanced human IgE
production results from exposure to the aromatic hydrocarbons from diesel exhaust:
Direct effects on B-cell IgE production. J Allergy Clin Immunol 95:103–115.
102. Nordenhall C, Pourazar J, Ledin MC, Levin JO, Sandstrom T, Adelroth E. 2001.
Diesel exhaust enhances airway responsiveness in asthmatic subjects. Eur Respir J
17:909–915.
103. Takano H, Ichinose T, Miyabara Y, Yoshikawa T, Sagai M. 1998. Diesel exhaust particles
enhance airway responsiveness following allergen exposure in mice. Immunopharmacol
Immunotoxicol 20:329–336.
104. Walters DM, Breysse PN, Wills-Karp M. 2001. Ambient urban Baltimore particulate-
induced airway hyperresponsiveness and inflammation in mice. Am J Respir Crit Care
Med 164:1438–1443.
105. Wichmann HE. 2007. Diesel exhaust particles. Inhal Toxicol 19(Suppl 1):241–244.
106. Lim HB, Ichinose T, Miyabara Y, Takano H, Kumagai Y, Shimojyo N et al. 1998.
Involvement of superoxide and nitric oxide on airway inflammation and hyper-
responsiveness induced by diesel exhaust particles in mice. Free Radic Biol Med
25:635–644.
107. Li N, Wang M, Oberley TD, Sempf JM, Nel AE. 2002. Comparison of the pro-oxidative
and proinflammatory effects of organic diesel exhaust particle chemicals in bronchial
epithelial cells and macrophages. J Immunol 169:4531–4541.
108. Doornaert B, Leblond V, Galiacy S, Gras G, Planus E, Laurent V et al. 2003. Negative
impact of DEP exposure on human airway epithelial cell adhesion, stiffness, and repair.
Am J Physiol Lung Cell Mol Physiol 284:L119–L132.
109. Ichinose T, Takano H, Miyabara Y, Sagai M. 1998. Long-term exposure to diesel exhaust
enhances antigen-induced eosinophilic inflammation and epithelial damage in the
murine airway. Toxicol Sci 44:70–79.
110. Nel AE, Diaz-Sanchez D, Li N. 2001. The role of particulate pollutants in pulmonary
inflammation and asthma: Evidence for the involvement of organic chemicals and
oxidative stress. Curr Opin Pulm Med 7:20–26.
111. Kreyling WG, Semmler-Behnke M, Moller W. 2006. Ultrafine particle-lung interac-
tions: Does size matter? J Aerosol Med 19:74–83.
112. Peters A, Veronesi B, Calderon-Garciduenas L, Gehr P, Chen LC, Geiser M et al. 2006.
Translocation and potential neurological effects of fine and ultrafine particles a critical
update. Part Fibre Toxicol 3:13.
113. Von Mutius E, Schwartz J, Neas LM, Dockery D, Weiss ST. 2001. Relation of body mass
index to asthma and atopy in children: The National Health and Nutrition Examination
Study III. Thorax 56:835–838.
114. Figueroa-Munõz JI, Chinn S, Rona RJ. 2001. Association between obesity and asthma
in 4–11 year old children in the UK. Thorax 56:133–137.
115. James AL, Knuiman MW, Divitini ML, Hui J, Hunter M, Palmer LJ et al. 2010. Changes
in the prevalence of asthma in adults since 1966: The Busselton Health Study. Eur
Respir J 35:273–278.
116. Hong SJ, Lee MS, Lee SY, Ahn KM, Oh JW, Kim KE et al. 2006. High Body mass
index and dietary pattern are associated with childhood asthma. Pediatr Pulmonol
41:1118–1124.
117. Chu YT, Chen WY, Wang TN, Tseng HI, Wu JR, Ko YC. 2009. Extreme BMI predicts
higher asthma prevalence and is associated with lung function impairment in school-
aged children. Pediatr Pulmonol 44:472–479.
118. Mai XM, Nilsson L, Axelson O, Bråbäck L, Sandin A, Kjellman NI et al. 2003. High
body mass index, asthma and allergy in Swedish schoolchildren participating in the
International Study of Asthma and Allergies in Childhood: Phase II. Acta Pediatr
92:1144–1148.
119. Oddy WH, Sherriff JL, de Klerk NH, Kendall GE, Sly PD, Beilin LJ et al. 2004. The
relation of breastfeeding and body mass index to asthma and atopy in children: A pro-
spective cohort study to age 6 years. Am J Public Health 94:1531–1537.
120. Eijkemans M, Mommers M, de Vries SI, van Buuren S, Stafleu A, Bakker I et al. 2008.
Asthmatic symptoms, physical activity, and overweight in young children: A cohort
study. Pediatrics 121:666–672.
121. Flaherman V, Rutherford GW. 2006. A meta-analysis of the effect of high weight on
asthma. Arch Dis Child 91:334–339.
122. Beuther DA, Sutherland ER. 2007. Overweight, obesity and incident asthma: A meta-
analysis of prospective epidemiologic studies. Am J Respir Crit Care Med 175:661–666.
123. Dixon AE, Holguin F, Sood A, Salome CM, Pratley RE, Beuther DA et al. 2010.
An Official American Thoracic Society Workshop Report: Obesity and Asthma.
Subcommittee on Obesity and Lung Disease. Proc Am Thorac Soc 7:325–325.
124. Noal RB, Menezes AM, Macedo SE, Dumith SC. 2011. Childhood body mass index and
risk of asthma in adolescence: A systematic review. Obes Rev 12:93–104.
125. Sideleva O, Black K, Dixon AE. 2012. Effects of obesity and weight loss on airway
physiology and inflammation in asthma. Pulm Pharmacol Ther 26:455–458.
126. Shore SA, Johnston RA. 2006. Obesity and asthma. Pharmacol Ther 110:83–102.
127. de Groot EP, Duiverman EJ, Brand PL. 2010. Comorbidities of asthma during child-
hood: Possibly important, yet poorly studied. Eur Respir J 36:671–678.
128. Guerra S, Wright AL, Morgan WJ, Sherill DL, Holberg CJ, Martinez FD. 2004.
Persistence of asthma symptoms during adolescence. Role of obesity and age at the
onset of puberty. Am J Respir Crit Care Med 170:78–85.
129. Lessard A, Turcotte H, Cormier Y, Boulet LP. 2008. Obesity and asthma: A specific
phenotype? Chest 134:317–323.
130. Mahadev S, Farah CS, King GG, Salome CM. 2012. Obesity, expiratory flow limitation
and asthma symptoms. Pulm Pharmacol Ther 26:438–443.
131. Johnston RA, Zhu M, Rivera-Sanchez YM, Lu FL, Theman TA, Flynt L et al. 2007.
Allergic airway responses in obese mice. Am J Respir Crit Care Med 176:650–658.
132. Calixto MC, Lintomen L, Schenka A, Saad MJ, Zanesco A, Antunes E. 2010. Obesity
enhances eosinophilic inflammation in a murine model of allergic asthma. Br J
Pharmacol 159:617–625.
133. Saraiva SA, Silva AL, Xisto DG, Abreu SC, Silva JD, Silva PL et al. 2011. Impact of
obesity on airway and lung parenchyma remodeling in experimental chronic allergic
asthma. Respir Physiol Neurobiol 177:141–148.
134. Scott HA, Gibson PG, Garg ML, Wood LG. 2011. Airway inflammation is augmented
by obesity and fatty acids in asthma. Eur Resp J 38:594–602.
135. Jensen ME, Collins CE, Gibson PG, Wood LG. 2011. The obesity phenotype in children
with asthma. Pediatr Resp Rev 12:152–159.
136. Scott HA, Gibson PG, Garg ML, Pretto JJ, Morgan PJ, Callister R et al. 2013. Dietary
restriction and exercise improve airway inflammation and clinical outcomes in over-
weight and obese asthma: A randomized trial. Clin Exp Allergy 43:36–49.
137. Li N, Xia T, Nel AE. 2008. The role of oxidative stress in ambient particulate matter-
induced lung diseases and its implications in the toxicity of engineered nanoparticles.
Free Radic Biol Med 44(9):1689–1699.
15 Muscle and Immune
Function
Anthony Thomas and David Heber
CONTENTS
Introduction............................................................................................................. 245
Function of Myokines: Cytokines Secreted by Muscle Cells.................................246
Visceral/Subcutaneous Fat and Inflammation......................................................... 250
Skeletal Muscle, Sarcopenia, and Inflammation..................................................... 252
Conclusion.............................................................................................................. 254
References............................................................................................................... 254
INTRODUCTION
The global obesity epidemic is associated with an array of cardiometabolic perturba-
tions and increased risk of chronic diseases including type 2 diabetes (T2D), cardio-
vascular disease (CVD), and various cancers. Specifically, excess intra-abdominal
fat accumulation (visceral obesity) is believed to be more important than total body
fat in predicting morbidities traditionally associated with obesity, and various fac-
tors (e.g., age, gender, physical activity/fitness, hormones, and ethnicity/genetics)
are believed to influence depot-specific adipose tissue distribution [1]. Visceral obe-
sity contributes to the chronic low-grade inflammation that plays a causative role
in obesity-induced insulin resistance and the pathophysiology of obesity-associated
diseases [2].
Both typical aging in association with sedentary lifestyles and inadequate pro-
tein intake are associated with sarcopenic obesity, a condition of reduced muscle
and increased body fat even when body weight is normal or low [3], leading to an
increase in visceral fat. In countries such as China and India, metabolic syndrome
and T2D are frequently observed at normal or low body weights due to the presence
of increased visceral fat [4]. In addition, sarcopenic obesity is very common in
postmenopausal women [5]. Magnetic resonance imaging studies demonstrated that
45% of women and 60% of men surveyed from the general population in London
had increased intra-abdominal fat even when body weight was normal and waist
circumference was normal [6]. Conversely, Matsuzawa [7] showed that very obese
active sumo wrestlers with low visceral adiposity are quite insulin sensitive com-
pared to retired sedentary sumo wrestlers displaying greater amounts of visceral adi-
pose tissue. Thus, both metabolically healthy obese and metabolically obese normal
245
weight individuals have been described with excess visceral fat and physical activity
as likely underlying factors determining differential disease risk [1].
The major modifier of intra-abdominal fat in the aforementioned studies was the
observation that fit people have reduced visceral fat [6]. Anti-inflammatory effects of
regular physical activity/exercise could be mediated in part via reduction in visceral
fat mass [7]. Contracting skeletal muscles release myokines, which can work in an
endocrine fashion on visceral fat as well as have direct anti-inflammatory effects
on cells in other organs and tissues [8]. At the same time, myokines function within
the muscle via autocrine/paracrine mechanisms, exerting effects on signaling path-
ways involved in fat oxidation and glucose metabolism. Regular exercise protects
against a number of chronic diseases associated with chronic inflammation, and
visceral obesity is associated with chronic inflammation [9]. Furthermore, physi-
cal inactivity is associated with increased chronic disease risk independent of body
weight, and acute periods of physical inactivity (no change in body composition) are
associated with negative metabolic/physiologic changes (e.g., reduced insulin sensi-
tivity, skeletal muscle atrophy, and increased visceral fat) [10].
Myokines may in part mediate the protective effects of exercise with regard to
sedentary lifestyle–related chronic diseases. It is likely that regular physical activity/
exercise is an essential component of any diet and lifestyle program aiming to reduce
intra-abdominal fat, inflammation, and related chronic disease risk.
At the other end of the nutritional spectrum, it is known that reductions in body
cell mass below 50% of preillness levels are incompatible with life regardless of the
cause of malnutrition including starvation, tumor, infection, or surgery [11]. Kotler
[12] was able to accurately estimate the date of death from AIDS using total body
potassium measures of lean body mass. Some research suggests that loss of specific
immune function with malnutrition may be secondary to loss of immune stimulation
by muscle cells [13].
The concept of skeletal muscle as an endocrine and immune organ parallels the
same concept in adipose tissue and fat cells. Therefore, muscle/myocyte immune
function, adipose/adipocyte immune function, exercise, and nutrition are all inti-
mately linked with immune function systemically.
FUNCTION OF MYOKINES: CYTOKINES

SECRETED BY MUSCLE CELLS
Cytokines and other peptides produced, expressed, and released by muscle fibers
have been termed myokines [14]. These proteins can exert both local (i.e., autocrine/
paracrine) and remote (i.e., endocrine) effects via the circulation (see Figure 15.1).
Muscle can play a central role in metabolism through interaction with other organs
and may mediate benefits of a healthy active lifestyle, including reducing abdomi-
nal visceral fat. The endocrine function of muscle is stimulated by exercise, and so
sedentary lifestyle may be considered a disease that is caused by the absence of the
beneficial secreted factors emanating from contracting muscle.
While a number of myokines have been described, including interleukin
(IL)-6, IL-8, IL-15, brain-derived neurotrophic factor, leukemia inhibitory factor,
fibroblast growth factor-21, and follistatin-like-1 [15–18], with many more yet to
Muscle and Immune Function 247
Proinflammatory Anti-inflammatory
IL-6
TNF
IL-1ra
TNF-R IL-10
Sepsis
Anti-inflammatory
IL-6
IL-1ra
IL-10
Exercise
FIGURE 15.1 (See color insert.) During sepsis, there is a marked and rapid increase in
circulating TNF-α, which is followed by an increase in IL-6. In contrast, during exercise,
the marked increase in IL-6 is not preceded by elevated TNF-α. (From Pedersen, B.K.
and Febbraio, M.A., Physiol. Rev., 88, 1379, 2008. With permission from the American
Physiological Society.)
be identified from the muscle cell secretome [19], the most studied of these is the
cytokine IL-6. IL-6 was the first myokine found to be secreted into circulation as
a result of muscle contraction; levels increase proportional to exercise duration
and the amount of muscle mass engaged in the exercise/exercise intensity [20],
and levels can acutely increase up to 100-fold from basal levels during exercise
(lesser increases are more frequently observed) [21]. It was previously believed that
increased IL-6 during exercise was due to an immune response evoked by local
skeletal muscle tissue damage with macrophages hypothesized to be the cellular
origin [22]; however, circulating IL-6 increases during exercise without observed
muscle damage [21], and muscle cells per se are now recognized as the predomi-
nant source during exercise.
With exercise, IL-6 mRNA levels increase markedly within 30 min of initiation
[23,24] with evidence suggesting that the concentration of IL-6 within contracting
skeletal muscle is potentially 5- to 100-fold higher than levels in the blood [25],
despite large amounts being secreted into circulation. Liver extraction of IL-6 from
the blood following exercise may limit potentially negative metabolic effects of pro-
longed systemic elevations in IL-6, as levels quickly decrease toward preexercise
levels following exercise. Increased basal systemic IL-6 levels are positively associ-
ated with physical inactivity and the metabolic syndrome, while there is a negative
association between regular physical activity and basal systemic IL-6 [21]. In fact,
the magnitude of exercise-induced acute increases in plasma IL-6 is diminished over
time with training, but basal IL-6 receptor (IL-6R) expression is robustly increased
in the trained skeletal muscle [26].
Thus, adaptations to regular exercise may increase sensitivity to IL-6 in trained
skeletal muscle despite systemically reduced basal and blunted acute exercise-
induced elevations. Exercise-induced muscle-derived IL-6 is thought to primarily
play a metabolic versus inflammatory role as it can be produced during contrac-
tion independent of TNF-α with expression increased due to glycogen depletion
and attenuated IL-6 release from contracting muscle with glucose ingestion dur-
ing exercise. With muscle contractions of both type I and type II muscle fibers,
IL-6 acts locally to activate AMP kinase and/or phosphatidylinositol 3 (PI3) kinase,
which increase fat oxidation and glucose uptake. IL-6 may also work in an endo-
crine fashion to increase hepatic glucose production during exercise and lipolysis in
adipose tissue as well as from intramuscular lipid stores [20]. It appears that IL-6
works in concert with catecholamines and cortisol to redistribute energy substrates
available to working muscles during exercise by enhancing the release of glucose
from the liver and fatty acids from adipose tissue while simultaneously increas-
ing uptake of glucose and fat oxidation in the working muscle (IL-6 antagonism
of insulin action in hepatocytes and adipocytes may contribute to these effects).
IL-6 released from contracting muscles may promote a general anti-inflammatory
response. IL-1 receptor antagonist (IL-1ra: attenuates proinflammatory actions
of IL-1), IL-10 (anti-inflammatory cytokine), soluble TNF-α receptor (sTNF-R:
antagonizes cellular TNF-α signaling), cortisol (glucocorticoid with potent anti-
inflammatory effects), and C-reactive protein (may enhance IL-1ra release from
monocytes) are all increased in response to infusion of rhIL-6 [21]. Additionally,
IL-6 attenuates TNF-α produced in response to lipopolysaccharide (LPS; circulat-
ing LPS is increased with high- fat feeding and obesity) in cultured human mono-
cytes as well as in vivo [27,28].
Mice that are deficient in IL-6, either due to neutralizing antibody treatment or
genetically deficient due to IL-6 gene knockout, have elevated TNF-α levels in their
circulation [29]. The activation of IL-6 signaling in macrophages is triggered by cal-
cium/NFAT and glycogen/p38 mitogen-activated protein kinase (MAPK) pathways
unlike the triggering of IL-6 in immune cells, which is linked to the activation of
nuclear factor-κB ( NF-κB). Differences in the cellular signaling pathways may be
needed since IL-6 derived from contracting muscle appears to have anti-inflammatory
activity, which is the opposite of what is found with IL-6 from immune cells (see
Figure 15.2). These observations are consistent with the idea that regular exercise exerts
IL-6
IL-6Rα/gp130Rβ
P13-K p-STAT3
p-Akt p-AMPK Blood vessel
Glucose uptake Fat oxidation IL-6

Liver
IL-6
Increased hepatic
Contraction glucose production
during exercise
IL-6 IL-6
IL-6 IL-6
IL-6
IL-6
Adipose tissue
IL-6
Increased lipolysis
FIGURE 15.2 (See color insert.) Skeletal muscle expresses and releases myokines into the
circulation. In response to muscle contractions, both type I and type II muscle fibers express
the myokine IL-6, which subsequently exerts its effects both locally within the muscle
(e.g., through activation of AMPK) and—when released into the circulation—peripherally
in several organs in a hormone-like fashion. Specifically, in skeletal muscle, IL-6 acts in
an autocrine or paracrine manner to signal through a gp130Rβ/IL-6Rα homodimer, result-
ing in the activation of AMP kinase and/or PI3 kinase to increase glucose uptake and fat
oxidation. IL-6 is also known to increase hepatic glucose production during exercise or lipol-
ysis in adipose tissue. (Modified from Pedersen, B.K. and Febbraio, M.A., Physiol. Rev.,
88, 1379, 2008. With permission from the American Physiological Society; Reprinted from
Curr. Opin. Clin. Nutr. Metab. Care., 10(3), Pedersen, B.K. and Fischer, C.P., Physiological
roles of muscle-derived interleukin-6 in response to exercise, 265–271, Copyright 2007, with
permission from Elsevier.)
anti-inflammatory effects capable of protecting against the systemic low-grade inflam-

mation triggered by visceral fat both by decreasing visceral fat and by counteracting
the proinflammatory action of visceral fat–derived adipocytokines. The situation is
more complex, as exercise also stimulates increases in the levels of catecholamines,
cortisol, growth hormone, prolactin, and other factors with immunomodulatory prop-
erties [30]. Therefore, myokines are only part of a larger coordinated temporal exercise
response with both proinflammatory and anti-inflammatory components.
Despite the complexity of the exercise endocrine and cytokine response, exer-
cise is ultimately anti-inflammatory and promotes healthy aging. As mentioned
earlier, aging is associated with declines in skeletal muscle mass and immune
function and increases in visceral fat accumulation, which promote sarcopenic
obesity and increased disease risk and all-cause mortality [31]. IL-15 is highly
expressed in skeletal muscle, declines with age, and is reported to transiently
increase with both resistance [32] and aerobic exercise. Evidence suggests that
IL-15 has protective effects against sarcopenic obesity to promote healthy aging.
IL-15 accumulates in muscle with strength training and is a factor both promoting
muscle hypertrophy (decreased muscle protein degradation) and also reducing
body fat [33]. Following strength training, IL-15 mRNA levels are upregulated
in human skeletal muscle [34]. In mice, a decrease in visceral, but not subcu-
taneous, fat was observed when IL-15 was genetically overexpressed in muscle
[35]. Additionally, IL-15-deficient mice become obese, insulin resistant, and glu-
cose intolerant in the absence of increased energy consumption; exogenous IL-15
administration reversed this obesity as well as diet-induced obesity and associated
metabolic perturbations [36,37].
Human adipocyte differentiation in culture was inhibited by IL-15 [36]. Increased
IL-15 in the circulation has been associated with lower body fat and increased bone
mineral content in humans. In these studies, there was no significant effect on lean
body mass or other cytokines [38]. IL-15 is required for the development and survival
of natural killer lymphocytes as well as other T-lymphocyte subsets, which suggests
that exercise/skeletal muscle is critical to the maintenance of proper immune func-
tion with aging to protect against diseases of the elderly including infections and
cancer [31]. Taken together, data from human and animal studies to date support the
concept that IL-15 secretion from muscle during or following exercise has favorable
effects on body composition and is a myokine that protects against age-related sar-
copenic obesity and declines in immune function.
Recently, a novel myokine termed Irisin (after the Greek messenger goddess
Iris, to highlight the role as a protein messenger derived from skeletal muscle to
other tissues) is increased in skeletal muscle and blood in response to exercise with
beneficial metabolic effects that partially manifest via regulation of adiposity/
adipocyte function [39]. Specifically, Irisin stimulates fat cells within white
adipose tissue to function more like highly thermogenic brown fat cells, thus
increasing energy expenditure to promote a lean phenotype. Boström et al. [39]
showed that modestly elevating plasma Irisin similar to increases observed with
regular exercise protects mice from diet-induced obesity and associated metabolic
derangements.
VISCERAL/SUBCUTANEOUS FAT AND INFLAMMATION

While all excess body fat has been associated with increased risk factors for age-
related chronic diseases, over the past two decades, visceral and not subcutaneous
fat has been more closely related to disease risk. A large body of evidence has estab-
lished the proinflammatory and systemically damaging effects of abdominal vis-
ceral fat and ectopic fat in the liver and muscle [40], while subcutaneous adipose
tissue may be protective via the secretion of adiponectin. The accumulation of fat
in ectopic sites such as the liver is much more common than is typically appreciated
and is acutely sensitive to changes in energy intake (e.g., short-term overnutrition can
increase hepatic fat accumulation and influence liver insulin sensitivity to impact
glucose homeostasis without significant changes in body composition). Visceral
fat is associated with increased risks of a number of age-related chronic diseases,
including dementia [41], CVD [42], T2D [43], and common forms of cancer such as
colon [44] and breast cancer [45], as well as all-cause mortality even in people with
a normal body weight (independent of BMI) [40]. Thus, the health consequences of
intra-abdominal adiposity and physical inactivity are similar. Moreover, both physi-
cal inactivity [10] and visceral obesity [46] are associated with persistent systemic
low-grade inflammation [47].
Models of lipodystrophy suggest that if the subcutaneous fat becomes inflamed
and adipocytes undergo apoptosis/necrosis, the fat storing capacity is impaired;
hence, fat is diverted from adipose (tissue specialized for energy storage as tri-
glycerides) and deposited as ectopic fat subjecting other tissues to the cytotoxic
effects of excess fatty acids. Given the anti-inflammatory effects of regular exercise
[48], physical inactivity may lead to inflammation of subcutaneous adipose tissue
and impaired ability to store fat, also in people who do not fulfill the criteria for
lipodystrophy.
Evidence exists that visceral fat is more susceptible to immune cell infiltration
and production of proinflammatory mediators with positive energy balance than
subcutaneous fat and constitutes an important source of chronic low-grade systemic
inflammation [46]. A number of studies point to an independent effect of exercise on
intra-abdominal adiposity. A recent review highlighted the notion that repeated bouts
of exercise have a major impact on visceral obesity [8]. Thus, most studies show that
increased physical activity is associated with significant reductions in waist circum-
ference (as an indirect correlated measure of intra-abdominal fat accumulation) and/
or visceral fat, despite either no change in body weight or a change of <3%, regard-
less of sex or age [8]. In a couple of studies [35,36], men and women with abdominal
obesity exercised under supervision and were required to consume additional food
calories to prevent exercise-induced weight loss. The objective was to determine
whether chronic exercise (40–60 min of daily exercise for 12–16 weeks) without
weight loss was associated with reductions in obesity. The results from these studies
illustrate that considerable reductions in total fat, visceral fat in particular (which
decreased by 12%–18%), and waist circumference can be achieved in the absence of
weight loss. In addition to marked reductions in these measures of obesity, increases
were also observed in skeletal muscle mass and cardiorespiratory fitness (both inde-
pendently associated with reduced disease risk).
Subcutaneous fat serves both as an energy store for other tissues such as working
muscles and also releases the anti-inflammatory adipokine, adiponectin (blood lev-
els are inversely associated with adiposity). Additionally, fatty acids derived from
fat stores during exercise are believed to play a role in skeletal muscle adaptations
to exercise. Innate immune receptors (i.e., Toll-like receptors 2 and 4 (TLR-2/4)) are
expressed on the cell surface of innate immune cells as well as various other cell
types including myocytes. Specific fatty acid species can modulate cell signaling
mediated through these receptors (e.g., MAPK family members and NF-κB) dif-
ferentially in various cells expressing TLR-2 and -4 on their cell surface. Exercise
regulates various signaling pathways including differential activation of MAPK
family members to influence cell metabolism, growth, proliferation, and differen-
tiation depending on type, intensity, duration, and nutritional status; however, the
mechanisms of activation are incompletely understood.
Exercise increases extracellular nonesterified fatty acids (NEFAs), and it was

recently shown that mice lacking TLR-2 or TLR-4 had reduced activation (phos-
phorylation) of p38 MAPK and c-Jun NH2-terminal kinase (JNK) (MAPK family
members modulating exercise-induced skeletal muscle adaptations) without influ-
ence on NF-κB signaling in skeletal muscle after performing an endurance exercise
protocol previously shown to enhance adipose tissue lipolysis or heparin injection
(similarly increased plasma NEFAs outside the context of exercise) [49]. TLR-4 has
also been implicated in skeletal muscle energy substrate utilization and substrate
inflexibility associated with metabolic disorders such as obesity/insulin resistance.
Frisard et al. [50] demonstrated that low doses of the TLR-4 agonist LPS (referred to
as metabolic endotoxemia as circulating levels are modestly increased in the blood
with obesity, T2D, or high-fat feeding) increased skeletal muscle glucose utiliza-
tion and lactate production concurrently with reduced oxidation of fatty acids in
mice. Mice lacking functional TLR-4 maintained skeletal muscle oxidative capacity
in the face of metabolic endotoxemia. The authors suggest that increased levels of
circulating LPS along with enhanced TLR-4 sensitivity (increased TLR-4 expres-
sion in skeletal muscle of obese and type 2 diabetic humans [51]) contribute to the
metabolic derangements associated with these pathological disease states. It should
be noted that energy substrate availability (e.g., glucose ingestion during exercise
and depletion of glycogen stores), generation of reactive oxygen species, lactate
accumulation/cellular acidification, hypoxia, and mechanical stress, which can be
influenced by the exercise paradigm, diet composition/timing, and physiological
state, interact in highly complex ways to influence tissue and immune responses to
contracting skeletal muscles. Although beyond the scope of this chapter, these excit-
ing concepts suggest direct, bidirectional interactions between exercising muscle and
the immune/nutritional state of the host with potential long-term benefits for many
different organ systems negatively affected by lifestyle factors that encourage excess
body fat accumulation. The dynamic and temporal (acute vs. chronic) nature of these
highly complex interactions is an active area of current research aimed at better
understanding how cellular stressors evoked by exercise bring about beneficial car-
diometabolic adaptations that are often protective against cellular stressors evoked
in different physiological contexts.
SKELETAL MUSCLE, SARCOPENIA, AND INFLAMMATION

Muscle wasting is the nutritional hallmark of the deadly progression of a number
of serious diseases associated with chronic systemic inflammation including AIDS,
common forms of cancer, pulmonary failure, sepsis, and chronic heart failure. Aging
and disuse common to a sedentary lifestyle are the most common causes of sarcope-
nia or inadequate muscle mass within the general population. Muscle atrophy is char-
acterized by a reduction in cross-sectional area of myofibers together with reduced
myofibrillar protein content and secondary loss of muscle strength and increased
fatigability [52,53]. Skeletal muscle atrophy results from increased protein degrada-
tion and/or reduced protein synthesis. Various different triggers of muscle atrophy
may be operative simultaneously to result in sarcopenia. A lack of muscle loading
with bed rest initiates atrophy through mechanisms related to reduced tension [54],
while in a number of common diseases characterized by increased systemic inflam-

mation and elevated levels of proinflammatory cytokines, glucocorticosteroids,
tumor-derived factors, and endotoxins can induce muscle atrophy [55,56]. Distinct
biochemical signaling pathways are involved in the regulation of skeletal muscle
mass [52,54]. The activation of the PI3K/Akt pathway that occurs in response to
loading and growth factors such as insulin and insulin-like growth factor (IGF) pre-
vents the loss of skeletal muscle mass [57], while activation of NF-κB, activator
protein-1 (AP-1), p53, Foxo transcription factors, and p38 MAPK signaling pathways
are implicated in skeletal muscle atrophy [58].
Chronic activation of the ubiquitously expressed transcription factor
NF-κB is most closely linked to skeletal muscle atrophy in aforementioned
pathophysiological conditions. A large number of genes are regulated by NF-κB
including many critical regulators of immune/inflammatory responses and several
key enzymes of the ubiquitin-proteasome system (UPS) that are involved in the
selective degradation of regulatory and structural muscle proteins during atrophy
[59]. Specifically, two inducible E3 ubiquitin ligases of the UPS directly regulated
by NF-κB, atrogin-1, and muscle RING finger protein 1 (MuRF1) are responsible
for the bulk of skeletal muscle protein degradation in atrophy promoting patho-
physiologic states. NF-κB activation also negatively regulates myogenesis, which
is essential for skeletal muscle growth as well as maintenance/repair of myofibers;
however, there are reports that it may positively influence myogenic differentiation
to support myogenesis under certain cellular context possibly due to the type of
stimulus (i.e., IGF-II), intensity/duration of NF-κB activation, and NF-κB pathway
(i.e., alternative vs. classical) activated. Resting energy expenditure is directly
linked to lean body mass, so sarcopenia results in a reduction in resting energy
expenditure of about 14 cal/lb of lost lean body mass. Individuals who continue to
consume their usual calories in the face of reduced energy expenditure are more
likely to accumulate visceral fat and associated systemic inflammation to further
exacerbate the condition.
In animals, reductions in skeletal muscle mass and strength result from hind limb
unloading and immobilization reflective of disuse as the most common cause of
muscle atrophy in the general population. Hind limb unloading–induced muscle
atrophy in rats was associated with increased NF-κB DNA binding and reporter gene
activities in the soleus muscle [60]. Analysis of the NF-κB/DNA complex showed
that it mainly contained p50, c-Rel, and Bcl-3, but not other members of the NF-κB/
IκB family [61]. There was no evidence for the activation of classical p50/p65 NF-κB
dimers, which are generally activated in response to proinflammatory cytokines and
implicated in the regulation of skeletal muscle wasting associated with various dis-
ease states characterized by increased inflammation. Therefore, NF-κB-mediated
muscle atrophy that results from disuse appears to be distinct from other inflam-
matory pathways. Despite differential activation of classical and alternative NF-κB
pathways in skeletal muscle depending on the stimulus, both can result in muscle
atrophy [60,61].
Given the pivotal role of activated NF-κB signaling in disease states hallmarked
by skeletal muscle atrophy, inhibition of NF-κB activity and/or downstream targets
mediating atrophy (e.g., MuRF1) holds therapeutic potential.
CONCLUSION
A sedentary lifestyle, especially when its effects are amplified by a Western diet,
appears to lead to the accumulation of visceral fat, which is a source of systemic
inflammation. Chronic inflammation is involved in the pathogenesis of many dis-
eases, including insulin resistance, atherosclerosis, neurodegeneration, and tumor
growth. Therefore, the protective effects of exercise may be due in part to the anti-
inflammatory effects of regular exercise, which can be mediated via a reduction in
visceral fat mass and/or by induction of an anti-inflammatory environment with each
bout of exercise. The finding that muscles produce and release myokines has opened
up a whole new area for investigations to understand the mechanisms whereby exer-
cise influences metabolism and exerts anti-inflammatory effects. Much work remains
to determine the interactions between adipokines and myokines as well as potential
muscle—fat crosstalk. These investigations should lead to new insights useful in the
prevention and treatment of obesity and its associated diseases.
REFERENCES
1. Tchernof A, Després JP. 2013. Pathophysiology of human visceral obesity: An update.
Physiol Rev 93:359–404.
2. Lumeng CN, Saltiel AR. 2011. Inflammatory links between obesity and metabolic dis-
ease. J Clin Invest 121:2111–2117.
3. Li Z, Heber D. 2012. Sarcopenic obesity in the elderly and strategies for weight manage-
ment. Nutr Rev 70:57–64.
4. Gordon-Larsen P, Adair LS, Meigs JB, Mayer-Davis E, Herring A, Yan SK et al. 2013.
Discordant risk: Overweight and cardiometabolic risk in Chinese adults. Obesity (Silver
Spring) 21:E166–E174.
5. Messier V, Rabasa-Lhoret R, Barbat-Artigas S, Elisha B, Karelis AD, Aubertin-Leheudre
M. 2011. Menopause and sarcopenia: A potential role for sex hormones. Maturitas
68:331–336.
6. Thomas EL, Frost G, Taylor-Robinson SD, Bell JD. 2012. Excess body fat in obese and
normal-weight subjects. Nutr Res Rev 25:150–161.
7. Matsuzawa Y. 1997. Pathophysiology and molecular mechanisms of visceral fat syn-
drome: The Japanese experience. Diabetes Metab Rev 13:3–13.
8. Ross R, Bradshaw AJ. 2009. The future of obesity reduction: Beyond weight loss. Nat
Rev Endocrinol 5:319–325.
9. Pedersen BK. 2011. Exercise-induced myokines and their role in chronic diseases.
Brain Behav Immun 25:811–816.
10. Pedersen BK, Febbraio MA. 2012. Muscles, exercise and obesity: Skeletal muscle as a
secretory organ. Nat Rev Endocrinol 8:457–465.
11. Jeejeebhoy KN. 2012. Malnutrition, fatigue, frailty, vulnerability, sarcopenia and
cachexia: Overlap of clinical features. Curr Opin Clin Nutr Metab Care 15:213–219.
12. Kotler DP. 2000. Body composition studies in HIV-infected individuals. Ann N Y Acad
Sci 904:546–552
13. Lightfoot A, McArdle A, Griffiths RD. 2009. Muscle in defense. Crit Care Med
37:S384–S390.
14. Pedersen BK, Steensberg A, Fischer C, Keller C, Keller P, Plomgaard P et al. 2003.
Searching for the exercise factor: Is IL-6 a candidate? J Muscle Res Cell Motil
24:113–119.
15. Broholm C, Mortensen OH, Nielsen S, Akerstrom T, Zankari A, Dahl B et al. 2008.
Exercise induces expression of leukaemia inhibitory factor in human skeletal muscle.
J Physiol 586:2195–2201.
16. Izumiya Y, Bina HA, Ouchi N, Akasaki Y, Kharitonenkov A, Walsh K. 2008. FGF21 is
an Akt-regulated myokine. FEBS Lett 582:3805–3810.
17. Ouchi N, Oshima Y, Ohashi K, Higuchi A, Ikegami C, Izumiya Y et al. 2008. Follistatin-
like 1, a secreted muscle protein, promotes endothelial cell function and revasculariza-
tion in ischemic tissue through a nitric-oxide synthase-dependent mechanism. J Biol
Chem 283:32802–32811.
18. Hojman P, Pedersen M, Nielsen AR, Krogh-Madsen R, Yfanti C, Akerstrom T et al.
2009. Fibroblast growth factor-21 is induced in human skeletal muscles by hyperinsu-
linemia. Diabetes 58:2797–2801.
19. Bortoluzzi S, Scannapieco P, Cestaro A, Danieli GA, Schiaffino S. 2006. Computational
reconstruction of the human skeletal muscle secretome. Proteins 62:776–792.
20. Pedersen BK, Febbraio MA. 2008. Muscle as an endocrine organ: Focus on muscle-
derived interleukin-6. Physiol Rev 88:1379–1406.
21. Fischer CP. 2006. Interleukin-6 in acute exercise and training: What is the biological
relevance? Exerc Immunol Rev 12:6–33.
22. Nehlsen-Cannarella SL, Fagoaga OR, Nieman DC, Henson DA, Butterworth DE,
Schmitt RL et al. 1997. Carbohydrate and the cytokine response to 2.5 h of running.
J Appl Physiol 82:1662–1667.
23. Keller C, Steensberg A, Pilegaard H, Osada T, Saltin B, Pedersen BK et al. 2001.
Transcriptional activation of the IL-6 gene in human contracting skeletal muscle:
Influence of muscle glycogen content. FASEB J 15:2748–2750.
24. Steensberg A, Keller C, Starkie RL, Osada T, Febbraio MA, Pedersen BK. 2002. IL-6
and TNF-alpha expression in, and release from, contracting human skeletal muscle. Am
J Physiol Endocrinol Metab 283:E1272–E1278.
25. Rosendal L, Søgaard K, Kjaer M, Sjøgaard G, Langberg H, Kristiansen J. 2005. Increase
in interstitial interleukin-6 of human skeletal muscle with repetitive low-force exercise.
J Appl Physiol 98:477–481.
26. Keller C, Steensberg A, Hansen AK, Fischer CP, Plomgaard P, Pedersen BK. 2005.
Effect of exercise, training, and glycogen availability on IL-6 receptor expression in
human skeletal muscle. J Appl Physiol 99:2075–2079.
27. Schindler R, Mancilla J, Endres S, Ghorbani R, Clark SC, Dinarello CA. 1990.
Correlations and interactions in the production of interleukin-6 (IL-6), IL-1, and tumor
necrosis factor (TNF) in human blood mononuclear cells: IL-6 suppresses IL-1 and
TNF. Blood 75:40–47.
28. Starkie R, Ostrowski SR, Jauffred S, Febbraio M, Pedersen BK. 2003. Exercise and
IL-6 infusion inhibit endotoxin-induced TNF-alpha production in humans. FASEB J
17:884–886.
29. Mizuhara H, O’Neill E, Seki N, Ogawa T, Kusunoki C, Otsuka K et al. 1994. T cell
activation-associated hepatic injury: Mediation by tumor necrosis factors and protection
by interleukin 6. J Exp Med 179:1529–1537.
30. Handschin C, Spiegelman BM. 2008. The role of exercise and PGC1[alpha] in inflam-
mation and chronic disease. Nature 454:463–469.
31. Lutz CT, Quinn LS. 2012. Sarcopenia, obesity, and natural killer cell immune senes-
cence in aging: Altered cytokine levels as a common mechanism. Aging (Albany NY)
4:535–546.
32. Riechman SE, Balasekaran G, Roth SM, Ferrell RE. 2004. Association of interleukin-15
protein and interleukin-15 receptor genetic variation with resistance exercise training
responses. J Appl Physiol 97:2214–2219.
33. Nielsen AR, Pedersen BK. 2007. The biological roles of exercise-induced cytokines:
IL-6, IL-8, and IL-15. Appl Physiol Nutr Metab 32:833–839.
34. Nielsen AR, Mounier R, Plomgaard P, Mortensen OH, Penkowa M, Speerschneider T,
Pilegaard H et al. 2007. Expression of interleukin-15 in human skeletal muscle effect of
exercise and muscle fibre type composition. J Physiol 584:305–312.
35. Nielsen AR, Hojman P, Erikstrup C, Fischer CP, Plomgaard P, Mounier R et al. 2008.
Association between IL-15 and obesity: IL-15 as a potential regulator of fat mass. J Clin
Endocrinol Metab 93:4486–4493.
36. Barra NG, Reid S, MacKenzie R, Werstuck G, Trigatti BL, Richards C et al. 2010.
Interleukin-15 contributes to the regulation of murine adipose tissue and human adipo-
cytes. Obesity (Silver Spring) 18:1601–1607.
37. Barra NG, Chew MV, Holloway AC, Ashkar AA. 2012. Interleukin-15 treatment
improves glucose homeostasis and insulin sensitivity in obese mice. Diabetes Obes
Metab 14:190–193.
38. Quinn LS, Anderson BG, Strait-Bodey L, Stroud AM, Argiles JM. 2009. Oversecretion
of interleukin-15 from skeletal muscle reduces adiposity. Am J Physiol Endocrinol
Metab 296:E191–E202.
39. Boström P, Wu J, Jedrychowski MP, Korde A, Ye L, Lo JC et al. 2012. A PGC1-α-
dependent myokine that drives brown-fat-like development of white fat and thermogen-
esis. Nature 481:463–468.
40. Pischon T, Boeing H, Hoffmann K, Bergmann M, Schulze MB, Overvad K et al.
2008. General and abdominal adiposity and risk of death in Europe. New Engl J Med
359:2105–2120.
41. Whitmer RA, Gustafson DR, Barrett-Connor E, Haan MN, Gunderson EP, Yaffe K.
2008. Central obesity and increased risk of dementia more than three decades later.
Neurology 71:1057–1064.
42. Haffner SM. 2007. Abdominal adiposity and cardiometabolic risk: Do we have all the
answers? Am J Med 120:S10–S16.
43. Bays HE. 2009. “Sick fat”, metabolic disease, and atherosclerosis. Am J Med
122:S26–S37.
44. Giovannucci E. 2007. Metabolic syndrome, hyperinsulinemia, and colon cancer: A
review. Am J Clin Nutr 86:s836–s842.
45. Xue F, Michels KB. 2007. Diabetes, metabolic syndrome, and breast cancer: A review
of the current evidence. Am J Clin Nutr 86:s823–s835.
46. Yudkin JS. 2007. Inflammation, obesity, and the metabolic syndrome. Horm Metab Res
39:707–709.
47. Festa A, D’Agostino Jr R, Tracy RP, Haffner SM. 2002. Elevated levels of acute phase
proteins and plasminogen activator inhibitor-1 predict the development of type 2 diabe-
tes: The insulin resistance atherosclerosis study. Diabetes 51:1131–1137.
48. Petersen M, Pedersen BK. 2005. The anti-inflammatory effect of exercise. J Appl
Physiol 98:1154–1162.
49. Zbinden-Foncea H, Raymackers JM, Deldicque L, Renard P, Francaux M. 2012. TLR2
and TLR4 activate p38 MAPK and JNK during endurance exercise in skeletal muscle.
Med Sci Sports Exerc 44:1463–1472.
50. Frisard MI, McMillan RP, Marchand J, Wahlberg KA, Wu Y, Voelker KA et al. 2010.
Toll-like receptor 4 modulates skeletal muscle substrate metabolism. Am J Physiol
Endocrinol Metab 298(5):E988–E998.
51. Reyna SM, Ghosh S, Tantiwong P, Meka CS, Eagan P, Jenkinson CP et al. 2008. Elevated
toll-like receptor 4 expression and signaling in muscle from insulin-resistant subjects.
Diabetes 57:2595–2602.
52. Jackman RW, Kandarian SC. 2004. The molecular basis of skeletal muscle atrophy. Am
J Physiol Cell Physiol 287:C834–C843.
53. Kandarian SC, Jackman RW. 2006. Intracellular signaling during skeletal muscle atro-
phy. Muscle Nerve 33:155–165.
54. Kandarian SC, Stevenson EJ. 2002. Molecular events in skeletal muscle during disuse
atrophy. Exerc Sport Sci Rev 30:111–116.
55. Ventadour S, Attaix D. 2006. Mechanisms of skeletal muscle atrophy. Curr Opin
Rheumatol 18:631–635.
56. Tisdale MJ. 2004. Cancer cachexia. Langenbecks Arch Surg 389:299–305.
57. McKinnell IW, Rudnicki MA. 2004. Molecular mechanisms of muscle atrophy. Cell
119:907–910.
58. Guttridge DC. 2004. Signaling pathways weigh in on decisions to make or break skel-
etal muscle. Curr Opin Clin Nutr Metab Care 7:443–450.
59. Kumar A, Takada Y, Boriek AM, Aggarwal BB. 2004. Nuclear factor-kappaB: Its role in
health and disease. J Mol Med (Berl) 82:434–448.
60. Hunter RB, Stevenson E, Koncarevic A, Mitchell-Felton H, Essig DA, Kandarian SC.
2002. Activation of an alternative NF-kappa B pathway in skeletal muscle during disuse
atrophy. FASEB J 16:529–538.
61. Cai D, Frantz JD, Tawa NE Jr, Melendez PA, Oh BC, Lidov HG et al. 2004. IKKbeta/
NF-kappaB activation causes severe muscle wasting in mice. Cell 119:285–298.
62. Pedersen BK, Fischer CP. 2007. Physiological roles of muscle-derived interleukin-6 in
response to exercise. Curr Opin Clin Nutr Metab Care 10(3):265–271.
16 Approaches to Reducing
Abdominal Obesity
CONTENTS
Introduction............................................................................................................. 259
Significance of Visceral Fat....................................................................................260
Origins of Visceral Fat............................................................................................ 261
Hormonal Influences on Abdominal Visceral Fat................................................... 262
Testosterone........................................................................................................ 262
Estrogen............................................................................................................. 262
Cortisol...............................................................................................................264
Endocannabinoids.............................................................................................. 265
Growth Hormone...............................................................................................266
Dietary Fructose......................................................................................................266
Correcting Underlying Hormonal Abnormalities................................................... 267
Iatrogenic Hormones and Obesity: Insulin and Glucocorticoids....................... 267
Testosterone........................................................................................................ 267
Growth Hormone............................................................................................... 268
Exercise and Diet Interventions to Reduce Visceral Adiposity............................... 268
Conclusion.............................................................................................................. 269
References............................................................................................................... 269
INTRODUCTION
Excess intra-abdominal adipose tissue accumulation, often termed visceral obesity,
is the critical target for medical and public health efforts to reduce the systemic
inflammation related to the global epidemic of obesity-associated metabolic disor-
ders. Visceral obesity is characterized by ectopic triglyceride (TG) storage closely
related to clustering risk factors for chronic age-related disorders including heart
disease, diabetes, liver disease, and common forms of cancer. Hypertriglyceridemia,
liver insulin resistance, inflammation of the liver, increased liver VLDL synthesis
and secretion, reduced TG-rich lipoprotein clearance, small dense LDL particles,
reduced HDL cholesterol, and increased circulating adipocytokines are among the
many metabolic alterations that characterize this common condition. Age, gender,
genetics, and ethnicity contribute to the observed variations in visceral adipose tis-
sue accumulation in different populations globally. Efforts to bring all obesity under
259
the rubric of body mass index have failed in much of Asia, where there will be a sig-
nificant expansion of the epidemic of type 2 diabetes mellitus among individuals not
classified as either overweight or obese by Western BMI criteria. Attempts to adjust
BMI criteria to lower levels have largely been without success since they do not deal
with the underlying problem of visceral obesity.
Specific mechanisms involved in increasing visceral fat storage when there is
positive energy balance include angiogenesis, sex hormone imbalance, local enzy-
matic cortisol production in abdominal adipose tissues, endocannabinoid actions,
growth hormone (GH) deficiency, and excessive dietary fructose. Physiological char-
acteristics of abdominal adipose tissues such as adipocyte size and number, lipolytic
responsiveness, lipid storage capacity, and inflammatory cytokine production are
significant correlates and even possible determinants of the increased chronic disease
risk factors associated with visceral obesity.
Estrogen replacement in postmenopausal women, and testosterone replacement
in androgen-deficient men, has been shown to favorably impact body fat distribution
while also reducing risk factors. However, hormonal therapies have been associated
with serious side effects, making them useful for only some individuals. However,
they cannot be endorsed as widespread public health answers to the problem of
visceral obesity. Lifestyle interventions leading to weight loss generally induce
preferential mobilization of visceral fat.
In clinical practice, measuring waist circumference in addition to the body mass
index is somewhat helpful for the identification and management of a subgroup of
overweight or obese patients at higher risk of chronic diseases. However, newer
methods of visualizing intra-abdominal fat, including magnetic resonance imaging,
have shown that even individuals with normal body weight and waist circumference
can have excess intra-abdominal fat. Recognizing the beneficial impacts of mus-
cle lipoprotein lipase on circulating TGs and of muscle on the insulin-independent
uptake of glucose from the circulation, a combined program of resistance exercise to
build muscle combined with balanced nutrition including adequate protein to main-
tain muscle mass may represent the best approach to reducing intra-abdominal fat.
However, documenting the benefits of this approach will require extensive and care-
fully designed clinical trials.
SIGNIFICANCE OF VISCERAL FAT

Obesity is defined by an excess of body fat. Population studies have clearly estab-
lished the link between obesity defined by the BMI and comorbidities associated
with obesity as well as mortality risk. Several organizations use categories of BMI to
define underweight, normal weight, overweight, and various classes of obesity [1,2].
Although the BMI is an adequate tool to report population trends in the prevalence
of obesity [3], it has serious limitations when applied to individuals. Overweight or
obese subjects as a group are clearly at greater risk for comorbid conditions com-
pared with normal weight individuals, but physicians have been perplexed by the
fact that while some obese patients clearly show complications associated with their
excess of body fat, some other equally obese patients do not display expected meta-
bolic abnormalities despite their significant excess of body fat [4].
Approaches to Reducing Abdominal Obesity 261
A striking example of the limitations of the BMI relates to individuals with sar-
copenic obesity, a common condition associated with low protein intake, sedentary
lifestyle, inflammation, and premature aging [5]. Another example of this concept is
the metabolically obese, normal weight (MONW) subject [6,7]. These MONW indi-
viduals, who have normal BMI values, nonetheless suffer from metabolic compli-
cations commonly found in obese people. Conversely, metabolically healthy obese
(MHO) individuals described by other research groups have a BMI above 30 kg/m2
but are not characterized by insulin resistance or dyslipidemia [8–10]. These obser-
vations suggest that high CVD risk may be observed even below the normal BMI
cutoff of 25 kg/m2. A key factor underpinning the difference in CVD risk between
MONW and MHO individuals is the likely presence of excess visceral adipose tissue
[11–13]. Matsuzawa [14] has demonstrated that very obese individuals with a small
amount of visceral adiposity—active sumo wrestlers, for example—are MHO with
normal insulin sensitivity, whereas retired sedentary sumo wrestlers with greater
amounts of visceral adipose tissue tend to be insulin-resistant, dyslipidemic and have
a high prevalence of metabolic complications such as type 2 diabetes and CVD.
This condition has been called hypertriglyceridemic waist, a simple clinical phe-
notype predictive of excess visceral adiposity [15,16]. Men with both low waist and
TG values were at low risk of being viscerally obese (<20%), while >80% of indi-
viduals with hypertriglyceridemic waist had excess visceral adiposity and related
metabolic abnormalities, including elevated glucose and insulin concentrations as
well as altered blood lipid profiles. The strategies needed to reduce visceral fat, a
condition often invisible to the clinician, are vital to the efforts to curb the global
epidemic of overweight and obesity-associated disorders linked to chronic low-grade
inflammation.
ORIGINS OF VISCERAL FAT

It was once thought that individuals were born with a certain number of fat cells and
that this number could increase until puberty [17]. Following puberty, it was once
thought that these cells could get larger or smaller, but never die. We now know that
humans can grow more fat cells as an adult and that fat tissue is one of the few tissues
in the human body with the ability to considerably expand or shrink after puberty. In
order to grow, the abdominal fat, like other tissues, must grow its own blood supply.
While the pioneer in this field, Dr. Judah Folkman, primarily studied angiogenesis
in tumor growth, he did one study in genetically obese (ob/ob) mice and prevented
the growth of fat tissue with an angiogenesis inhibitor [18].
An extensive vasculature supplies the adipose tissue, and a capillary network sur-
rounds essentially every adipocyte. Like the growth of other tissues, adipose tissue
expansion during increased caloric intake requires angiogenesis. The potential of
adipose tissue to promote angiogenesis is well established and has been used thera-
peutically for at least half a century [19]. Conversely, there is compelling evidence
that treatment of obese mice with pharmacological angiogenesis inhibitors decreases
adipose tissue mass and prevents obesity [18,20,21]. Diet-induced adipose tissue
expansion has been noted in transgenically modified Id3-deficient mice presumably
due to altered angiogenesis.
As abdominal fat grows as the result of an obesogenic diet, the expanding adi-
pose tissue becomes hypoxic. This poor oxygenation results from the tortuous nature
of the new blood vessels formed, a phenomenon that also occurs in tumor tissue.
Both differentiation and hypoxia induce vascular endothelial growth factor (VEGF)
expression by adipocytes [22–24]. Inhibition of VEGF signaling prevents adipose
tissue expansion during diet-induced obesity [25]. Furthermore, diet-induced VEGF
secretion from adipose tissue is attenuated in Id3-deficient mice. These studies sup-
port a defined molecular mechanism by which Id3 can regulate VEGF expression by
the E-protein E12 (see Figure 16.1). Id3 functions to repress the activity of E-proteins
through direct protein binding, preventing the association of E-proteins with target
DNA. These studies provide support for the concept that angiogenesis is critical in
the formation of abdominal visceral fat, and its inhibition may be one strategy to
reduce abdominal fat by inhibiting its formation during weight regain.
HORMONAL INFLUENCES ON ABDOMINAL VISCERAL FAT

Testosterone
While testosterone is a critical steroid hormone in reproductive and sexual func-
tioning, it is also an important hormone in energy balance, metabolic pathways,
maintenance of lean body mass, nitrogen retention, and regulation of adipogenesis
including the visceral fat depot [26–28].
Low testosterone levels are clearly associated with sarcopenic obesity and
increased risk of insulin resistance, metabolic syndrome, and type 2 diabetes in men
at middle age who suffer from hypogonadism [29,30].
The potential of testosterone to improve the metabolic situation in hypogonadal
men has been met with some caution by primary care physicians and urologists due
to the perceived increased disease risk associated with testosterone treatment. For
many decades, testosterone has been perceived by the medical community to play a
key role in the development of prostate cancer (PCa) [31], and the higher prevalence
of coronary heart disease in men, compared with women, has been attributed to
testosterone [32], however recent studies have not found evidence that testosterone
promotes either heart disease [32] or PCa [31]. These perceptions are so common in
the medical community that they are difficult to dispel. Therefore, in this chapter, a
role for testosterone in the treatment of visceral obesity and its associated conditions
in men with testosterone deficiency will necessarily need to be balanced by patients
and doctors with some consideration and discussion of potential perceived risks of
testosterone administration.
Estrogen
Menopause leads to a decline in both estrogen and progesterone production from ova-
ries. These hormonal changes are associated with changes in body composition and
body fat including increased visceral fat [33,34]. Together with the decline in estra-
diol levels, a smaller decline in testosterone levels occurs, leading to an increased
testosterone to estrogen ratio [35]. Women with polycystic ovarian syndrome have a
Lean Obese
Id3 Expression
Id3
E12 E12 binding
Weight gain Capillaries Release of repression

VEGF promoter
E-box
Approaches to Reducing Abdominal Obesity
Angiogenesis
VEGF
Adipose tissue expansion
FIGURE 16.1 Regulation of adipose tissue vascular endothelial growth factor (VEGF) expression and angiogenesis by inhibitor of differentiation-3
(Id3) during diet-induced obesity. In response to high-fat diet feeding, endothelial cells of capillaries within adipose tissue express Id3. Id3 associates
with the E-box protein E12, which prevents E12 binding and transrepression of the VEGF promoter. The ensuing increase in VEGF promoter activity
induces transcription and VEGF secretion, which subsequently facilitates angiogenesis and adipose tissue expansion during obesity.
263
hormonal profile of androgen excess that is related to a high prevalence of cardiovas-

cular risk factors and metabolic syndrome [36–40]. The Study of Women’s Health
across the Nation investigators have shown that the progressive androgenicity (i.e.,
increased levels of bioavailable testosterone and/or decreased levels of sex-hormone-
binding globulin [SHBG]) during the postmenopausal years is a strong predictor
of visceral fat accumulation and may increase the risk for metabolic syndrome and
cardiovascular diseases [39–41]. Serum SHBG level, which determines the bioavail-
ability of sex hormones, decreases after menopause [42,43].
Estrogen promotes the accumulation of subcutaneous fat [44], and the loss of
estrogen with menopause is associated with an absolute and relative increase in
visceral fat to subcutaneous fat [45,46]. Visceral fat varies inversely with estrogen
levels [47]. When estrogen levels become sufficiently low, visceral fat accumula-
tion occurs in females, possibly due to direct effects of estrogen, especially since
progesterone and androgen receptors (PR and AR) as well as estrogen receptor (ER)
are expressed in adipose tissues [48]. Subcutaneous adipose tissue has higher con-
centrations of ER and PR; however, visceral adipose tissue has higher concentrations
of AR [49]. Additionally, subcutaneous adipose tissue has few ARs, and estrogen
downregulates AR expression in subcutaneous fat [50]. Adipose tissue–specific AR
knockout mice have increased intra-adipose estradiol levels, which lead to increased
subcutaneous obesity and hyperleptinemia [51].
Cortisol
Chronic psychological stress can lead to increased cortisol secretion. Low socio-
economic status and job stress, two indicators of chronic stress, are associated with
greater abdominal adiposity in cross-sectional and prospective studies [52–54].
Stress can impact abdominal adiposity through repeated activation of the hypotha-
lamic–pituitary–adrenal (HPA) axis, resulting in hypersecretion of cortisol. Cortisol
can then bind to glucocorticoid receptors (GRs) on fat cells, activating lipoprotein
lipase leading to fat storage in adipocytes [55]. Increases in cortisol in combination
with increased levels of insulin mobilize amino acids and fatty acids from peripheral
to abdominal regions for immediate use by the liver for gluconeogenesis and ketones
for energy use by the brain [56,57]. A greater density of GRs are found on visceral
compared to peripheral fat cells partly explaining why fat stores are redistributed to
intra-abdominal regions in the presence of elevated cortisol [58–60].
Healthy volunteers who exhibit increased cortisol reactivity in response to labo-
ratory stress tasks have greater abdominal adiposity [61–63], and among depressed
postmenopausal women, those with higher morning cortisol have greater levels of
visceral fat as measured by computed tomography compared to those with lower
cortisol levels [64] and healthy controls [65].
In addition to direct effects of chronic stress on abdominal obesity, psychological
stress can also trigger consumption of high- fat and sweet food, leading to overall
weight gain [66–76]. Stress eating may also increase visceral adiposity independent
of total weight gain. The combination of chronic stress and a high fat and sugar
diet markedly increases visceral adipose tissue through stress-mediated upregulation
of neuropeptide Y and its receptors in fat tissues of rodents [77]. Neuropeptide Y
promotes fat angiogenesis and the proliferation and differentiation of new adipo-
cytes. In humans, self-identified stress eaters tend to gain more abdominal fat during
stressful periods compared to nonself-identified stress eaters [78]. However, attempts
to use behavioral therapies such as mindfulness training to reduce stress have not
been successful in also reducing visceral fat while having some impact on stress eat-
ing and cortisol secretion patterns [79].
There are also clinical similarities between obesity and the overactivity of gluco-
corticoids in Cushing’s syndrome. A buffalo hump, which is one of the key findings
in Cushing’s syndrome, also occurs in severe obesity, but circulating cortisol levels
are not elevated in obesity.
There is increasing evidence that altered cortisol metabolism may influence the
development of visceral fat and the metabolic syndrome [80–82]. The key enzyme
responsible for the reversible activation of cortisol, 11b-hydroxysteroid dehydro-
genase (11b-HSD), has been extensively explored with its isoforms, types 1 and 2,
being differently expressed in different organs indicating that cortisol action is tissue
specific. Increased adipose tissue 11b-HSD type 1 expression and activity [83,84]
have been observed in obese individuals.
Correlations between glucocorticoid secretion and other components of the meta-
bolic syndrome, that is, insulin sensitivity, hypertension, and HDL cholesterol, have
also been seen in cross-sectional studies [80,85]. Comprehensive studies have shown
that measurement of urinary-free cortisol and cortisone provides the best estimate of
11b-HSD type 2 activity. This enzyme is mainly expressed in kidneys, protecting the
mineral corticoid receptor against excessive cortisol levels [86].
Urinary cortisol output and serum cortisol concentrations in the steady state mea-
sured under field conditions and during standardized inhibitory and stimulatory tests
in premenopausal obese women have been analyzed in relation to adipose tissue dis-
tribution [87]. Urinary cortisol output was increased under field conditions in women
with an elevated waist-to-hip circumference ratio and, in particular, in women with
a large abdominal sagittal diameter, indicating visceral fat accumulation. However,
dexamethasone inhibition of cortisol secretion was normal. Stimulation with cor-
ticotropin analogue and with physical (cold pressor test) or mental (color-word or
mathematic) stress tests also showed elevated responses of serum cortisol, but not of
prolactin or GH concentrations. It is suggested that women with visceral fat accumu-
lation have elevated cortisol secretion due to an increased sensitivity along the HPA
axis and that this may be causing their abnormal fat depot distribution.
Endocannabinoids
The extensive distribution of the receptors in the endocannabinoid system reflects
a profound complexity of the system regarding human physiology. The effects of
endocannabinoids are mediated via the endocannabinoid receptor 1 (CB1) and 2
(CB2). CB1 is found in the brain, liver, and muscle tissue, whereas CB2 is present in
monocytes as well as other cells of the immune system [88]. Both animal and human
studies have linked the endocannabinoid system with food intake, lipogenesis, and
addictive behavior [89]. Furthermore, the activation of the CB1 may play a role in
the development of insulin resistance in skeletal muscle [90]. In placebo-controlled
clinical trials, treatment with the CB1-selective antagonist rimonabant has resulted
in reduction in body weight and waist circumference and improvement of risk factors
including insulin sensitivity and lipid profile [91–94], and the appetite-suppressant
effect of rimonabant has been shown to depend on CB1 [95]. In contrast, treatment
with a CB1 agonist is associated with an increase in hepatic lipogenesis in wild-type
mice [96].
Growth Hormone
Aging is associated with a progressive decline in GH and IGF-I availability [97–99].
Healthy older individuals with relative reductions in GH and patients with patho-
logical GH deficiency share a similar physical phenotype marked by osteopenia,
sarcopenia, visceral adiposity, insulin resistance, hyperlipidemia, and increased risk
of cardiovascular disease.
In women with increased abdominal adiposity, physiological GH secretion is
impaired and peak stimulated GH response is decreased [99,100]. GH plays a role in
modulating body composition, and GH deficiency in women with hypopituitarism is
associated with increased body fat, including visceral adiposity, and decreased lean
body mass [101]. Visceral adiposity has also been shown to be a major determinant
of GH secretion in nonobese adults [102]. IGF-1, an important modulator of body
composition, is secreted by the liver and other organs in response to GH. As IGF-1
is an important determinant of body composition, particularly critical for the main-
tenance of muscle mass [103], reduced levels in obese premenopausal women may
exert deleterious effects on body.
DIETARY FRUCTOSE
Fructose was in the ancient human diet primarily in fruits, which also contain antioxi-
dants and dietary fiber. With the introduction of cane sugar in the 1600s into the human
diet, table sugar, which is 50% fructose within the sucrose molecule, became a staple
in the human diet. In the 1980s with the subsidization of corn crops, high-fructose corn
syrup (HFCS) with either 42% or 55% fructose was introduced into the diet in large
amounts as a substitute for sucrose. HFCS is added to many foods including ketchup
and breads where consumers do not expect to find added sugar. The food industry has
found that sweetening many foods increases their appeal and sales of processed foods.
As a result, the intake of fructose has increased in parallel to the rise of obesity over
the last 40 years. The intake of fructose has risen from about 37 g/day in the 1970s to
an average of 55 g/day in the 1990s. Adolescents are by far the highest fructose con-
sumers, consuming on average 70 g/day or about 12% of total calories. About one-fifth
of adolescents consume 25% of their total calories as fructose [104,105].
HFCS and sucrose have similar endocrine and metabolic effects [106]. Unlike
the hepatic metabolism of glucose, which principally leads to glycogen synthesis,
the hepatic metabolism of fructose results in sustained elevations in postprandial TG
levels [107–111]. Importantly, increased fructose consumption, particularly in the
form of sugar-sweetened beverages, has been implicated in promoting weight gain
[112–114], and visceral adiposity [115,116], as well as hepatic steatosis [117–120].
CORRECTING UNDERLYING HORMONAL ABNORMALITIES

Iatrogenic Hormones and Obesity: Insulin and Glucocorticoids
Physician’s administration of hormones that commonly increase abdominal fat
include insulin, insulinotropic hypoglycemic drugs, and long-acting glucocorticoids
including prednisone and its analogs. Insulin is commonly overused in the control of
blood sugar in type 2 diabetes mellitus. Excess insulin leads to adipogenesis and accu-
mulation of visceral fat [121]. The important strategy here is to utilize the lowest pos-
sible doses of insulin by employing dietary restriction and exercise as detailed later.
Type 2 diabetes mellitus is a progressive and complex disorder that is difficult to
treat effectively in the long term. The majority of patients are overweight or obese at
diagnosis and will be unable to achieve or sustain near normoglycemia without oral
antidiabetic agents; a sizeable proportion of patients will eventually require insulin
therapy to maintain long-term glycemic control, either as monotherapy or in con-
junction with oral antidiabetic therapy. The frequent need for escalating therapy is
held to reflect progressive loss of islet beta-cell function, usually in the presence of
obesity-related insulin resistance.
There are a number of oral antidiabetic drugs for type 2 diabetes [122]. These
classes include agents that stimulate insulin secretion (e.g., sulfonylureas), enhance
the biological activity of insulinotropic peptides (e.g., DPP4 inhibitors), reduce hepatic
glucose production (biguanides), delay digestion and absorption of intestinal carbo-
hydrate (alpha-glucosidase inhibitors), or improve insulin action (thiazolidinediones).
The United Kingdom Prospective Diabetes Study (UKPDS) demonstrated the benefits
of intensified glycemic control on microvascular complications in newly diagnosed
patients with type 2 diabetes. However, there was no reduction observed in macro-
vascular disease (i.e., cardiovascular events) with either sulfonylureas or insulin. In
the UKPDS, overweight and obese patients randomized to initial monotherapy with
metformin experienced significant reductions in myocardial infarction and diabetes-
related deaths. Metformin did not promote weight gain and had beneficial effects on
several cardiovascular risk factors. Metformin is widely regarded as the drug of choice
for most patients with type 2 diabetes as it reduces or can even eliminate the use of
insulin in many patients. Moreover, intensive lifestyle intervention can be more effec-
tive than even metformin drug therapy, at least in the setting of interventional clinical
trials. Therefore, combining metformin with lifestyle intervention may be an excellent
strategy to reduce visceral fat in patients with type 2 diabetes or prediabetes.
Long-acting glucocorticoids are used in a number of autoimmune disorders
to induce remissions of disease [123]. These drugs lead to muscle atrophy and an
increase in visceral fat. Again, the best strategy is to utilize diet and lifestyle when
possible, especially during remissions from autoimmune disease flares, to minimize
the amounts of glucocorticoids being used.
Testosterone
Testosterone replacement therapy decreases visceral fat area [124,125] and visceral
fat accumulation [126] in middle-aged obese men and aging men with low-normal
testosterone levels.
Growth Hormone
Administration of GH or its secretagogues in older adults for intervals of one to
six months elevates GH and IGF-I concentrations, increases lean body mass, and
decreases intra-abdominal adiposity [127–129]. It appears that while GH decreases
with age, the IGF-1 response to GH is maintained in the liver.
Although the effects of GH administration to decrease visceral adiposity, increase
muscle mass, and improve cardiovascular risk markers, including hsCRP, are well
established in patients with GH deficiency due to hypothalamic/pituitary disorders
[130–132], few studies have been performed administering low-dose GH in other-
wise healthy obese subjects [133–136]. Administration of low-dose GH to obese
men [137,138] and postmenopausal women [133] resulted in decreased visceral fat
mass and improved lipid profiles, suggesting a possible beneficial effect of GH in
healthy subjects with visceral obesity. Abdominal obesity and insulin resistance are
central findings in metabolic syndrome. Since treatment with recombinant human
growth hormone (rhGH) can reduce body fat mass in patients with GH deficiency,
rhGH therapy may also have favorable effects on patients with metabolic syndrome.
However, due to the highly increased risk for type 2 diabetes in these patients, strat-
egies are needed to reduce the antagonistic effect of rhGH against insulin. One
strategy that has been successful in HIV–AIDS patients has been the coadministra-
tion of thiazolidinediones to increase subcutaneous fat and counteract the GH effects
on glucose tolerance [137].
EXERCISE AND DIET INTERVENTIONS TO

REDUCE VISCERAL ADIPOSITY
Interventions that include both diet and physical activity are superior to pharmaceu-
tical interventions in reducing visceral fat in overweight individuals [138]. A num-
ber of randomized studies of aerobic exercise, resistance exercise, or combinations
of both have demonstrated the safety and efficacy of physical activity in reducing
metabolic abnormalities that increase the risk of chronic diseases [139–151]. When
compared, resistance training including weight lifting has been found to be more
effective than aerobic endurance training in reducing chronic disease risk factors
[144,146]. Moreover, combined resistance and aerobic exercise have yielded better
results than either modality alone [141,145].
In a recent study, 100 participants, aged 50–70 years, were provided diets that
restricted calories but provided a protein intake of 1.2 g/kg/day with a high exer-
cise volume of 15–20 h/week. Subjects were randomized to three training groups:
moderate resistance/moderate endurance, high resistance/moderate endurance, or
moderate resistance/high endurance. A total of 78 subjects completed the one-year
program. Visceral fat loss was greatest in the high-resistance/moderate-endurance
group (−18%, p < 0.0001) and higher in the moderate-resistance/high-endurance
group than in the moderate-resistance/moderate-endurance group (−12% vs. −7%,
p < 0.0001). In this study, increased intensity in high-volume training was more effi-
cient in improving visceral fat loss, and high-intensity resistance training induced a
faster visceral fat loss than moderate interventions [152].
There is inconsistency in the literature on the effects of exercise combined with

diet, and this may be due in part to the low intensity of exercises prescribed in these
studies [153]. It has been known for some time that simple walking or mild aerobic
exercise suitable for cardiac rehabilitation during diet restriction does not enhance
short-term weight loss [154]. However, regular exercise for 200 min/week is the most
reliable correlate of long-term weight maintenance. These effects of consistent daily
exercise of 30–60 min/day as recommended by many institutions and governments
may simply be effective by increasing adherence once subjects are acclimated to
regular exercise [155]. However, it is clear that to reduce visceral fat optimally, a
combination of high-intensity aerobic and resistance training is needed.
CONCLUSION
Visceral fat is a key organ in the genesis of the chronic low-grade inflammation
associated with obesity and diabetes and that has so many organ-specific disease
implications outlined in this text. The correction of hormonal factors, dietary fac-
tors, physical activity, and inhibition of angiogenesis are all reasonable targets for the
prevention of increased visceral fat. The measurement of excess fat and its targeting
as the goal of weight management efforts promises to have a major impact on the
global epidemic of obesity-associated inflammatory diseases discussed in this text.
REFERENCES
1. Expert Consultation. 2004. Appropriate body-mass index for Asian populations and its
implications for policy and intervention strategies. Lancet. 363:157–163.
2. World Health Organization. 2000. Obesity: Preventing and Managing the Global
Epidemic: Report of a WHO Consultation. Geneva, Switzerland: World Health
Organization (WHO technical report series 894).
3. Keys A, Fidanza F, Karvonen MJ, Kimura N, Taylor HL. 1972. Indices of relative weight
and obesity. J Chronic Dis. 25:329–343.
4. Wildman RP, Muntner P, Reynolds K et al. 2008. The obese without cardiometabolic
risk factor clustering and the normal weight with cardiometabolic risk factor cluster-
ing: Prevalence and correlates of 2 phenotypes among the US population (NHANES
1999–2004). Arch Intern Med. 168:1617–1624.
5. Heber D, Ingles S, Ashley JM, Maxwell MH, Lyons RF, Elashoff RM. 1996. Clinical
detection of sarcopenic obesity by bioelectrical impedance analysis. Am J Clin Nutr.
64:472S–477S.
6. Ruderman NB, Berchtold P, Schneider S. 1982. Obesity-associated disorders in normal
weight individuals: Some speculations. Int J Obes. 6(Suppl. 1):151–157.
7. Ruderman NB, Schneider SH, Berchtold P. 1981. The “metabolically-obese,” normal
weight individual. Am J Clin Nutr. 34:1617–1621.
8. Bonora E, Kiechl S, Willeit J et al. 1998. Prevalence of insulin resistance in metabolic
disorders: The Bruneck Study. Diabetes. 47:1643–1649.
9. Ferrannini E, Natali A, Bell P, Cavallo-Perin P, Lalic N, Mingrone G. 1997. Insulin resis-
tance and hypersecretion in obesity. European Group for the Study of Insulin Resistance
(EGIR). J Clin Invest. 100:1166–1173.
10. Karelis AD, St-Pierre DH, Conus F, Rabasa-Lhoret R, Poehlman ET. 2004. Metabolic
and body composition factors in subgroups of obesity: What do we know? J Clin
Endocrinol Metab. 89:2569–2575.
11. Després JP, Nadeau A, Tremblay A et al. 1989. Role of deep abdominal fat in the asso-
ciation between regional adipose tissue distribution and glucose tolerance in obese
women. Diabetes. 38:304–309.
12. Fujioka S, Matsuzawa Y, Tokunaga K, Tarui S. 1987. Contribution of intra-abdominal
fat accumulation to the impairment of glucose and lipid metabolism in human obesity.
Metabolism. 36:54–59.
13. Sparrow D, Borkan GA, Gerzof SG, Wisniewski C, Silbert CK. 1986. Relationship of
fat distribution to glucose tolerance. Results of computed tomography in male partici-
pants of the Normative Aging Study. Diabetes. 35:411–415.
14. Matsuzawa Y. 1997. Pathophysiology and molecular mechanisms of visceral fat syn-
drome: The Japanese experience. Diabetes Metab Rev. 13:3–13.
15. Lemieux I, Pascot A, Couillard C et al. 2000. Hypertriglyceridemic waist: A marker of
the atherogenic metabolic triad (hyperinsulinemia; hyperapolipoprotein B; small, dense
LDL) in men? Circulation. 102:179–184.
16. Lemieux I, Poirier P, Bergeron J et al. 2007. Hypertriglyceridemic waist: A useful
screening phenotype in preventive cardiology? Can J Cardiol. 23:23B–31B.
17. Salans LB, Cushman SW, Weismann RE. 1973. Studies of human adipose tissue.
Adipose cell size and number in nonobese and obese patients. J Clin Invest. 52:
929–941.
18. Rupnick MA, Panigrahy D, Zhang CY et al. 2002. Adipose tissue mass can be regulated
through the vasculature. Proc Natl Acad Sci USA. 99:10730–10735.
19. Vineberg AM, Baichwal KS, Myers J. 1965. Treatment of acute myocardial infarction
by epicardiectomy and free omental graft. Surgery. 57:836–838.
20. Cutchins A, Harmon DB, Kirby JL et al. 2012. Inhibitor of differentiation-3 medi-
ates high fat diet-induced visceral fat expansion. Arterioscler Thromb Vasc Biol.
32:317–324.
21. Lyden D, Young AZ, Zagzag D et al. 1999. Id1 and Id3 are required for neurogenesis,
angiogenesis and vascularization of tumour xenografts. Nature. 401:670–677.
22. Kabon B, Nagele A, Reddy D et al. 2004. Obesity decreases perioperative tissue oxy-
genation. Anesthesiology. 100:274–280.
23. Claffey KP, Wilkison WO, Spiegelman BM. 1992. Vascular endothelial growth factor.
Regulation by cell differentiation and activated second messenger pathways. J Biol
Chem. 267:16317–16322.
24. Zhang QX, Magovern CJ, Mack CA, Budenbender KT, Ko W, Rosengart TK. 1997.
Vascular endothelial growth factor is the major angiogenic factor in omentum:
Mechanism of the omentum-mediated angiogenesis. J Surg Res. 67:147–154.
25. Tam J, Duda DG, Perentes JY, Quadri RS, Fukumura D, Jain RK. 2009. Blockade of
VEGFR2 and not VEGFR1 can limit diet-induced fat tissue expansion: Role of local
versus bone marrow-derived endothelial cells. PLoS One. 4:e4974.
26. Traish AM, Feeley RJ, Guay A. 2009. Mechanisms of obesity and related pathologies:
Androgen deficiency and endothelial dysfunction may be the link between obesity and
erectile dysfunction. FEBS J. 276:5755–5767.
27. Singh R, Artaza JN, Taylor WE et al. 2006. Testosterone inhibits adipogenic differ-
entiation in 3T3-L1 cells: Nuclear translocation of androgen receptor complex with
beta-catenin and T-cell factor 4 may bypass canonical Wnt signaling to down-regulate
adipogenic transcription factors. Endocrinology. 147:141–154.
28. Traish AM, Toselli P, Jeong SJ, Kim NN. 2005. Adipocyte accumulation in penile cor-
pus cavernosum of the orchiectomized rabbit: A potential mechanism for veno-occlusive
dysfunction in androgen deficiency. J Androl. 26:242–248.
29. Grossmann M, Thomas MC, Panagiotopoulos S et al. 2008. Low testosterone levels are
common and associated with insulin resistance in men with diabetes. J Clin Endocrinol
Metab. 93:1834–1840.
30. Bhasin S, Cunningham GR, Hayes FJ et al. 2010. Testosterone therapy in men with
androgen deficiency syndromes: An Endocrine Society clinical practice guideline.
J Clin Endocrinol Metab. 95:2536–2559.
31. Morgentaler A, Traish AM. 2009. Shifting the paradigm of testosterone and prostate
cancer: The saturation model and the limits of androgen-dependent growth. Eur Urol.
55:310–320.
32. Fernandez-Balsells MM, Murad MH, Lane M et al. 2010. Clinical review 1: Adverse
effects of testosterone therapy in adult men: A systematic review and meta-analysis.
33. Ley CJ, Lees B, Stevenson JC. 1992. Sex- and menopause-associated changes in body-
fat distribution. Am J Clin Nutr. 55:950–954.
34. Tchernof A, Desmeules A, Richard C et al. 2004. Ovarian hormone status and abdominal
visceral adipose tissue metabolism. J Clin Endocrinol Metab. 89:3425–3430.
35. Lamberts SW, van den Beld AW, van der Lely AJ. 1997. The endocrinology of aging.
Science. 278:419–424.
36. Giallauria F, Orio F, Palomba S, Lombardi G, Colao A, Vigorito C. 2008. Cardiovascular
risk in women with polycystic ovary syndrome. J Cardiovasc Med (Hagerstown).
9:987–992.
37. Hoffman LK, Ehrmann DA. 2008. Cardiometabolic features of polycystic ovary
syndrome. Nat Clin Pract Endocrinol Metab. 4:215–222.
38. Guthrie JR, Dennerstein L, Taffe JR et al. 2003. Central abdominal fat and endogenous
hormones during the menopausal transition. Fertil Steril. 79:1335–1340.
39. Janssen I, Powell LH, Crawford S, Lasley B, Sutton-Tyrrell K. 2008. Menopause and
the metabolic syndrome: The Study of Women’s Health across the Nation. Arch Intern
Med. 168:1568–1575.
40. Janssen I, Powell LH, Kazlauskaite R, Dugan SA. 2010. Testosterone and visceral fat in
midlife women: The Study of Women’s Health across the Nation (SWAN) fat patterning
study. Obesity (Silver Spring). 18:604–610.
41. Sutton-Tyrrell K, Wildman RP, Matthews KA et al. 2005. Sex-hormonebinding globulin
and the free androgen index are related to cardiovascular risk factors in multiethnic
premenopausal and perimenopausal women enrolled in the Study of Women across the
Nation (SWAN). Circulation. 111:1242–1249.
42. Burger HG, Dudley EC, Cui J, Dennerstein L, Hopper JL. 2000. A prospective
longitudinal study of serum testosterone, dehydroepiandrosterone sulfate, and sex
hormone-binding globulin levels through the menopause transition. J Clin Endocrinol
Metab. 85:2832–2838.
43. Maggio M, Lauretani F, Basaria S et al. 2008. Sex hormone binding globulin levels
across the adult lifespan in women V the role of body mass index and fasting insulin.
J Endocrinol Invest. 31:597–601.
44. Krotkiewski M, Bjorntorp P, Sjostrom L, Smith U. 1983. Impact of obesity on metabo-
lism in men and women. Importance of regional adipose tissue distribution. J Endocrinol
Invest. 72:1150–1162.
45. Lee CG, Carr MC, Murdoch SJ et al. 2009. Adipokines, inflammation, and visceral
adiposity across the menopausal transition: A prospective study. J Clin Endocrinol
Metab. 94:1104–1110.
46. Poehlman ET, Toth MJ, Gardner AW. 1995. Changes in energy balance and body com-
position at menopause: A controlled longitudinal study. Ann Intern Med. 123:673–675.
47. Bouchard C, Despres JP, Mauriege P. 1993. Genetic and nongenetic determinants of
regional fat distribution. Endocrine Rev. 14:72–93.
48. Crandall DL, Busler DE, Novak TJ, Weber RV, Kral JG. 1998. Identification of estro-
gen receptor beta RNA in human breast and abdominal subcutaneous adipose tissue.
Biochem Biophys Res Commun. 248:523–526.
49. Lu SF, McKenna SE, Cologer-Clifford A, Nau EA, Simon NG. 1998. Androgen receptor
in mouse brain: Sex differences and similarities in autoregulation. Endocrinology.
139:1594–1601.
50. Bjorntorp P. 1997. Hormonal control of regional fat distribution. Human Reprod. 12
(Suppl 1):21–25.
51. Yu IC, Lin HY, Liu NC et al. 2008. Hyperleptinemia without obesity in male mice lack-
ing androgen receptor in adipose tissue. Endocrinology. 149:2361–2368.
52. Brunner EJ, Marmot MG, Nanchahal K et al. 1997. Social inequality in coronary risk:
Central obesity and the metabolic syndrome. Evidence from the Whitehall II study.
Diabetologia. 40:1341–1349.
53. Brunner EJ, Chandola T, Marmot MG. 2007. Prospective effect of job strain on general
and central obesity in the Whitehall II study. Am J Epidemiol. 165:828–837.
54. Rosmond R, Bjorntorp P. 2000. Occupational status, cortisol secretory pattern, and vis-
ceral obesity in middle-aged men. Obes Res. 8:445–450.
55. Rosmond R. 2003. Stress induced disturbances of the HPA axis: A pathway to type 2
diabetes? Med Sci Monitor. 9:RA35–RA39.
56. Dallman MF, Pecoraro NC, La Fleur SE. 2005. Chronic stress and comfort foods: Self-
medication and abdominal obesity. Brain Behav Immun. 19:275–280.
57. Peters A, Schweiger U, Pellerin L et al. 2004. The selfish brain: Competition for energy
resources. Neurosci Biobehav Rev. 28:143–180.
58. Dallman MF, Pecoraro N, Akana SF et al. 2003. Chronic stress and obesity: A new view
of “comfort food.” Proc Nat Acad Sci USA. 100:11696–11701.
59. Rebuffe-Scrive M, Walsh UA, McEwen B, Rodin J. 1991. Effect of chronic stress and
exogenous glucocorticoids on regional fat distribution and metabolism. Physiol Behav.
52:583–590.
60. Rebuffe-Scrive M, Bronnegard M, Nilsson A, Eldh J, Gustafsson JA, Bjorntorp P.
1990. Steroid hormone receptors in human adipose tissues. J Clin Endocrinol Metab.
71:1215–1219.
61. Epel ES, McEwen B, Seeman T et al. 2000. Stress and body shape: Stress-induced cor-
tisol secretion is consistently greater among women with central fat. Psychosom Med.
62:623–632.
62. Epel EE. 1999. Stress-induced cortisol, mood, and fat distribution in men. Obes Res.
7:9–15.
63. Gluck ME, Geliebter A, Lorence M. 2004. Cortisol stress response is positively cor-
related with central obesity in obese women with Binge Eating Disorder (BED) before
and after cognitive-behavioral treatment. Ann NY Acad Sci. 1032:202–207.
64. Weber-Hamann B, Hentschel F, Kniest A et al. 2002. Hypercortisolemic depression is
associated with increased intraabdominal fat. Psychosom Med. 64:274–277.
65. Thakore JH, Richards PJ, Reznek RH, Martin A, Dinan TG. 1997. Increased intra-
abdominal fat deposition in patients with major depressive illness as measured by com-
puted tomography. Biol Psychiatry. 41:1140–1142.
66. Ng DM Jeffery RW. 2003. Relationships between perceived stress and health behaviors
in a sample of working adults. Health Psychol. 22:638–642.
67. McCann BS, Warnick GR, Knopp RH. 1990. Changes in plasma lipids and dietary
intake accompanying shifts in perceived workload and stress. Psychosom Med.
52:97–108.
68. Grunberg NE, Straub RO. 1992. The role of gender and taste class in the effects of stress
on eating. Health Psychol. 11:97–100.
69. Cartwright M, Wardle J, Steggles N, Simon AE, Croker H, Jarvis MJ. 2003. Stress and
dietary practices in adolescents. Health Psychol. 22:362–369.
70. Oliver G, Wardle J, Gibson EL. 2000. Stress and food choice: A laboratory study.
Psychosom Med. 62:853–865.
71. Epel E, Lapidus R, McEwen B, Brownell K. 2001. Stress may add bite to appetite
in women: A laboratory study of stress induced cortisol and eating behavior.
Psychoneuroendocrinology. 26:37–49.
72. Roemmich JN, Wright SM, Epstein LH. 2002. Dietary restraint and stress-induced
snacking in youth. Obes Res. 10:1120–1126.
73. Kivimaki M, Head J, Ferrie JE et al. 2006. Work stress, weight gain and weight loss:
Evidence for bidirectional effects of job strain on body mass index in the Whitehall II
study. Int J Obes. 30:982–987.
74. Korkeila M, Kaprio J, Rissanen A, Koskenvuo M, Sorensen TIA. 1998. Predictors of
major weight gain in adult Finns: Stress, life satisfaction and personality traits. Int J
Obes. 22:949–957.
75. Kouvonen A, Kivimaki M, Cox SJ, Cox T, Vahtera J. 2005. Relationship between work
stress and body mass index among 45,810 female and male employees. Psychosom.
Med. 67:577–583.
76. Pickett KE, Kelly S, Brunner E, Lobstein T, Wilkinson RG. 2005. Wider income gaps,
wider waistbands? An ecological study of obesity and income inequality. J Epidemiol
Comm Health. 59:670–674.
77. Kuo LE, Kitlinska JB, Tilan JU et al. 2007. Neuropeptide Y acts directly in the periph-
ery on fat tissue and mediates stress induced obesity and metabolic syndrome. Nat
Med.13:803–811.
78. Epel E, Jimenez S, Brownell K, Stroud L, Stoney C, Niaura R. 2004. Are stress eaters at
risk for the metabolic syndrome? Ann NY Acad Sci. 1032:208–210.
79. Daubenmier J, Kristeller J, Hecht FM et al. 2011. Mindfulness intervention for stress
eating to reduce cortisol and abdominal fat among overweight and obese women: An
exploratory randomized controlled study. J Obes. 2011:651936.
80. Shen W, Punyanitya M, Silva AM et al. 2009. Sexual dimorphism of adipose tissue dis-
tribution across the lifespan: A cross-sectional whole-body magnetic resonance imaging
study. Nutr Metab. 16:6.
81. Rask E, Olsson T, Soderberg S et al. 2001. Tissue-specific dysregulation of cortisol
metabolism in human obesity. J Clin Endocrinol Metab. 86:1418–1421.
82. Walker BR, Soderberg S, Lindahl B, Olsson T. 2000. Independent effects of obesity
and cortisol in predicting cardiovascular risk factors in men and women. J Int Med.
247:198–204.
83. Rask E, Walker BR, Soderberg S et al. 2002. Tissue-specific changes in peripheral corti-
sol metabolism in obese women: Increased adipose 11 beta-hydroxysteroid dehydroge-
nase type 1 activity. J Clin Endocrinol Metab. 87:3330–3336.
84. Livingstone DEW, Jones GC, Smith K et al. 2000. Understanding the role of gluco-
corticoids in obesity: Tissue-specific alterations of corticosterone metabolism in obese
Zucker rats. Endocrinology. 141:560–563.
85. Hughes KA, Webster SP, Walker BR. 2008. 11-Beta-hydroxysteroid dehydrogenase
type 1 (11 beta-HSD1) inhibitors in type 2 diabetes mellitus and obesity. Exp Opin
Invest Drugs. 17:481–496.
86. Andersson T, Simonyte K, Andrew R et al. 2009. Tissue-specific increases in 11 beta-
hydroxysteroid dehydrogenase type 1 in normal weight postmenopausal women. PLoS
One. 29:12.
87. Ljung T, Andersson B, Bengtsson BA, Björntorp P, Mårin P. 1996. Inhibition of cortisol
secretion by dexamethasone in relation to body fat distribution: A dose-response study.
Obes Res. 4:277–282.
88. Howlett AC, Barth F, Bonner TI et al. 2002. International Union of Pharmacology.
XXVII. Classification of cannabinoid receptors. Pharmacol Rev. 54:161–202.
89. Matias I, Di Marzo V. 2007. Endocannabinoids and the control of energy balance.
Trends Endocrinol Metab. 18:27–37.
90. Eckardt K, Sell H, Taube A et al. 2009. Cannabinoid type 1 receptors in human skeletal
muscle cells participate in the negative crosstalk between fat and muscle. Diabetologia.
52:664–674.
91. Van Gaal LF, Scheen AJ, Rissanen AM, Rossner S, Hanotin C, Ziegler O. 2008. Long-
term effect of CB1 blockade with rimonabant on cardiometabolic risk factors: Two year
results from the RIO-Europe Study. Eur Heart J. 29:1761–1771.
92. Despres JP, Golay A, Sjostrom L. 2005. Effects of rimonabant on metabolic risk factors
in overweight patients with dyslipidemia. N Engl J Med. 353:2121–2134.
93. Pi-Sunyer FX, Aronne LJ, Heshmati HM, Devin J, Rosenstock J. 2006. Effect of
rimonabant, a cannabinoid-1 receptor blocker, on weight and cardiometabolic risk fac-
tors in overweight or obese patients: RIO-North America: A randomized controlled trial.
JAMA. 295:761–775.
94. Scheen AJ, Finer N, Hollander P, Jensen MD, Van Gaal LF. 2006. Efficacy and tolerabil-
ity of rimonabant in overweight or obese patients with type 2 diabetes: A randomised
controlled study. Lancet. 368:1660–1672.
95. Di Marzo V, Goparaju SK, Wang L, Liu J, Bátkai S, Járai Z et al. 2001. Leptin-regulated
endocannabinoids are involved in maintaining food intake. Nature. 410:822–825.
96. Osei-Hyiaman D, DePetrillo M, Pacher P et al. 2005. Endocannabinoid activation at
hepatic CB1 receptors stimulates fatty acid synthesis and contributes to diet-induced
obesity. J Clin Invest. 115:1298–1305.
97. Finkelstein JW, Roffwarg HP, Boyar RM, Kream J, Hellman L. 1972. Age related change
in the twenty-four-hour spontaneous secretion of growth hormone. J Clin Endocrinol
Metab. 35:665–670
98. Weltman A, Weltman JY, Hartman ML et al. 1994. Relationship between age, percent-
age body fat, fitness, and 24-hour growth hormone release in healthy young adults:
Effects of gender. J Clin Endocrinol Metab. 78:543–548.
99. Pijl H, Langendonk JG, Burggraaf J, Frolich M, Cohen AF, Veldhuis JD et al. 2001.
Altered neuroregulation of GH secretion in viscerally obese premenopausal women.
100. Utz AL, Yamamoto A, Hemphill L, Miller KK. 2008. Growth hormone deficiency by
growth hormone releasing hormone-arginine testing criteria predicts increased cardio-
vascular risk markers in normal young overweight and obese women. J Clin Endocrinol
Metab. 93:2507–2514.
101. Weaver JU, Monson JP, Noonan K, John WG, Edwards A, Evans KA et al. 1995.
The effect of low dose recombinant human growth hormone replacement on regional
fat distribution, insulin sensitivity, and cardiovascular risk factors in hypopituitary
adults. J Clin Endocrinol Metab. 80:153–159.
102. Vahl N, Jorgensen JO, Skjaerbaek C, Veldhuis JD, Orskov H, Christiansen JS. 1997.
Abdominal adiposity rather than age and sex predicts mass and regularity of GH secre-
tion in healthy adults. Am J Physiol. 272:E1108–E1116.
103. Rommel C, Bodine SC, Clarke BA et al. 2001. Mediation of IGF-1-induced skeletal
myotube hypertrophy by PI(3)K/Akt/mTOR and PI(3)K/Akt/GSK3 pathways. Nat Cell
Biol. 3:1009–1013.
104. Marriott BP, Olsho L, Hadden L, Connor P. 2010. Intake of added sugars and selected
nutrients in the United States, National Health and Nutrition Examination Survey
(NHANES) 2003–2006. Crit Rev Food Sci Nutr. 50:228–258.
105. Vos MB, Kimmons JE, Gillespie C, Welsh J, Blanck HM. 2008. Dietary fructose con-
sumption among US children and adults: The Third National Health and Nutrition
Examination Survey. Medscape J Med. 10:160.
106. Stanhope KL, Havel PJ. 2008. Endocrine and metabolic effects of consuming beverages
sweetened with fructose, glucose, sucrose, or high-fructose corn syrup. Am J Clin Nutr.
88:1733S–1737S.
107. Abraha A, Humphreys SM, Clark ML, Matthews DR, Frayn KN. 1998. Acute effect
of fructose on postprandial lipaemia in diabetic and non-diabetic subjects. Br J Nutr.
80:169–175.
108. Cohen JC, Schall R. 1988, Reassessing the effects of simple carbohydrates on the serum
triglyceride responses to fat meals. Am J Clin Nutr. 48:1031–1034.
109. Teff KL, Elliott SS, Tschöp M et al. 2004. Dietary fructose reduces circulating insulin
and leptin, attenuates postprandial suppression of ghrelin, and increases triglycerides in
women. J Clin Endocrinol Metab. 89:2963–2972.
110. Stanhope KL, Griffen SC, Bair BR, Swarbrick MM, Keim NL, Havel PJ. 2008. Twenty-
four-hour endocrine and metabolic profiles following consumption of high-fructose
corn syrup-,sucrose-, fructose-, and glucose-sweetened beverages with meals. Am J Clin
Nutr. 87:1194–1203.
111. Swarbrick MM, Stanhope KL, Elliott SS et al. 2008. Consumption of fructose-sweetened
beverages for 10 weeks increases postprandial triacylglycerol and apolipoprotein-B
concentrations in overweight and obese women. Br J Nutr. 100:947–952.
112. Havel PJ. 2005. Dietary fructose: Implications for dysregulation of energy homeostasis
and lipid/carbohydrate metabolism. Nutr Rev. 63:133–157.
113. Bray GA, Nielsen SJ, Popkin BM. Consumption of high-fructose corn syrup in beverages
may play a role in the epidemic of obesity. Am J Clin Nutr. 79:537–543.
114. Malik VS, Schulze MB, Hu FB. 2006. Intake of sugar-sweetened beverages and weight
gain: A systematic review. Am J Clin Nutr. 84:274–288.
115. Stanhope KL, Havel PJ. 2008. Fructose consumption: Potential mechanisms for its
effects to increase visceral adiposity and induce dyslipidemia and insulin resistance.
Curr Opin Lipidol. 19:16–24.
116. Stanhope KL, Schwarz JM, Keim NL et al. 2009. Consuming fructose-sweetened, not
glucose-sweetened, beverages increases visceral adiposity and lipids and decreases
insulin sensitivity in overweight/obese humans. J Clin Invest. 119:1322–1334.
117. Thuy S, Ladurner R, Volynets V et al. 2008. Nonalcoholic fatty liver disease in humans
is associated with increased plasma endotoxin and plasminogen activator inhibitor 1
concentrations and with fructose intake. J Nutr. 138:1452–1455.
118. Collison KS, Saleh SM, Bakheet RH et al. 2009. Diabetes of the liver: The link between
nonalcoholic fatty liver disease and HFCS-55. Obesity. 17:2003–2013.
119. Lê KA, Ith M, Kreis R et al. 2009. Fructose overconsumption causes dyslipidemia and
ectopic lipid deposition in healthy subjects with and without a family history of type 2
diabetes. Am J Clin Nutr. 89:1760–1765.
120. Lim JS, Mietus-Snyder M, Valente A, Schwarz JM, Lustig RH. 2010. The role of fruc-
tose in the pathogenesis of NAFLD and the metabolic syndrome. Nat Rev Gastroenterol
Hepatol. 7:251–264.
121. McNay EC, Teske JA, Kotz CM et al. 2013. Long-term, intermittent, insulin-induced
hypoglycemia produces marked obesity without hyperphagia or insulin resistance:
A model for weight gain with intensive insulin therapy. Am J Physiol Endocrinol Metab.
304:E131–E138.
122. Krentz AJ, Bailey CJ. 2005. Oral antidiabetic agents: Current role in type 2 diabetes
mellitus. Drugs. 65:385–411.
123. Manaboriboon B, Silverman ED, Homsanit M, Chui H, Kaufman M. 2013. Weight
change associated with corticosteroid therapy in adolescents with systemic lupus
erythematosus. Lupus. 22:164–170.
124. Schroeder ET, Zheng L, Ong MD et al. 2004. Effects of androgen therapy on adipose
tissue and metabolism in older men. J Clin Endocrinol Metab. 89:4863–4872.
125. Svartberg J, Agledahl I, Figenschau Y et al. 2008. Testosterone treatment in elderly men
with subnormal testosterone levels improves body composition and BMD in the hip. Int
J Impot Res. 20:378–387.
126. Allan CA, Strauss BJ, Burger HG et al. 2008. Testosterone therapy prevents gain in
visceral adipose tissue and loss of skeletal muscle in nonobese aging men. J Clin
127. Bowers CY, Granda R, Mohan S, Kuipers J, Baylink D, Veldhuis JD. 2004. Sustained
elevation of pulsatile growth hormone (GH) secretion and insulin like growth factor I
(IGF-I), IGF-binding protein-3 (IGFBP-3), and IGFBP-5 concentrations during 30-day
continuous subcutaneous infusion of GH releasing peptide-2 in older men and women.
128. Veldhuis JD, Patrie J, Frick K, Weltman JY, Weltman AL. 2005. Administration of
recombinant human GHRH-1, 44-amide for three months reduces abdominal visceral
fat mass and increases physical-performance measures in postmenopausal women. Eur
J Endocrinol. 153:669–677.
129. Harman SM, Blackman MR. 2003. The effects of growth hormone and sex steroid on
lean body mass, fat mass, muscle strength, cardiovascular endurance and adverse events
in healthy elderly women and men. Horm Res. 60:121–124.
130. Beauregard C, Utz AL, Schaub AE, Nachtigall L, Biller BM, Miller KK et al. 2008.
Growth hormone decreases visceral fat and improves cardiovascular risk mark-
ers in women with hypopituitarism: A randomized, placebo-controlled study. J Clin
131. Sesmilo G, Biller BM, Llevadot J, Hayden D, Hanson G, Rifai N, Klibanski A. 2000.
Effects of growth hormone administration on inflammatory and other cardiovascular
risk markers in men with growth hormone deficiency. A randomized, controlled clinical
trial. Ann Int Med. 133:111–122.
132. Baum HB, Biller BM, Finkelstein JS et al. 1996. Effects of physiologic growth hormone
therapy on bone density and body composition in patients with adult-onset growth hor-
mone deficiency. A randomized, placebo-controlled trial. Ann Int Med. 125:883–890.
133. Franco C, Brandberg J, Lonn L, Andersson B, Bengtsson BA, Johannsson G. 2005. Growth
hormone treatment reduces abdominal visceral fat in postmenopausal women with abdomi-
nal obesity: A 12-month placebo-controlled trial. J Clin Endocrinol Metab. 90:1466–1474.
134. Johannsson G, Marin P, Lonn L et al. 1997. Growth hormone treatment of abdominally
obese men reduces abdominal fat mass, improves glucose and lipoprotein metabolism,
and reduces diastolic blood pressure. J Clin Endocrinol Metab. 82:727–734.
135. Pasarica M, Zachwieja JJ, Dejonge L, Redman S, Smith SR. 2007. Effect of growth
hormone on body composition and visceral adiposity in middle-aged men with visceral
obesity. J Clin Endocrinol Metab. 92:4265–4270.
136. Tomlinson JW, Crabtree N, Clark PM, Holder G, Toogood AA, Shackleton CH et al.
2003. Low-dose growth hormone inhibits 11 beta-hydroxysteroid dehydrogenase type 1
but has no effect upon fat mass in patients with simple obesity. J Clin Endocrinol Metab.
88:2113–2118.
137. Han TS, van Leer EM, Seidell JC, Lean ME. Waist circumference action levels in the
identification of cardiovascular risk factors: Prevalence study in a random sample. BMJ.
311:1401–1405.
138. Matsuzawa Y, Funahashi T, Nakamura T. 2011. The concept of metabolic syndrome:
Contribution of visceral fat accumulation and its molecular mechanism. J Atheroscler
Thromb. 18:629–639.
139. Balducci S, Zanuso S, Nicolucci A et al. 2010. Effect of an intensive exercise interven-
tion strategy on modifiable cardiovascular risk factors in subjects with type 2 diabe-
tes mellitus: A randomized controlled trial: The Italian Diabetes and Exercise Study
(IDES). Arch Intern Med. 170:1794–1803.
140. Balducci S, Zanuso S, Nicolucci A et al. 2010. Anti-inflammatory effect of exercise train-
ing in subjects with type 2 diabetes and the metabolic syndrome is dependent on exercise
modalities and independent of weight loss. Nutr Metab Cardiovasc Dis. 20:608–617.
141. Bateman LA, Slentz CA, Willis LH et al. 2011. Comparison of aerobic versus resistance
exercise training effects on metabolic syndrome (from the Studies of a Targeted Risk
Reduction Intervention through Defined Exercise—STRRIDE-AT/RT). Am J Cardiol.
108:838–844.
142. Camhi SM, Stefanick ML, Ridker PM, Young DR. 2010. Changes in C-reactive protein
from low-fat diet and/or physical activity in men and women with and without metabolic
syndrome. Metabolism. 59:54–61.
143. Castaneda C, Layne JE, Munoz-Orians L et al. 2002. A randomized controlled trial of
resistance exercise training to improve glycemic control in older adults with type 2 dia-
betes. Diabetes Care. 25:2335–2341.
144. Cauza E, Hanusch-Enserer U, Strasser B et al. 2005. The relative benefits of endurance
and strength training on the metabolic factors and muscle function of people with type 2
diabetes mellitus. Arch Phys Med Rehabil. 86:1527–1533.
145. Cuff DJ, Meneilly GS, Martin A et al. 2003. Effective exercise modality to reduce insu-
lin resistance in women with type 2 diabetes. Diabetes Care. 26:2977–2982.
146. Eriksson J, Tuominen J, Valle T et al. 1998. Aerobic endurance exercise or circuit-type
resistance training for individuals with impaired glucose tolerance? Horm Metab Res.
30:37–41.
147. Irving BA, Davis CK, Brock DW et al. 2008. Effect of exercise training intensity on
abdominal visceral fat and body composition. Med Sci Sports Exerc. 40:1863–1872.
148. Kalter-Leibovici O, Younis-Zeidan N, Atamna A et al. 2010. Lifestyle intervention in
obese Arab women: A randomized controlled trial. Arch Intern Med. 170:970–976.
149. Kim CJ, Kim DJ, Park HR.2011. Effects of a cardiovascular risk reduction intervention
with psychobehavioral strategies for Korean adults with type 2 diabetes and metabolic
syndrome. J Cardiovasc Nurs. 26:117–128.
150. Maruyama C, Kimura M, Okumura H et al. 2010. Effect of a worksite-based inter-
vention program on metabolic parameters in middle-aged male white-collar workers:
A randomized controlled trial. Prev Med. 51:11–17.
151. Seligman BG, Polanczyk CA, Santos AS et al. 2011. Intensive practical lifestyle inter-
vention improves endothelial function in metabolic syndrome independent of weight
loss: A randomized controlled trial. Metabolism. 60:1736–1740.
152. Dutheil F, Lac G, Lesourd B et al. 2013. Different modalities of exercise to reduce vis-
ceral fat mass and cardiovascular risk in metabolic syndrome: The RESOLVE* random-
ized trial. Int J Cardiol. pii: S0167-5273(13)00921-2. doi: 10.1016/j.ijcard.2013.05.012.
[Epub ahead of print] PubMed PMID: 23714599.
153. Alberga AS, Frappier A, Sigal RJ, Prud’homme D, Kenny GP. 2013. A review of ran-
domized controlled trials of aerobic exercise training on fitness and cardiometabolic risk
factors in obese adolescents. Phys Sportsmed. 41:44–57.
154. Vissers D, Hens W, Taeymans J, Baeyens JP, Poortmans J, Van Gaal L. 2013. The effect
of exercise on visceral adipose tissue in overweight adults: A systematic review and
meta-analysis. PLoS One. 8:e56415.
155. Jakicic JM, Clark K, Coleman E et al. 2001. American College of Sports Medicine posi-
tion stand. Appropriate intervention strategies for weight loss and prevention of weight
regain for adults. Med Sci Sports Exerc. 33:2145–2156.
17 Barriers to Fruit and
Vegetable Consumption
and Practical Strategies
for Increasing Fruit
and Vegetable Intake
Susan Bowerman
CONTENTS
Introduction............................................................................................................. 279
Fruit and Vegetable Intakes in the US Population..................................................280
Barriers to Fruit and Vegetable Consumption.........................................................280
Cost as a Barrier to Fruit and Vegetable Consumption...................................... 281
Lack of Access as a Barrier to Fruit and Vegetable Consumption..................... 282
Inconvenience as a Barrier to Consumption...................................................... 283
Taste as a Barrier to Intake................................................................................. 283
Increasing Fruit and Vegetable Consumption......................................................... 283
Predictors of Fruit and Vegetable Intake............................................................ 283
Addressing Barriers to Fruit and Vegetable Intake............................................284
Simple Strategies for Increasing Fruit and Vegetable Intake............................. 286
References............................................................................................................... 287
INTRODUCTION
Fruit and vegetable consumption is a cornerstone of a well-balanced diet, and the link
between fruit and vegetable intake and health is well established. Yet, while the
majority of Americans acknowledge the importance of fruits and vegetables in the
diet, most are not meeting recommended intakes [1].
Consumers cite numerous barriers to meeting the recommended number of daily
servings of fruits and vegetables, including taste preferences both individually and
within the family unit, cost, perishability of fresh produce, limited access, as well as
lack of time and knowledge with regard to preparation.
In order to encourage consumers to increase their intake of fruits and vegetables,
health-care providers must first be familiar with the challenges faced by their patients.
279
With a better understanding of the barriers, recommendations can be individually

tailored to help patients more easily meet the recommended intakes.
FRUIT AND VEGETABLE INTAKES IN THE US POPULATION

General recommendations for fruit and vegetable intake from the 2005 US Dietary
Guidelines were that adults aim for five to nine servings of fruits and vegetables per
day [2], and current dietary guidelines suggest filling “half your plate with fruits and
vegetables at each meal or eating occasion” with an emphasis on dark green, red, and
orange vegetables [3]. With the introduction of the USDA MyPyramid in 2005,
updated recommendations based on age, gender, and activity level were introduced,
which, in many cases, suggested increases for most people beyond the five to nine
daily servings, with total recommendations for fruit and vegetable intake ranging
from 2 to 6½ cups per day [4].
In a 2010 study commissioned by the Produce for Better Health Foundation,
average fruit and vegetable intake among American adults was compared to the
USDA recommendations. The study found that the average adult consumes a total
of just 1.8 cups of fruits and vegetables in a typical day and that only 6.4% of the
population achieves the target for vegetable consumption in an average day. With
regard to fruit intake, the report stated that 20% of individuals do not consume even
10% of the recommended amount and that only 7.6% achieve their fruit target in a
typical day [5].
Further support that Americans are not meeting their recommended target
intakes comes from the 2003–2004 National Health and Nutrition Examination
Survey (NHANES), which indicated that fewer than 10% of Americans met their
specific fruit and vegetable recommendations [6]. The report also noted that orange
juice and potatoes were the largest single contributors to fruit and vegetable intake,
respectively.
Healthy People 2010, an initiative of the Centers for Disease Control and
Prevention, had as one of its goals that at least 75% of Americans would eat at
least two daily servings of fruit and at least three daily servings of vegetables, with
1/3 of those servings from deep green or orange vegetables. And yet, in the final
report, which compared intake trends over 10 years, little progress toward these
goals was reported [7]. Only 40% of adults met the recommendation for fruit intake,
fewer than half met the recommendations for total vegetable intake, and only 9%
consumed 1/3 of their vegetables as deep green or orange varieties.
BARRIERS TO FRUIT AND VEGETABLE CONSUMPTION

Despite the issuance of dietary guidelines and the promotion of fruit and vegetable
intake by various agencies, consumers cite numerous barriers to meeting their target
intakes, even though they are well aware of the health benefits associated with fruit
and vegetable consumption [8]. These barriers include, but are not limited to, the
relative cost of fruits and vegetables and concerns over food waste since fruits and
vegetables are perishable, lack of access, lack of time required for preparation, and
lack of knowledge related to selection, storage, and preparation.
Barriers to Fruit and Vegetable Consumption 281
Cost as a Barrier to Fruit and Vegetable Consumption

Cost is one of the primary drivers in food selection, second only to taste, and is par-
ticularly influential among those in lower socioeconomic groups [9,10]. Energy-dense
foods—composed of refined grains, added sugars, or fats—are often the lowest-cost
option to the consumer [11], while higher fruit and vegetable intake and lower intake
of solid fats, sugars, and alcohol are strongly associated with higher food costs [12].
In addition to the high cost of fresh produce, the fact that it is perishable acts as a
deterrent to intake, since the concern is that the food will spoil before it is consumed
and, therefore, money will be wasted [8].
In an analysis of over 1300 foods, Drewnowski examined the relationship between
food costs and energy density [13]. Using existing databases for food costs, nutritive
value, and federally established serving sizes, the analysis indicated that on a per-
calorie basis, grains and sugars supplied the most calories for the least cost and were
less expensive than vegetables and fruit and that the energy cost ($/kcal) was highest
for fruits and vegetables. These price differentials across food groups support the
observation that lower incomes are associated with increased intake of energy-dense,
nutrient-poor foods. Other studies in Europe have reported similar findings, empha-
sizing that the high cost of fruits and vegetables is a significant impediment to their
regular inclusion in the diet [14–16].
Further evidence of the relationship between food costs and diet quality comes
from a cross-sectional study of the monetary costs of diets consumed by participants
in the 2001–2002 NHANES study compared to Healthy Eating Index (HEI-2005)
values [12]. Higher intakes of total vegetable and fruit servings, as well as dark
green/orange vegetables, were strongly associated with higher food costs. In addi-
tion, higher diet costs were also associated with higher HEI-2005 scores.
Related to the issue of cost, the high spoilage rate of fresh fruits and vegetables
has been cited as a deterrent to their consumption. Yeh et al. [8] conducted focus
groups with 147 multiethnic adults in two states and found that the high cost of fruits
and vegetables was the most prevalent concern, with many citing frustration with
food wastage due to the perishable nature of fresh fruits and vegetables.
When price reduction strategies are applied to targeted healthy foods, it can pro-
mote intake. French [17] studied the impact of a 50% price reduction on fresh fruit
and baby carrots in the school cafeteria setting. Compared with normal price con-
ditions, the price reduction led to a 400% increase in fresh fruit sales and a 200%
increase in sales of carrots.
Despite perceptions that increasing fruit and vegetable intake substantially
increases food costs, a report from the Economic Research Service/USDA concluded
that consumers can meet the recommended three fruit servings and four vegetable
servings for 64 cents per person per day [18].
The Women’s Healthy Eating and Living (WHEL) study was a large, randomized,
controlled trial, which examined whether the adoption of a plant-based diet would
affect prognosis in women who had been diagnosed with early-stage breast cancer.
As part of the analysis, the impact of adopting such a diet on grocery costs over a
one-year period was also examined. At baseline, women in both groups reported
similar dietary intakes and food costs, but at the one-year mark the intervention
group reported increases in total fruit and vegetable intake from 6.3 to 8.9 servings
per day, with a modest increase in food costs of $1.22 per person per week [19].
Lack of Access as a Barrier to Fruit and Vegetable Consumption

Access to fresh fruits and vegetables is another frequently cited barrier to adequate
consumption. Produce selection in certain regions of the country, particularly rural
areas and low-income areas, is often limited. There may be limited outlets such as
supermarkets or farmer’s markets in these areas. In addition, many individuals do
not live within walking distance of a supermarket and may lack transportation to
retail outlets where fresh fruits and vegetables could be purchased.
Larger-sized food stores and grocery chains are more likely to stock healthy foods
at competitive prices [20,21], and the availability of supermarkets is associated with
increased fruit and vegetable intake [22–24]. In addition, shopping at supermarkets,
as compared with shopping at independent grocers, is associated with more frequent
fruit and vegetable intake [25].
Powell et al. conducted a comprehensive analysis of the availability of differ-
ent types of food stores by zip codes nationwide, covering a population of over
280,000,000 living in 28,050 zip codes [26] and found significant disparities across
racial and income groups. The availability of chain supermarkets in predominantly
African-American neighborhoods was only 52% of that in predominantly white
communities, and Hispanic neighborhoods had about one-third as many chain
supermarkets compared with non-Hispanic neighborhoods. Residents in zip codes
with the lowest income quintile had the least availability of chain supermarkets and
greater numbers of nonchain food outlets.
Michimi and Wimberly examined the interaction between supermarket acces-
sibility and fruit and vegetable intake nationally [27]. Using fruit and vegetable con-
sumption data from the Behavioral Risk Factor Surveillance System for 2002–2006
and distance to supermarkets from the 2006 Census Zip Code Business Patterns
data, the authors found that the odds of consuming fruits and vegetables five or more
times daily decreased as distance to supermarkets increased in metropolitan areas.
In an examination of fruit and vegetable intake in a multiethnic urban popula-
tion, Zenk et al. [28] found that fruit and vegetable intake was higher when a large
grocery store was present in the neighborhood. The sample comprised 919 African-
American, Latino, and white adults living in urban Detroit. Among the entire sam-
ple, daily fruit and vegetable intake averaged 3.4 servings. In those neighborhoods
with a large grocery store, residents averaged 0.7 more daily fruit and vegetable serv-
ings. The authors concluded that presence of a grocery store in the immediate neigh-
borhood was associated with increased intake, but the relationship between distance
to the nearest supermarket and fruit and vegetable consumption was nonsignificant.
Despite the perception that residents in poor urban areas lack access to fresh
foods, a survey of low-income residents in Boston found a high degree of mismatch
between perceived and actual supermarket access which, in turn, influenced fruit
and vegetable consumption [29]. Survey data was collected from over 800 residents
in low-income housing in urban Boston, and actual distance to the nearest supermar-
ket was compared to the residents’ reported distance. The results suggested that the
residents did not, in fact, lack access, as the average distance to a supermarket was
less than half a mile. Despite the fact that there was a supermarket within an average
of 0.6 miles, those who reported that they did not have a supermarket within walking
distance consumed significantly fewer fruits and vegetables (0.56 servings/day) than
those who reported a supermarket within walking distance from home.
Inconvenience as a Barrier to Consumption

Convenience is another major factor in food choice decisions, and those who eat few
fruits and vegetables often do so because they perceive them to be inconvenient [9,30].
The fact that fresh fruits and vegetables are perishable means that they need to be
purchased more frequently, which means more time needs to be available to purchase
food, and many consumers feel they have limited time to shop [31]. For vegetables,
preparation time is also considered an additional barrier to increased consumption [32].
In focus groups, inconvenience is often cited as an impediment to fruit and veg-
etable intake. Participants comment that they are less likely to serve foods that are
considered time consuming to prepare, while foods that are easy to eat, such as
bananas, are cited as more convenient than messy items, such as oranges, that require
peeling [30,33]. Consumers also acknowledge that prepared items such as precut
vegetables or prewashed and bagged salad greens, while more expensive, are appeal-
ing due to the fact that they save preparation time. In one study of nearly 500 urban
black males, the primary reported barrier to eating fruits and vegetables was that
they depended upon others to prepare these foods for them [34].
Taste as a Barrier to Intake

Along with cost and convenience, taste is one of the primary factors in food choice
[35]. Since eating food is a source of pleasure, the taste, texture, quality, odor, and
appearance all play important roles in determining whether a food will be eaten. For
the most part, good taste is seen as a benefit of increasing fruit intake, but taste is
also considered a barrier for increased consumption of certain vegetables, particu-
larly cruciferous vegetables [32].
Thiourea-containing compounds are bitter tasting and naturally occur in many
fruits, vegetables, and bitter-tasting foods. Sensitivity to bitter tastes is genetically
influenced, and highly sensitive individuals report dislike of cruciferous and green
leafy vegetables, as well as citrus [36]. Additionally, it has been reported that those
who are highly sensitive to bitter tastes consume less fruit [37] and vegetables [38,39]
than those who are not. Palatability can be improved with the addition of fat, sugar,
or salt, and certain cooking methods such as sautéing, braising, or pickling of veg-
etables can diminish the bitter taste and improve palatability [40].
INCREASING FRUIT AND VEGETABLE CONSUMPTION

Predictors of Fruit and Vegetable Intake
There are few studies on the behavioral predictors of fruit and vegetable intake,
but what is known is that attitudes and beliefs play a role and that individuals who
believe that vegetable consumption is important to good health make an extra effort
to do so and that taste preferences, particularly for raw vegetables, is positively cor-
related with increased intake. In a survey of 838 adults in Washington state, the most
common behaviors related to increased vegetable consumption were the inclusion
of a vegetable at the evening meal, snacking on raw vegetables, and selecting mixed
dishes that were vegetable based such as vegetable soups and vegetable stir-fries [41].
In a cross-sectional survey of 1138 adult women in Melbourne, Australia,
those who enjoyed the process of meal planning, shopping, and preparation were
more likely to consume two or more servings of vegetables per day, while those
who reported that cooking was a chore or who spent less than 15 min preparing a
meal were less likely to consume two daily vegetable servings. Fruit consumption
also increased as the number of meals prepared at home and the number of packed
lunches increased. Planning, cooking, and packing meals ahead of time, as well as
the enjoyment of the process of meal preparation and consuming meals at home with
family were all associated with higher intakes [42].
As might be expected, those who grow their own vegetables report higher intakes
of fruits and vegetables than those who do not [43]. Similarly, farm-to-consumer
approaches (farmers’ markets, pick-your-own produce stands, community-supported
agriculture programs) may also improve diet quality [44,45] and may also have the
potential to increase access to fruits and vegetables in low-income areas that have
limited access.
Addressing Barriers to Fruit and Vegetable Intake

Clearly, time management is a significant barrier [46], and consumers need practical
guidance with regard to meal planning, food shopping, and food preparation in order
to streamline the process. Planning meals ahead of time and getting family mem-
bers involved in the process can be helpful, since having to cater to individual tastes
within the family can be an impediment to healthful eating [47]. Planning menus for
the week, preparing foods in advance, and then freezing or portioning out for later
meals, as well as having quick, healthy recipes and methods for preparation have all
been cited as strategies for improving diet quality and increasing consumption of
vegetables.
One of the simplest and most practical strategies for increasing consumption of
fruits and vegetables is simply to ensure that the items are available in the home.
Home availability of fruits and vegetables is positively associated with intake among
children, adolescents, and adults, and the association between availability and intake
is maintained over time [48]. It has been suggested that simple external cues, such as
the sight of freely available fruits and vegetables, could lead to increased consumption
[48], which is why it is often suggested, for example, that fresh fruit be kept on a
kitchen counter, or that cut vegetables be stored prominently in the refrigerator.
Similarly, availability of fruits and vegetables in settings outside the home can
also lead to increased intake. In a Danish study, five work-site cafeterias served as
study sites in which cafeteria staff received training sessions to encourage the offer-
ings of fruits and vegetables and to track changes in fruit and vegetable consumption
from baseline to the 1-year endpoint. All sites reported significant increases in intake,
amounting to an average increase of 70 g/day. Follow-up data collected 4 months

after the endpoint indicated that this level of intake was maintained or, at some sites,
significantly increased, with intakes averaging 95 g higher than baseline across all
five sites [49].
Incorporating more vegetables into meals and dishes is often recommended as
a way to decrease energy density and increase vegetable intake, but can be diffi-
cult to implement in cases where individuals do not care for the taste of vegetables.
However, pureed vegetables can be successfully hidden in dishes without affecting
palatability. Rolls et al. [50] enrolled 41 subjects in a crossover design with repeated
measures within subjects. The subjects were provided all of their foods once a week
for 3 weeks, and the entrées varied in energy density. The standard meal served as
the reference, and the two test meals provided 85% and 75% of the calories of the
standard meals as a result of the covert addition of pureed vegetables to the entrée.
Regardless of energy density of the meal, subjects consumed a consistent weight of
food, so that energy intake decreased as the amount of vegetable puree in the entrée
increased. Total equivalent vegetable servings increased by one in the 85% meal and
by two servings in the 75% meal, and all meals were rated similar in palatability.
Simply serving larger portions of both fruits and vegetables has been shown to
increase intake among adults and children. In another study by Rolls et al. [51],
the effect of varied portion size of vegetable servings within a meal was examined
relative to overall energy intake and total vegetable intake within the meal. Subjects
were served a meal consisting of a meat and a starch, along with varying amounts
of cooked broccoli (180, 270, or 360 g). Portion sizes of the meat and starch were
kept constant. As portion size increased, subjects consumed more vegetables, such
that those served 270 g consumed the equivalent of an additional half-serving of
vegetables, and those served 360 g consumed the equivalent of an additional three-
quarters of a serving of broccoli.
A similar experiment was conducted in 38 children aged four to six years [52]. In
order to participate in the study, children had to report that they liked the test fruit or
vegetable, but not necessarily both. Children were served a pasta and tomato sauce
entrée with varying amounts of canned peaches in light syrup and cooked broccoli.
When fruit portions were doubled from 75 to 150 g, children increased their fruit
intake by 70% and increased their vegetable intake by 37% when broccoli portions
were increased from 75 to 150 g. One limitation of this study was the fact that chil-
dren were prescreened for liking of the fruits and vegetables served, and therefore
strategies for increasing acceptance of fruits and vegetables may also need to be
employed when serving larger quantities to children who report disliking these foods.
A review of strategies designed to encourage children to increase their acceptance
of fruits and vegetables is beyond the scope of this chapter. However, it has been
demonstrated that increasing the variety of highly palatable foods leads to increased
consumption [53,54]. Whether the same strategy would lead to increased vegetable
consumption was the purpose of another study [55]. In a crossover design, 66 adults
were fed a meal of pasta and cooked vegetables once a week for four weeks, with
variations on the type of vegetables that were served. At three of the meals, subjects
were offered 600 g of either broccoli, carrots, or snap peas, and in one meal they
were offered 200 g of each of the three vegetables served side by side. When subjects
were offered three different vegetables on the plate, they consumed an additional
48 g of total vegetables, or the equivalent of more than a one-half serving.
The issue of cost as it relates to fresh fruits and vegetables could be addressed, in
part, with an encouragement to consumers to purchase frozen items, which would
help alleviate concern over food wastage. In a 2012 survey conducted by the Produce
for Better Health Foundation, almost two-thirds (65%) of the primary shoppers in
the family reported that they throw out at least some of the fresh fruit they buy, and
over 80% throw out at least some of the fresh vegetables they buy. Overall, food
purchasers considered fresh fruits and vegetables to be the most healthy form, but
acknowledged that frozen foods are easy to use, quick to prepare, and generally more
cost-effective [56].
Despite a common perception that freezing is destructive to the nutrients in fresh
produce, several studies have demonstrated otherwise. One study examined the
vitamin C content of frozen peas, broccoli, spinach, green beans, and carrots to that
of fresh and found that the nutritional value did not suffer as a result of freezing and
that, in some cases, the nutritional quality was superior to vegetables that had been
purchased fresh and stored in the home for several days [57].
In another study, the antioxidant activity of fresh vegetables was compared to the
same vegetables that had been frozen, jarred, or canned. It was noted that the anti-
oxidant activity of fresh vegetables declines significantly after harvest and during the
storage process, and that frozen vegetables exhibit antioxidant activity similar to the
same vegetables purchased in the fresh state from supermarkets [58], due, in part, to
the fact that processing vegetables into a frozen product is done relatively quickly
after harvest.
Vision plays an important role in food selection, and visual appearance and color
of foods is considered before taste, aroma, and texture [59]. Color is one of the more
important visual attributes in foods and can serve, for example, as an indicator of food
quality (such as the difference between a ripe yellow banana and an overripe brown
banana). In addition, different colors are known to influence taste perception [60].
The primary phytochemical pigments that impart color to foods are fat-soluble
chlorophylls, which contribute green colors, and carotenoids (e.g., lycopene,
lutein, α- and β-carotene), which impart yellow, orange, and red colors to foods.
Water-soluble compounds that contribute color include the anthocyanins (red and
blue colors), flavonoids (yellow), and betalains (red). The distinct colors provided
by these different pigments were used as the basis for the creation of a color-
coded system [61] aimed at helping consumers to increase their fruit and veg-
etable intake by including one serving from each of seven distinct color groups
each day (Table 17.1).
Simple Strategies for Increasing Fruit and Vegetable Intake

As a practical matter, consumers can be encouraged to increase their fruit and veg-
etable intake by employing a variety of simple strategies listed as follows:
• Make a point to have a fruit or vegetable at every meal or snack.

• Get in the habit of having fruit for dessert.
TABLE 17.1
Color Code Groups of Fruits and Vegetables
Color Group Phytochemical Representative Fruits and Vegetables
Red Lycopene Tomato and tomato products such as juice, soups, and
pasta sauces; pink grapefruit, watermelon, guava
Red-purple Anthocyanins Red grapes, blackberries, raspberries, blueberries,
pomegranate
Orange α- and β-Carotene Carrot, mango, pumpkin
Orange-yellow β-Cryptoxanthin and Tangerine, orange, pineapple, papaya
flavonoids
Yellow-green Lutein and zeaxanthin Spinach, avocado, kiwi, honeydew, yellow corn
Green Glucosinolates and indoles Broccoli, brussels sprouts, cabbage, cauliflower, kale
White-green Allyl sulfides Leeks, onions, garlic, chives
• Add vegetables to mixed dishes such as pasta and rice dishes and to pre-
pared soups.
• Add vegetables or fruits as an ingredient in foods such as pasta sauces, meat
loaves, omelets, smoothies, and sandwiches.
• Add vegetables to takeout foods such as Chinese or Indian dishes.
• Keep serving bowls of fruits, vegetables, and salads on the dinner table to
encourage consumption.
• Make time on the weekends to plan meals, shop, and prepare as much as
possible.
• Keep a fruit bowl on the kitchen counter and cut up vegetables in the refrig-
erator to encourage intake.
• Seek out local farmers’ markets or community-supported agriculture pro-
grams which may offer more fruit and vegetable variety at affordable prices.
• Add fresh fruit to cereals and yogurt.
• Increase variety by trying a new fruit or vegetable on a regular basis.
REFERENCES
1. Department of Health and Human Services, Centers for Disease Control and Prevention.
2009. CDC State Indicator Report on fruits and vegetables, 2009. http://www.cdc.gov/
nutrition/downloads/StateIndicatorReport2009.pdf (accessed on June 11, 2013).
2. U.S. Department of Health and Human Services and U.S. Department of Agriculture.
January 2005. Dietary Guidelines for Americans, 2005, 6th edn. Washington, DC: U.S.
Government Printing Office.
3. U.S. Department of Agriculture and U.S. Department of Health and Human Services.
December 2010. Dietary Guidelines for Americans, 2010, 7th edn. Washington,
DC: U.S. Government Printing Office.
4. United States Department of Agriculture. www.choosemyplate.gov (accessed on
June 10, 2013).
5. Produce for Better Health Foundation. 2010. State of the plate: 2010 study on America’s
consumption of fruits and vegetables, 2010. http://www.pbhfoundation.org (accessed on
June 1, 2013).
6. Kimmons J, Gillespie C, Seymour J et al. 2009. Fruit and vegetable intake among
adolescents and adults in the United States: Percentage meeting individualized
recommendations. Medscape J Med 11:26. http://www.ncbi.nlm.nih.gov/pmc/articles/
PMC2654704/
7. U. S. Department of Health and Human Services, Centers for Disease Control and
Prevention. 2010. Healthy people 2010 final review. http://www.cdc.gov/nchs/data/
hpdata2010/hp2010_final_review.pdf (accessed on June 11, 2013).
8. Yeh MC, Ickes SB, Lowenstein LM et al. 2008. Understanding barriers and facilitators
of fruit and vegetable consumption among a diverse multi-ethnic population in the USA.
Health Promot Int 23:42–51.
9. Glanz K, Basil M, Maibach E et al. 1998. Why Americans eat what they do: Taste, nutri-
tion, cost, convenience, and weight control concerns as influences on food consumption.
J Am Diet Assoc 98:1118–1126.
10. Wiig K and Smith C. 2009. The art of grocery shopping on a food stamp budget: Factors
influencing the food choices of low-income women as they try to make ends meet.
Public Health Nutr 12:1726–1734.
11. Drewnowski A and Specter SE. 2004. Poverty and obesity: The role of energy density
and energy costs. Am J Clin Nutr 79:6–16.
12. Rehm CD, Monsivais P, and Drewnowski A. 2011. The quality and monetary value of
diets consumed by adults in the United States. Am J Clin Nutr 94:1333–1339.
13. Drewnowski A. 2010. The cost of US foods as related to their nutritive value. Am J Clin
Nutr 92:1181–1188.
14. Pollard J, Kirk SF, and Cade JE. 2002. Factors affecting food choice in relation to fruit
and vegetable intake: A review. Nutr Res Rev 15:373–387.
15. Darmon N, Ferguson EL, and Briend A. 2002. A cost constraint alone has adverse
effects on food selection and nutrient density: An analysis of human diets by linear
programming. J Nutr 132:3764–3771.
16. Dibsdall LA, Lambert N, Bobbin RF, and Frewer LJ. 2003. Low-income consumers’
attitudes and behaviour towards access, availability and motivation to eat fruit and veg-
etables. Public Health Nutr 6:159–168.
17. French SA. 2003. Pricing effects on food choices. J Nutr 133:841S–843S.
18. Reed J, Frazao E, and Itskowitz R. 2004. How much do Americans pay for fruits and
vegetables? Agriculture Information Bulletin No. (AIB-790). Economic Research
Service/USDA, Washington, DC, July 2004.
19. Hyder JA, Thomson CA, Natarajan L et al. 2009. Adopting a plant-based diet minimally
increased food costs in WHEL Study. Am J Health Behav 33:530–539.
20. Sallid JF, Nader PR, Rupp JW, Atkins CJ, and Wilson WC. 1986. San Diego surveyed
for heart-healthy foods and exercise facilities. Public Health Rep 101:216–219.
21. Horowitz, CR, Colson KA, Hebert PL, and Lancaster K. 2004. Barriers to buying
healthy foods for people with diabetes: Evidence of environmental disparities. Am J
Public Health 94:1549–1554.
22. Morland K, Wing S, Diez Roux A, and Poole C. 2002. Neighborhood characteristics
associated with the location of food stores and food service places. Am J Prev Med
22:23–29.
23. Morland K, Diez Roux A, and Wing S. 2006. Supermarkets, other food stores, and
obesity: The atherosclerosis risk in communities study. Am J Prev Med 30:333–339.
24. Laraia BA, Siega-Riz AM, Kaurman JS, and Janes SJ. 2004. Proximity of supermarkets
is positively associated with diet quality index for pregnancy. Prev Med 39:869–875.
25. Zenk S, Schulz AJ, Hollis-Neely T et al. 2005. Fruit and vegetable intake in African
Americans: Income and store characteristics. Am J Prev Med 29:1–9.
26. Powell LM, Slater S, Mirtcheva D, Bao Y, and Chaloupka FJ. 2007. Food store availability
and neighborhood characteristics in the United States. Prev Med 44:189–195.
27. Michimi A and Wimberly MC. 2010. Associations of supermarket accessibility with
obesity and fruit and vegetable consumption in the conterminous United States. Int J
Health Geogr 8:9.
28. Zenk SN, Lachance LL, Schulz AJ et al. 2009. Neighborhood retail food environment
and fruit and vegetable intake in a multiethnic urban population. Am J Health Promot
23:255–264.
29. Caspi CE, Kawachi I, Subramanian SV, Adamkiewicz G, and Sorensen G. 2012. The
relationship between diet and perceived and objective access to supermarkets among
low-income housing residents. Soc Sci Med 75:1254–1262.
30. Maclellan DL, Gottschall-Pass K, and Larsen R. 2004. Fruit and vegetable consumption:
Benefits and barriers. Can J Diet Pract Res 65:101–105.
31. Hastmann TJ, Bopp M, Fallon EA, Rosenkranz RR, and Dzewaltowski DA. 2013.
Factors influencing the implementation of organized physical activity and fruit and
vegetable snacks in the HOP’N after-school obesity prevention program. J Nutr Educ
Behav 45:60–68.
32. Heimendinger J and Van Duyn MA. 1995. Dietary behavior change: The challenge
of recasting the role of fruit and vegetables in the American diet. Am J Clin Nutr
61:1397S–1401S.
33. Rolnick SJ, Calvi J, Heimendinger J et al. 2009. Focus groups inform a web-based
program to increase fruit and vegetable intake. Patient Educ Couns 77:314–318.
34. Wolf RL, Lepore SJ, Vandergrift JL et al. 2008. Knowledge, barriers, and stage of change
as correlates of fruit and vegetable consumption among urban and mostly immigrant
black men. J Am Diet Assoc 108:1315–1322.
35. Connors M, Bisogni CA, Sobal J et al. 2001. Managing values in personal food systems.
Appetite 36:189–200.
36. Drewnowski A. 1997. Taste preferences and food intake. Ann Rev Nutr 17:237–253.
37. Yackinous CA and Guinard JX. 2002. Relation between PROP (6-n-propylthiouracil)
taster status, taste anatomy and dietary intake measures for young men and women.
Appetite 38:201–209.
38. Dinehart ME, Hayes JE, Bartoshuk LM et al. 2006. Taste markers explain variability in
vegetable sweetness, bitterness, and intake. Physiol Behav 87:304–313.
39. Jerzsa-Latta M, Krondl M, and Coleman P. 1990. Use and perceived attributes of crucif-
erous vegetables in terms of genetically-mediated taste sensitivity. Appetite 15:127–134.
40. Drewnowski A and Gomez-Carneros C. 2000. Bitter taste, phytonutrients and the con-
sumer: A review. Am J Clin Nutr 72:1424–1435.
41. Satia JA, Kristal AR, Patterson RE, Neuhouser ML, and Trudeau E. 2002. Psychosocial
factors and dietary habits associated with vegetable consumption. Nutrition 18:247–254.
42. Crawford D, Ball K, Mishra G, Salmon J, and Timperio A. 2007. Which food-related
behaviours are associated with healthier intakes of fruits and vegetables among women?
43. Billson H, Pryer JA, and Nichols R. 1999. Variation in fruit and vegetable consumption
among adults in Britain. An analysis from the dietary and nutritional survey of British
adults. Eur J Clin Nutr 53:946–952.
44. Blanck HM, Thompson OM, Nebeling L, and Yaroch AL. 2011. Improving fruit and
vegetable consumption: Use of farm-to-consumer venues among US adults. Prev
Chronic Dis 8:A49.
45. McCormack LA, Laska MN, Larson NI, and Story M. 2010. Review of the nutrition
implications of farmers’ markets and community gardens: A call for evaluation and
research efforts. J Am Diet Assoc 110:399–408.
46. Welch N, McNaughton SA, Hunter W, Hume C, and Crawford D. 2009. Is the percep-
tion of time pressure a barrier to healthy eating and physical activity among women?
47. Chopra V, Gold A, and Reicks M. Barriers to healthful eating among midlife women during
eating occasions focused on nourishing family. http://ncsu.edu/ffci/publications/2012/ v17-
n2–2012-summer-fall/index-v17-n2-december-2012.php (accessed on June 10, 2013).
48. Jago R, Baranowski T, and Baranowski JC. 2007. Fruit and vegetable availability:
A micro environmental mediating variable? Public Health Nutr 10:681–689.
49. Lassen A, Thorsen AV, Trolle E, Elsig M, and Ovesen L. 2004. Successful strategies to
increase the consumption of fruits and vegetables: Results from the Danish ‘6 a day’
Work-site Canteen Model Study. Public Health Nutr 7:263–270.
50. Blatt AD, Roe LS, and Rolls BJ. 2011. Hidden vegetables: An effective strategy to reduce
energy intake and increase vegetable intake in adults. Am J Clin Nutr 93:756–763.
51. Rolls BJ, Roe LS, and Meengs JS. 2010. Portion size can be used strategically to increase
vegetable consumption in adults. Am J Clin Nutr 91:913–922.
52. Mathias KC, Rolls BJ, Birch LL et al. 2012. Serving larger portions of fruits and veg-
etables together at dinner promotes intake of both foods among young children. J Acad
Nutr Diet 112:266–270.
53. Rolls BJ, Rowe EA, Rolls ET et al. 1981. Variety in a meal enhances food intake in man.
Physiol Behav 26:215–221.
54. Brondel L, Romer M, Van Wymelbeke V et al. 2009. Variety enhances food intake in
humans: Role of sensory-specific satiety. Physiol Behav 97:44–51.
55. Meengs JS, Roe LS, and Rolls BJ. 2012. Vegetable variety: An effective strategy to
increase vegetable intake in adults. J Acad Nutr Diet 112:1211–1215.
56. Produce for Better Health Foundation: Primary Shoppers. Moms with kids 10 & under
study report—March 9, 2012. http://www.pma.com/system/files/2012%20Primary%
20Shoppers%20Moms%20Study.pdf (accessed on June 18, 2013).
57. Favell DJ. 1998. A comparison of the vitamin C content of fresh and frozen vegetables.
Food Chem 62:59–64.
58. Hunter KJ and Fletcher KM. 2002. The antioxidant activity and composition of fresh,
frozen, jarred and canned vegetables. Innov Food Sci Emerg Technol 3:399–406.
59. Barrett DM, Beaulieu JC, and Shewfelt R. 2010. Color, flavor, texture, and nutritional
quality of fresh-cut fruits and vegetables: Desirable levels, instrumental and sensory
measurement, and the effects of processing. Crit Rev Food Sci Nutr 50:369–389.
60. Cardello A. 1996. The role of the human senses in food acceptance. In: Food Choice,
Acceptance and Consumption, H. Meiselman and H. Macfie (eds.), pp. 1–82. London,
U.K.: Blackie.
61. Heber D and Bowerman S. 2001. What Color Is Your Diet? New York: Regan Books/
Harper-Collins.
18 Balancing Omega-3 and
Healthy Fats and Oils
Omega-6 Acids in Tissues

Bill Lands
CONTENTS
Introduction............................................................................................................. 291
Converting Dietary Fats into Tissue Lipids............................................................ 292
Converting Tissue HUFA into Receptor-Mediated Signals.................................... 295
Recognizing How Nutrients Cause Harm and What Is a Valid Surrogate..............300
Tools to Create Balanced Omega-3 and Omega-6 Acids in Tissues.......................304
Health Risk Assessment.....................................................................................304
Quantitative Link of Foods with HRA Values................................................... 305
Ways to “NIX the 6 and EAT the 3”..................................................................308
Vegetables and Fruits.........................................................................................308
Food Oils and Fats.............................................................................................308
Beans Are Legumes............................................................................................309
Unexpected Values and Other Surprises............................................................309
Key Foods for Americans...................................................................................309
Conclusions............................................................................................................. 311
Acknowledgment.................................................................................................... 311
References............................................................................................................... 311
INTRODUCTION
Daily food habits can provide imbalanced nutrients in ways that impair health and
cause the signs and symptoms of many chronic diseases that health professionals
treat. Effective health care is more likely when preventing the cause rather than just
treating symptoms created by the cause. Also, it seems unethical to remove symp-
toms and create a sense of benefit while leaving the primary cause unchanged to
continue harming individuals and their future generations. This chapter considers
some chronic immune–inflammatory processes modulated by foods. The challenge
is to identify explicit molecular connections by which nutrient imbalances cause the
clinical conditions so that we can prevent the causal imbalance and maintain health.
Healthy people do not need treatments.
A complex web of signals occurs during immune–inflammatory processes as
diverse cells like neutrophils, macrophages, monocytes, lymphocytes, and eosinophils
291
are recruited to tissues where they participate in immune processes and create both
inflammatory and anti-inflammatory conditions. The signaling web includes over a
hundred gene-defined regulatory proteins called cytokines (including chemokines and
interleukins), which act on specific cellular receptors that activate the transcription
of genes which alter the migration, adhesion, growth, differentiation, and behavior of
the responding cells. Science can-not measure the purpose of the molecular interac-
tions involved, but it can measure how the interactions produce health-related conse-
quences and how nutrient intakes alter the outcomes. Healthy nutrition must provide
enough of the needed essential amino acids for synthesis of the cytokine-regulatory
proteins. Fortunately, most foods contain all of the essential and nonessential amino
acids needed to maintain the complex web of regulatory protein signals. As a result,
current nutrition habits permit cytokine-mediated events to proceed in a normal
balanced manner in accord with inherent gene-defined specificities.
In contrast to this situation, another aspect of healthy nutrition involves essential
omega-3 (n-3) and omega-6 (n-6) fatty acids that form bioactive lipid mediators acting
in a different web of signals that alter the immune–inflammatory cytokine-mediated
network [1]. Those lipid mediators can be present in mammalian tissues only from
nutrients that are eaten, and the relative amounts of n-3 and n-6 nutrients differ widely
among the foods that we eat. Because n-3 and n-6 fatty acids have different physi-
ological actions, food choices can create unintended consequences. This chapter will
focus on how ill-informed food choices create a balance of tissue-selective signals
that produce harmful health outcomes. People recognizing this risk can make vol-
untary food choices to create a balance that prevents the unwanted outcomes. This
chapter discusses how n-3 and n-6 polyunsaturated fatty acids (PUFAs) are converted
to highly unsaturated fatty acids (HUFAs) and accumulated in tissue membrane lipids
from which they are mobilized and converted into potent bioactive mediator actions.
Their different actions on cellular migration, adhesion, growth, differentiation, and
behavior have a profound impact on human physiology and pathophysiology.
For example, the balance among HUFAs accumulated in a tissue sets the stage
for creating chronic inflammatory conditions by recruiting neutrophils and macro-
phages to the site. The n-6 mediator, LTB4, is a much more potent chemotactic agent
than the n-3 mediator, LTB5 [2–4]. As a result, tissues with more n-6 HUFA than
n-3 HUFA develop more intense inflammatory conditions. In contrast, tissues that
have accumulated more n-3 HUFA than n-6 HUFA develop less harmful conditions.
Some similarities and differences in the conversion of n-3 and n-6 nutrients into
health consequences are noted in the following sections.
CONVERTING DIETARY FATS INTO TISSUE LIPIDS

Digestion of dietary fats involves hydrolysis by gut lipases and esterases to form
nonesterified acids, which are absorbed into intestinal cells and esterified into
triglycerides that are transported to the blood system. There the esters are once again
hydrolyzed, and the nonesterified acids enter tissues where they may be incorporated
into tissue lipids. The liver plays a major role in converting 18-carbon PUFAs into
20- and 22-carbon HUFAs by the action of enzymes that desaturate and elongate
the acids as shown in Figure 18.1. The result of this process is a supply of HUFA
Healthy Fats and Oils 293
Omega-3 acids Omega-6 acids
18-carbon polyunsaturated fatty acids (PUFAs)

α-Linolenic acid (18:3n-3) ALA Linoleic acid (18:2n-6) LA
Desaturase action
Stearidonic acid (18:4n-3) SDA γ-Linolenic acid (18:3n-6) GLA
Elongase action
20- and 22-carbon highly unsaturated fatty acids (HUFAs)

(20:4n-3) Dihomo-γ-linolenate (20:3n-6) DGLA
Desaturase action
Eicosapentaenoic acid (20:5n-3) EPA Arachidonic acid (20:4n-6) AA
Elongase action
(22:5n-3) n-3DPA Adrenic acid (22:4n-6)
Sprecher shunt
Docosahexaenoic (22:6n-3) DHA (22:5n-6) n-6DPA
FIGURE 18.1 Conversion of PUFA into HUFA. The corresponding n-3 and n-6 homologs
react similarly with the enzymes noted, making the relative abundance of substrates a major
determining factor in the relative amount of products formed. The number notations describe
the length of the carbon chain and the number of double bonds in the fatty acid.
that accumulates in the liver phospholipids and triglycerides, which are secreted into
the bloodstream carried by very-low-density lipoproteins (VLDL) and high-density
lipoproteins (HDL).
All of the many enzyme-catalyzed steps noted have different reaction rates for
acids with different lengths of acyl chain and numbers of double bonds. However,
they have similar rates for n-3 and n-6 structures as these two types compete with
each other for binding to the catalytic site. The similar competitive action means
that the amounts of n-3 and n-6 in PUFA and HUFA of the diet are reflected in the
relative proportions of n-3 and n-6 HUFA accumulated in liver lipids. Further,
the proportions of HUFA secreted from the liver as VLDL components tend to
be reflected in the circulating plasma lipoproteins and erythrocytes as well as
other tissues following subsequent indiscriminate reactions of hydrolysis and
esterification.
The earliest quantitative evidence of similar competitive metabolic actions was
provided by Mohrhauer and Holman [5,6]. They showed (Figure 18.2) how the pro-
portion of n-6 or n-3 in HUFA rises and then plateaus as dietary amounts rise from
0 to 2% of food calories (0–2 en%). In those studies, the HUFA response and the
growth of young rats had a midpoint response to dietary supply near 0.1 en% for
either n-6 linoleic or n-3 linolenic acid. Such very low levels of PUFA intake allow
the oleic acid in the tissue to compete also in elongation and desaturation processes
and to form the HUFA 20:3n-9. That competition is clearly evident in panel c, which
shows equal competition of n-6 or n-3 PUFA (with a midpoint near 0.1 en%) in
displacing the n-9 acid from accumulating in HUFA. The results also show that
Very low intakes of n-3 or n-6 acids prevent EFA deficiency

Predicted
80 90 70
Observed Observed
70 Predicted 80 Growth
60
Growth 70
60
50
n-3 as % HUFA
n-9 as % HUFA
20:4 % HUFA
60
50 JLR΄63
50 40
40 JLR΄63 no fat
no fat 40 30
30
30 C3 = 0.08en%
20 20 C6 = 0.08en%
20
10 C6 = 0.1 en% 10 C3 = 0.1 10
0 0 0
0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 5
(a) Dietary en% 18:2n-6 (b) Dietary en% 18:3n-3 (c) en% n-6 or n-3 PUFA
FIGURE 18.2 Similar abilities for n-3 and n-6 PUFA in forming HUFA. The graphs show
similar growth responses of rats from 27 to 100 days of age (expressed as percent of maxi-
mum) with low intakes of (a) 18:2n-6 and (b) 18:3n-3. The observed HUFA proportions in liver
lipids are compared with those predicted by a simple model of competitive substrate interac-
tions. The proportion of (c) 20:3n-9 in HUFA was less when dietary PUFA was higher. (Data
from Mohrhauer, H. and Holman, R.T., J. Lipid Res., 4, 151, 1963 [as shown in Lands, B.,
Prog. Lipid Res., 47, 77, 2008].)
5%–10% n-9 in HUFA can occur in healthy animals. This fact will be discussed in
the context of using levels of n-9 HUFA as a surrogate for clinical status.
In 1963, quantitative results with human infants also showed a midpoint near
0.1 en% for n-6 linoleic acid efficacy in preventing signs of essential fatty acid defi-
ciency, which were absent at intakes of 1 en% or above [7]. Those valuable results
can never be repeated for ethical reasons, but they showed clearly how similar the
metabolic dynamics of n-3 and n-6 acids are for rats and humans. Later, Cuthbertson
affirmed that many healthy babies had been raised on intakes of 0.5 en% linoleic
acid [8]. These quantitative results are important as we consider further what a
healthy balance of n-6 and n-3 acids might be in dietary and tissue lipids.
Over 20 years after the early reports noted, my laboratory confirmed the quan-
titative competitive relationship in rats, describing it with an empirical hyperbolic
equation [9]. The equation was then extended to predict the likely impact of dietary
PUFA and HUFA on the proportions of n-3 and n-6 in the HUFA of humans [10,11].
Twenty years after that, the empirical equation was further confirmed as it predicted
successfully the impact of dietary essential fatty acids on tissue HUFA in an analysis
of data from 34 published studies of nearly 4000 people in 92 groups from 11 dif-
ferent countries [12].
We now recognize that the wide range of blood HUFA proportions (28%–88%
n-6 in HUFA) reported for different populations worldwide [12–14] is caused pri-
marily by the wide range of ethnic food habits that occur, and different combina-
tions of familiar foods can give a wide range of values. To a first approximation, the
general balance of n-3 and n-6 HUFA accumulated in human tissues is influenced
more by the relative proportions of n-3 and n-6 fats in foods eaten than by any gene-
defined selective preference for n-3 or n-6 structures. There is no evidence of a gene-
determined normal balance of n-3 and n-6 HUFA being maintained in most human
tissues. Rather, each person creates (knowingly or unknowingly) a balance in tissue
HUFA and thereby lives with the consequences. In the absence of a gene-defined
normal balance, those consequences shape our sense of what would be a desirable
healthy balance.
An interesting genetic form of fatty acid desaturase activity that gives more rapid
conversion of PUFA to HUFA occurs more frequently in people of African than
European or Asian origin [15,16]. Africans with fast alleles may synthesize n-3 and
n-6 HUFA sufficient for growth and development when eating a vegetarian diet with
balanced amounts of n-3 and n-6 PUFA and no HUFA. However, when they eat
typical American foods with much more n-6 linoleic than the n-3 linolenate, they
accumulate higher proportions of n-6 arachidonate in their HUFA and have a higher
risk of diseases mediated by n-6 eicosanoids than do Americans of European or
Asian origin. The elevated risk opens a broad concern about possible harm from the
current 7–10 en% of linoleic acid in the US diet. Further concern of harm from added
linoleic acid can be viewed in the context of Cuthbertson’s conclusion that intakes of
0.5 en% seem adequate for normal human growth and development [8]. We are all
free to pick the amounts of nutrients that best serve our desire for good health, and
we must decide when enough is enough.
CONVERTING TISSUE HUFA INTO RECEPTOR-MEDIATED SIGNALS

To understand the consequences of n-3 and n-6 HUFA accumulated in tissue lip-
ids, the following section reviews briefly some examples of events influenced by
20-carbon HUFA-based mediators called eicosanoids. While many active agents are
formed from PUFA and HUFA, two major types of eicosanoids, leukotrienes and
prostaglandins, give useful examples. Comprehensive reviews of these mediators
provide more details [17,18]. Figure 18.3 shows how HUFA form unstable intermedi-
ates, LTA and PGH, which are enzymatically converted to active mediators that act
at selective receptors. Leukotrienes are formed with 5-lipoxygenase (5-LO), and the
prostaglandins with cyclooxygenases 1 and 2 (COX-1 and COX-2). Fatty acid oxy-
genases add oxygen to the HUFA in an unusual reaction that forms a hydroperoxide
and also requires a hydroperoxide activator to reach a maximum rate of reaction
[19–21]. As a result, these enzymes develop an explosive type of positive feedback
loop that rapidly increases the rate once the reaction begins.
The requirement for a hydroperoxide activator means that the fatty acid oxygen-
ases can react more vigorously in the presence of HOOH and hydroperoxides. Such
conditions occur at inflammatory sites where hydrogen peroxide and superoxide are
abundant [22]. Paradoxically, both oxygenases catalyze their own inactivation as the
explosive reaction proceeds. Thereby they produce only a limited number of prod-
uct molecules per molecule of enzyme. Overall, competing n-3 substrates and low
hydroperoxide availability lower the rate of n-6 eicosanoid formation. Once formed,
the eicosanoids are rapidly inactivated by dehydrogenases, allowing each active
molecule to act only in a brief transient way. Anything slowing the rate of formation
causes fewer active molecules to act for a shorter time.
Some external events stimulate a cell and cause a rise in intracellular calcium,
which activates cellular phospholipases that hydrolyze HUFA from their major site
of accumulation, the 2-position of membrane phospholipids. As with the enzymes
mentioned previously, the phospholipases respond to chain length and number of
double bonds, but do not act appreciably differently with n-3 or n-6 structures. As a
result, the relative proportions of nonesterified n-3 and n-6 HUFA made available to
the fatty acid oxygenases reflect those in the cellular membranes.
Figure 18.3 notes that formation of LTA4 and LTA5 by 5-LO as well as LTB4
and LTB5 by LTA hydrolase occurs with no appreciable discrimination. If the BLT
receptor responded equally to the n-3 and n-6 active agents, there would be little
Tissue HUFA Phospholipid-AA (EPA)
cPLA2 1.0
Release HUFA sPLA2 1.0
AA (EPA)
O2
Oxidize HUFA 5-HPETE (5-HPEPE) 5-LO 1.0

to intermediates
LTA4 (LTA5)
LTAH LTCS
Form 1.0 0.2
bioactive GSH
mediators
LTB4 LTC4 LTD4 LTE4

(LTB5) (LTC5) (LTD5) (LTE5)
Selective BLT1 BLT2

receptor 0.02
actions CysLT1 CysLT2
(a)
FIGURE 18.3 Selective and nonselective events in eicosanoid formation and action:
(a) Leukotrienes. Release of arachidonic acid (AA) from membrane phospholipids by
cytosolic phospholipase A2 (cPLA2) and soluble phospholipase A2 (sPLA2) is similar with
AA and EPA as shown by a ratio of 1.0. The 5-lipoxygenase (5-LO) converts AA and EPA
similarly into hydroperoxy eicosatetraenoic acid (5-HPETE) and hydroperoxy eicosatetrae-
noic acid (5-HPETE) as well as to leukotriene A4 (LTA4) and leukotriene A5 (LTA5). Also,
LTA hydrolase (LTAH) forms LTB4 and LTB5 at similar rates. However, the receptors, BLT1
and BLT2, respond less markedly to LTB5 than LTB4 when forming intracellular signals.
Stimulus
Tissue HUFA
Phospholipid-AA (EPA)
cPLA2 1.0
Release HUFA sPLA2 1.0
AA (EPA)
O2
Oxidize HUFA PGG2 (PGG3) COX-1 <0.1

COX-2 0.3
to intermediates
PGH2 (PGH3)
L-PGDS mPGES-1 PGFS PGIS TXAS

Form 0.3 0.3 ND 1.0 1.0
bioactive H-PGDS
mediators 0.2
PGD2 PGE2 PGF2α PGI2 TXA2

(PGD3) (PGE3) (PGF3α) (PGI3) (TXA3)
Selective DP1 DP2 FP TP

receptor 1.7 1.0 0.2 0.8
actions EP1 EP2 EP3 EP4 IP
0.5 0.4 0.3 0.2 0.8
(b)
FIGURE 18.3 (continued) Selective and non-selective events in eicosanoid formation and
action: (b) Prostaglandins. Cycloxygenase-1 (COX-1) and cycloxygenase-2 (COX-2) react
more slowly with EPA than AA, giving ratios of 0.1 and 0.3, respectively. Other abbreviations
are L-PGDS, lipocalin prostaglandin D synthase; H-PGDS, hematopoietic prostaglandin D
synthase; m-PGES-1, microsomal prostaglandin E synthase-1; PGFS, prostaglandin F syn-
thase; PGIS, prostaglandin I synthase; TXAS, thromboxane synthase; DP, prostaglandin D
receptors (1–2); EP, prostaglandin E receptors (1–4); IP, prostaglandin I receptor; TP, throm-
boxane receptor. (Modified from Wada, M. et al., J. Biol. Chem., 282, 22254, 2007.)
consequence of whether or not we ate different amounts of n-3 or n-6 nutrients.

However, BLT1 receptor signaling with the n-6 LTB4 is much more potent than
with the n-3 LTB5 [23], and that makes more intense inflammatory conditions pos-
sible when the proportion of n-6 HUFA is high. In addition, the reaction of LTC
synthase with glutathione favors LTA4 over LTA5 [24,25] and forms LTC4 faster
than LTC5.
The receptor selectivities that make more vigorous actions with n-6 than n-3 eico-
sanoids give more importance to the initial choice of foods, and they provide a rea-
son for caution about the amount of n-6 nutrients put into the system. Wada et al.
[23] gave a comprehensive comparison of differences in the formation and action of
n-3 and n-6 prostanoids shown in Figure 18.3b. With slower rates of formation by the
oxygenases and similar rates of inactivation by the dehydrogenase, signaling is often
less vigorous with n-3 than n-6 homologs. The active thrombogenic TXA produced
by platelets is unstable and spontaneously decomposes in a few seconds. Slow action
of COX-1 during TXA5 formation from the n-3 EPA gives less potent platelet aggre-
gation compared to that for TXA2 from the n-6 AA [26].
Prostaglandins amplify cytokine signals and create more chemokines that recruit
more cells in a positive feedback loop that shifts a transient state of inflammation
to chronic inflammatory damage [27]. For example, PGE2 acting through the EP2
receptor activates NFkB, which induces formation of COX-2, which then produces
more PGE2 and amplifies inflammation. Also, the PGI2 signaling through the IP
receptor synergizes with the cytokine IL1β to increase expression of the CXCL7
chemokine and increase recruitment of neutrophils, fibroblasts, and endothelial cells
to inflamed sites in the vascular wall.
Rapid progress in defining important prostanoid signaling pathways has come
from use of gene knockout of specific receptors in mice to show the action of individ-
ual prostanoid mediators in acute thrombosis, atherosclerosis, hypertension, brain
injury, and vascular tone [28,29]. Also, the knockout of the BLT-1-receptor prevented
elevated glucose and insulin resistance associated with inflammation in adipose tis-
sue and liver of obese mice [30]. The BLT-1-mediated activation of intracellular JNK
and NF-kB in macrophages elevates release of inflammatory cytokines CCL2 and
IL-6 and further increases the inflammatory condition that underlies the rise in some
biomarkers noted at the right of Figure 18.4.
Prostanoid receptors can be grouped by their coupled G-proteins, which con-
vert mediator binding into intracellular signals [29]. The EP1, FP, and TP recep-
tors couple to Gq, which raises intracellular Ca2+. This amplifies signaling mediated
by the prenylated proinflammatory factor Rho [29]. As a result, the proinflam-
matory action of Rho is less when statin drugs lower the availability of required
isoprenoids and when tissue HUFA have lower proportions of competing n-6 HUFA
(see Figure 18.4). The receptors also promote muscle contraction. Relaxant receptors
(DP, EP2, EP4, IP) couple to Gs and raise intracellular cAMP levels, whereas EP3
couples to Gi and lowers intracellular cAMP levels. All four different PGE recep-
tors create more intensive signals with the n-6 PGE2 compared to the n-3 PGE3
[23]. Their selective location on different cells gives a wide range of physiological
responses to stimuli as they modify intracellular signaling networks. For example,
fever during illness is mediated by a pathway of interleukin-1 inducing COX-2 that
forms PGH2, which PGES converts to PGE2 that acts on the EP3 receptor, which
activates Gi-mediated lowering of intracellular cAMP levels. Alternatively, anorexia
from illness is mediated by PGE2 acting on EP4 receptors [31]. Also, the anxiogenic
effect of repeated social defeat (subjugation or frustration) is mediated by a signaling
pathway of COX-1 forming PGH2 that PGES converts to PGE2, which then activates
an EP1 receptor which activates Gq-mediated elevation of intracellular Ca2+ [32].
As the lipid mediators operate in lipid–cytokine–chemokine cascades, they
recruit and activate leukocytes, which produce even more cytokines and chemokines
and amplify further recruitment of leukocytes in an explosive type of positive feed-
back loop [1]. Importantly, nonlinear aspects of amplification by these mediators
can create more severe consequences than anticipated from the quiet initial state of
normal tissue. The context for interpreting these consequences is considered in the
next section.
Food
Work
Amino acids CO2 Exercise
Fatty acids Acetyl-CoA Synthesis
Sugars
Essential FA
Healthy Fats and Oils
HMG-CoA Fatty acyl-CoA

HUFA biomarker
n-3 and n-6 HUFA in Food

Food Energy toxicity
energy Toxicity
phospholipids Mevalonate Other biomarkers
VLDL and Triglyceridemia
Cholesterol Adipose/obesity
Isoprenoids and LDL lipoprotein
n-3 and n-6 HUFA release NEFA
Prenylated proteins
Insulin resistance
Elevated glucose
n-6 eicosanoids
Oxidant stress and
Postprandial
inflammation and
insults
Excessive n-6 signals proliferation and
Vessel wall
impaired nitric oxide
plaques
Platelet activation
Ischemia
Morbidity and
Mortality
Thrombosis Arrhythmia
FIGURE 18.4 Food-based problems. The role of n-3 and n-6 essential fatty acids is indicated on the left of the figure, and the flow of excess food
energy and its biomarkers are noted at the right.
299
RECOGNIZING HOW NUTRIENTS CAUSE HARM

AND WHAT IS A VALID SURROGATE
Food is a vital aspect in preventing disease and maintaining health. Unfortunately,
randomized clinical trials using physician diet advice to diminish mortality had a
relative risk of 0.97, suggesting that the imprecise advice offered was no better than
no advice at all ([33], as summarized in [14]). One example of that was the large
Multiple Risk Factor Intervention Trial, MRFIT, which studied 12,866 people for
10 years at a cost of over $115 million [34]. The intervention had been designed to
decrease several known predictive risk factors for cardiovascular disease (CVD),
although the factors were not rigorously shown to be a cause of CVD death. The
treated and control groups had no significant difference in mortality.
Fortunately, data collected from the untreated 6000 people in the MRFIT control
group gave results that showed the quintile of participants eating foods that give a
value near 60% n-6 in blood HUFA had a mortality rate nearly one half that of the
remaining four quintiles whose nutrient intake fitted a value near 80% n-6 in HUFA
[13,35]. Importantly, the MRFIT results fit very closely (r2 = 0.98) with evidence from
Canada, Japan, and Greenland, showing that people with less than 50% n-6 in HUFA
have a lower risk of CVD mortality than those with a value higher than 50% [13]. The
results, combined with the metabolic steps noted, indicate that the % n-6 in HUFA can
be a valuable health risk assessment biomarker. It is a valid surrogate biomarker that
has known molecular mediators connecting it to an important clinical outcome. The
results showed that some populations were eating foods that likely prevented CVD.
To avoid very large, expensive trials that measure the primary clinical endpoints of
saved lives or prevented disease, surrogate markers are used as convenient endpoints.
However, identifying valid surrogate markers for a trial is a major clinical concern
[36]. In the compromises made to study the diet–heart question, the director of the
National Heart, Lung, and Blood Institute confirmed that “surrogate endpoints are a
big issue” [34]. This issue remains a very serious one in today’s biomedical discus-
sions. While all risk factors are surrogates for predicting an impending problem, iden-
tifying and preventing the explicit causal factors is the most effective clinical action.
The biomedical community has a serious problem of considering whether efforts
to decrease a marker that is not a valid surrogate for the clinical endpoints of saved
lives or prevented disease might waste millions of dollars. Figure 18.4 sketches out a
logical chain of events that connect foods to some surrogate markers for the clinical
outcome of CVD. We should consider which surrogate is a causal mediator and which
is merely an associated biomarker. Treatments that lower the level of an associated
marker may remove a sign and create a sense of benefit while leaving the primary
cause unchanged. Earlier sections of this review noted explicit molecular pathways
and mediators by which n-3 and n-6 fats in foods affect health. This section views the
clinical context in which those mediators are perceived to cause food-based health
problems. It examines a hypothesis that a preventable imbalance between the n-3 and
n-6 nutrients combines with an imbalanced intake and expenditure of food energy to
create harmful conditions.
Experts in the 1984 Cholesterol Consensus Conference regarded CVD as a diet-
induced disease caused by imbalanced food energy, and they emphasized that maximal
diet therapy of caloric restriction and weight loss should be continued even when use
of drugs seems appropriate [37]. This gave a clear priority to preventing the disease
while treating an associated predictive sign. However, the committee also voted that
cholesterol was a causal mediator, and many people then regarded it as a valid surro-
gate for CVD. From that time, attention turned to treatments with statin drugs to lower
cholesterol, and clinicians and patients were distracted from preventing the causal
food energy imbalances. The following decades led to an obesity epidemic with little
change in the overall incidence and prevalence of CVD among Americans while bil-
lions were spent to lower blood cholesterol rather than prevent its food-based cause.
The Centers for Disease Control and Prevention continues to regard CVD as a pre-
ventable disease that profoundly affects mortality, disability, and health-care costs in
the United States [38]. While major efforts continue to try to reduce the prevalence
of CVD risk factors, concern remains whether the factors being monitored are major
causal mediators (i.e., valid surrogates for CVD) or associated markers caused by an
unrecognized causal factor. After decades of effort at preventing CVD, death rates
have decreased while an unprevented cause leaves the prevalence of CVD mainly
unchanged. An estimated 82,600,000 American adults (>1 in 3) currently have one
or more types of CVD. The American Heart Association noted that CVD accounted
for 1 of every 2.9 deaths in the United States in 2007 [39]. This high prevalence
seems inappropriate for a preventable disease.
For six decades, the biomedical community has regarded eating too much energy-
dense food to be a cause of cardiovascular disease ([37], reviewed in [14]), and it rec-
ognizes elevated plasma triglycerides (triglyceridemia) as a univariant predictive risk
factor for CVD morbidity [40]. In this context, people who maintain constant body
weight metabolize most food energy to CO2 and H2O (Figure 18.4) while some of the
nutrients add to or replace cellular components during growth and repair. The basal
rate of energy use for an average adult human sitting at a desk, watching computers
or television, or driving a car is near 200 cal per 3 h [41]. This rate means that a meal
of 900 cal has many more calories than would be used in the next few hours. Without
added work and exercise to remove the extra 700 cal from the body, the liver is likely
to convert much of it to circulating VLDL, which are mostly triglycerides. That is
the way that it responds to excess circulating food energy. Eating fewer calories per
meal seems wise.
The biomarkers for excess food energy at the right side of Figure 18.4 are widely
discussed by health professionals as predictive risk factors for CVD. These factors
accompany the hydrolysis in the bloodstream of VLDL, which produces nonesterified
acids (NEFAs) and low-density lipoproteins (LDL). Over the past few decades, billions
of dollars have been spent in studying LDL and the cholesterol that it carries, while
the simultaneously produced NEFA are seldom discussed. Nevertheless, the NEFAs
that always must occur whenever LDL is formed can cause transient local oxidative
inflammatory conditions [42–45] and inhibit the action of insulin [30,46]. Importantly,
the transient postprandial inflammation following every large meal may be converted
into chronic inflammatory vascular damage when amplified by excessive actions of
n-6 eicosanoids [27,30] in a type of food energy toxicity or metabolic syndrome.
Evidence from a 25-year follow-up of the pioneering Seven Countries Study
([47], reviewed in [14]) suggests that the excess postprandial food energy that
30
25
25-year CHD mortality (%)
%n-6 in HUFA
20 No. Europe–80%
USA–75%
15
Serbia–65%
10 So. Europe–60%
Crete–50%
5
Japan–40%
0
100 150 200 250 300 350
Serum TC (mg/dL)
FIGURE 18.5 Comparing mortality and cholesterol with different HUFA balances. The
figure is modified from Figure 13 in Reference 14 which describes results from the 25-year
follow-up of the Seven Countries Study. (From Verschuren, W.M. et al., JAMA, 274, 131,
1995.)
elevates circulating cholesterol may not cause fatalities in people who maintain bal-
anced proportions of n-3 and n-6 in their tissue HUFA (Figure 18.5). In Japan, where
the average proportion of n-6 in HUFA is near 50% or lower, blood cholesterol lev-
els did not predict mortality. In fact, recent results from 173,539 Japanese men and
women showed slightly lower mortality with higher cholesterol values [48]. Further,
the low proportion of n-6 in HUFA of the Japanese has long been associated with
a much lower incidence of CVD than that in the United States [49]. Such results
suggest that blood cholesterol levels may be a less valid surrogate biomarker for
clinical endpoints than the proportions of n-3 and n-6 HUFA. Certainly, n-6 eico-
sanoids have a more established role in mediating inflammation than does choles-
terol. Atherosclerosis is an inflammatory disease [50].
Recently, a large clinical trial named ezetimibe and simvastatin in hypercholester-
olemia enhances atherosclerosis regression (ENHANCE) lowered blood cholesterol
without lowering coronary heart disease (CHD) clinical events [51]. That prompted
public questions of whether cholesterol is a valid surrogate marker for CHD and
whether cholesterol drugs actually do any good [52–54]. Also, the Justification for the
Use of Statins in Primary Prevention: An Intervention Trial Evaluating Rosuvastatin
(JUPITER) trial [55] showed that statin treatment lowered elevated levels of an acute
stress protein that is released during inflammatory conditions. It reopened public
questions of whether cholesterol or inflammation is more important in mediating
CVD morbidity and mortality. More serious attention to the maxim “Association is
not causation!” may avoid overreactions that fuel continual efforts to lower blood
cholesterol. There is no clear gene-defined level of what is normal. Well-intended
efforts to define a normal level and to lower current noncausal surrogate levels need
rigorously logical measures that indicate when enough is enough. When a tissue has
lower proportions of n-6 in HUFA, it forms less n-6 prostanoids that amplify the
signaling of the proinflammatory prenylated Rho [29]. Independent of the choles-

terol level in the blood, Rho would be less active when statin drugs lower the avail-
ability of its required isoprenoid activator and provide less action that is amplified by
n-6 prostanoids (see Figure 18.3). It seems that assertive messages from drug market-
ers can distract clinicians and patients from identifying and preventing the ongoing
underlying causal imbalances in n-3 and n-6 nutrients, which are not prevented by
the anticholesterol drugs [56].
One rheumatology group [56] considered that the main barrier to clinician accep-
tance of rational nutrient intervention is the assertive promotion of drug use by the
pharmaceutical sales force. Without similar promotion of fish oil, rheumatologists
seem not inclined to consider, or even be aware of, the potential use of omega-3
nutrients as part of routine therapy for RA patients. Nevertheless, use of fish oil as
an alternative can avoid a serious NSAID-related increase in cardiovascular risk [57]
while it decreases the underlying cause of discomfort caused by high proportions of
n-6 in tissue HUFA. Also, the inescapable competitive metabolism of n-3 and n-6
acids means that decreasing dietary omega-6 fats allow added omega-3 fats to be
more effective in balancing tissue HUFA. Preventing high levels of nutrient-based
n-6 mediators of disease differs appreciably from treating symptoms caused by the
excessive actions of those mediators.
A different example of adding nutrients to diminish clinical symptoms occurs
when avoiding a clinical deficiency of essential fatty acids during parenteral feeding.
One surrogate marker used is the amount of n-9 HUFA (20:3n-9) in blood. While a
severe deficit of dietary PUFA elevates 20:3n-9, clinical signs are not apparent when-
ever the n-9 HUFA is less than 20% of the HUFA (reviewed in [14]). One longitudinal
study showed clinical signs only when 20:3 n-9 was equal to or greater than 20:4n-6
[58]. The clinical deficiency disorder is caused by a deficit of essential n-3 and n-6
mediators. It is not caused by the associated elevated levels of surrogate biomarker
20:3n-9. However, well-intentioned nutritionists may overreact and attempt to pre-
vent the appearance of any n-9 HUFA. The nearly undetectable amount of 20:3n-9
seen in current gas chromatographic analyses of blood samples from Americans
indicates that PUFA intakes are likely much more than needed.
Advice to substitute polyunsaturated fats for saturated fats is a key feature of
dietary guidelines for CHD risk reduction, and an American Heart Association com-
mittee report [59] regarded eating at least 5%–10% of energy from omega-6 PUFA
to reduce the risk of CHD relative to lower intakes. They suggested that even higher
intakes may be more beneficial. The report concluded with an opinion that reducing
omega-6 PUFA intakes from their current levels would be more likely to increase than
to decrease risk of CHD. However, an updated review of linoleic acid intervention tri-
als showed no evidence of cardiovascular benefit in substituting omega-6 linoleic acid
for saturated fats. The Sydney Diet Heart Study substituted saturated fats with linoleic
acid and observed increased rates of death from all causes, CHD, and cardiovascular
disease [60]. The recovery of those previously unused data from that trial followed
an alert observation that n-6 fatty acid–specific and mixed polyunsaturated dietary
interventions have different effects on CHD risk [61]. There is much merit in further
careful review of how an imbalanced intake of n-3 and n-6 fats affects human health
and how the imbalance can be prevented.
Consumers have been provided with much fragmentary and conflicting advice
on dietary lipids [14]. For instance, imprecise advice to replace saturated fats with
unsaturated fats still remains inadequately assessed. Repeated messages from phar-
maceutical marketers and food marketers often focus on a narrow part of the health
problem without a broader context, and the partial information is often misinter-
preted by the public. During the twentieth century, Americans had a striking rise
in consumption of vegetable oils rich in n-6 fats [62]. Whether this consumption
represents a fully informed food choice needs careful review, especially by people
suffering from preventable health disorders that are amplified by n-6 mediators.
Primary prevention of the cause of a preventable food-related disease works well
when individuals make voluntary choices to prevent early nutritional imbalances
before they develop into costly health disorders [63]. Figure 18.4 shows known events
in an overall context that allows people to make better informed decisions about how
to prevent imbalances in foods they eat that are affecting their health. Healthy people
do not need to pay for treatments.
The next section examines tools with which people can inform themselves how
to find foods that increase the intake of omega-3 nutrients, decrease the intake of
omega-6 nutrients, and have fewer calories per meal to maintain a desired balance in
their physiological processes.
TOOLS TO CREATE BALANCED OMEGA-3

AND OMEGA-6 ACIDS IN TISSUES
Explicit connections between n-3 and n-6 nutrients in the foods we eat, the media-
tors they produce, and the clinical context in which nutrients affect many disorders
and diseases are described in this review. The evidence supports the hypothesis that
a preventable imbalance between n-3 and n-6 nutrients combines with an imbalance
in intake and use of food energy to cause harmful health conditions in America. We
have a growing list of human health problems linked to a relative deficit of n-3 nutri-
ents with a resulting excessive action of n-6 bioactive mediators. The wide scope of
these problems reflects the fact that nearly every cell in every tissue of the human
body has eicosanoid receptors that influence cell function.
The health problems include heart attacks [13], atherosclerosis, thrombosis [14],
arrhythmia, stroke, immune–inflammatory disorders [64], asthma, arthritis [56],
Alzheimer’s disease [65], cancer proliferation [66], obesity [67], psychiatric disor-
ders, depression, suicide [68], homicide [69,70], oppositional behavior, unproduc-
tive workplace behaviors, length of stay in hospitals [71], and annual health-care
claim costs [72]. This section examines the contents of n-3 and n-6 nutrients in
foods, which can give our tissues the ability to maintain moderate physiological
responses.
Health Risk Assessment

Because there is no gene-defined normal omega-6 (or omega-3) status, an important
bit of health-related information is in knowing the current HUFA balance that each
individual actually has at the present. Gas chromatographic analyses easily measure
the proportions of n-3 and n-6 acids in blood HUFA. The assay results are valu-
able evidence of the balance that an individual is maintaining [73,74]. Such assays
showed similar relative proportions for n-6 and n-3 HUFA in the total HUFA of
liver, plasma, serum, and red blood cells (RBC) in rats [1–5,9] and plasma, serum,
and RBCs of humans [10,73,75,76]. Despite an average 125-day lifespan of RBCs,
continual remodeling of fatty acids in phospholipids gives proportions of HUFA in
RBCs similar to those in plasma and serum HUFA [75]. These similar proportions
make possible a simple, economical, direct assay of a fingertip blood spot that does
not need time-consuming efforts to separate blood fractions [73,77]. Reporting assay
results as either % n-3 in HUFA or % n-6 in HUFA confirms values predicted from
the causal dietary PUFA and HUFA consumed. Reporting the % n-6 in HUFA keeps
attention on the diet-induced capacity to form n-6 eicosanoids, which mediate so
many undesired disease processes [64].
Quantitative Link of Foods with HRA Values

To help researchers design effective studies of the impact of dietary n-3 and n-6 fats
on clinical conditions, the quantitative empirical equation noted earlier [10,11] was
embedded in a simple spreadsheet with the omega-3 and omega-6 acids grouped
in four categories: the 18-carbon n-3 and n-6 PUFA and the 20- and 22-carbon n-3
and n-6 HUFA. It is posted at the distance learning website for essential fatty acid
education, efaeducation.nih.gov [78]. While the simple calculator allows general
estimates for planning likely health risk assessment (HRA) outcomes, many investi-
gators asked for data on the abundance of n-3 and n-6 fatty acids in foods they might
use in the studies.
To meet this need, the equation was combined with data on thousands of foods
from the USDA Nutrient Database [79] to form a personalized interactive menu-
planning software program that manages all of the many numbers involved. It is
freely available from the efa education website [80]. The software combines for
each daily personal menu plan the number of selected food servings with their
weight, calories, and milligrams of 11 different essential fatty acids. The software
sets the 11 omega-3 and omega-6 acids in four categories: “short 6”: 18:2 and 18:3
and “short 3”: 18:3 and 18:4, plus “long 6”: 20:3, 20:4, 22:4 and 22:5, and “long 3”:
20:5, 22:5 and 22:6.
The software sums the milligrams of these four categories for all food servings
in the daily menu plan and expresses the four sums as a percent of the overall food
calories (en%). Then, it uses the equation to convert the en% values to the likely
resulting HRA value of the % n-6 in HUFA that would result from continued use of
the plan. The software manages all of the numbers involved to predict outcomes not
always expected by the consumer. Two plans in Figure 18.6 illustrate how 21 differ-
ent familiar foods in a daily menu plan can give HRA outcomes of either 30% or
80% n-6 in HUFA. The wide range from 28% to 88% n-6 in HUFA seen in different
populations [12–14] is caused by simple combinations that give different degrees of
primary prevention of a profound set of food-related problems.
Of course, people want to eat different foods from day to day, and the software
prediction with each daily menu plan gives only an estimate of the direction in which
the plan would influence future HRA outcomes. The software uses rigorous quan-
titative relationships of the nutrient interactions that are described in this chapter to
help dietitians and nutritionists develop and advise healthy food combinations for
their clients. However, those nutrition professionals need fast access to information
on explicit foods to put into the plans. Also, both clients and professionals found
that planning all of the different menu plans for a week or two is an unwanted,
tedious task. Rather, people asked for a faster way to identify individual desirable or
undesirable foods when shopping and planning meals. With a context of Americans
having average HRA values near 75%–80% n-6 in HUFA, we developed an omega
3–6 balance score [81] to help people increase their intake of omega-3 nutrients and
decrease their intake of omega-6 nutrients and move their HRA values toward a
more balanced value associated with lower incidence of CVD.
KIM Report by Meal Times For Sample: 30% 30% Plan # 4 Sample: 30%
Breakfast Serving Size grams kcals Servings Short 6 Short 3 Long 6 Long 3
Milk, reduced fat, fluid, 2% milkfat, with added 1 cup 244 122 1 105 68 0 0
Cereals ready-to-eat, KELLOGG, KELLOGG'S 0.75 cup (1 NLEA 29 92 1 297 21 1 0
Blueberries, raw 50 berries 68 38 1 67 46 0 0
Lunch Serving Size grams kcals Servings Short 6 Short 3 Long 6 Long 3
Oil, olive, salad or cooking 1 tbsp 14 239 2 2133 162 0 0
Pork, fresh, loin, tenderloin, separable lean only, 3 oz 85 159 1 417 9 34 0
Lettuce, looseleaf, raw 0.5 cup, shredded 28 10 2 26 63 0 0
Mushrooms, raw 0.5 cup pieces 35 18 2 93 1 0 0
Spinach, raw 1 cup 30 7 1 7 35 0 0
Bread, cracked-wheat 1 slice 25 130 2 324 17 0 0
Lemon juice, raw 1 floz 31 8 1 0 0 0 0
Dinner Serving Size grams kcals Servings Short 6 Short 3 Long 6 Long 3
Cheese, cottage, nonfat, uncreamed, dry, large or 4 oz 113 96 1 12 5 0 0
Finfish, salmon, coho, wild, cooked, dry heat 0.5 fillet 178 247 1 100 190 39 1885
Broccoli, cooked, boiled, drained, without salt 1 stalk, large 280 78 1 106 361 0 0
Cauliflower, cooked, boiled, drained, with salt 3 flowerets 54 12 1 27 90 0 0
Alcoholic beverage, wine, table, white 1 glass (3.5 fl oz) 103 70 1 0 0 0 0
Cheese, gouda 1 oz 28 101 1 75 112 0 0
Turnips, cooked, boiled, drained, with salt 1 cup, cubes 156 33 1 14 50 0 0
Snacks Serving Size grams kcals Servings Short 6 Short 3 Long 6 Long 3
Cheese, feta 1 oz 28 75 1 92 75 0 0
Apples, raw, with skin 1 medium (2-3/4" 138 81 1 120 25 0 0
Ice creams, chocolate 0.5 cup (4 fl oz) 66 285 2 330 198 0 0
Nuts, pine nuts, pignolia, dried 1 oz 28 80 0.5 2933 93 0 0
Total Energy Choice = 1981 kcals total mg = 7278 1619 74 1885
%Cal= 3.31% 0.74% 0.03% 0.86%
These overall choices will give
KIM notes: 30% long 6 in your body's long total
Your energy allowance is 1977 kcals
Your Weight seems OK % long 6 in long total ==>>> 47% 58% 78%
Heart attack deaths/100,000 ===>>> 50 90 200
FIGURE 18.6 Two daily menu plans prepared with KIM-2. Each plan has 21 different
familiar foods which lead to either 30% or 80% n-6 in HUFA.
KIM Report by Meal Times For Harold Jackson 80% Plan # 647 Harold
Breakfast Serving Size grams kcals Servings Short 6 Short 3 Long 6 Long 3
Butter, with salt 1 pat (1" sq, 1/3" 5 36 1 92 59 0 0
Milk, reduced fat, fluid, 2% milkfat, with added 1 cup 244 122 1 105 68 0 0
Applesauce, canned, unsweetened, without added 1 cup 244 105 1 29 7 0 0
Bread, cracked-wheat 1 slice 25 130 2 324 17 0 0
Lunch Serving Size grams kcals Servings Short 6 Short 3 Long 6 Long 3
Cheese, cottage, nonfat, uncreamed, dry, large or 4 oz 113 96 1 12 5 0 0
Oil, soybean, salad or cooking 1 tbsp 14 120 1 6936 925 0 0
Chicken, broilers or fryers, meat only, roasted 1 unit (yield from 1 146 277 1 2000 102 161 102
Lettuce, looseleaf, raw 0.5 cup, shredded 28 5 1 13 32 0 0
Mushrooms, raw 0.5 cup pieces 35 9 1 47 0 0 0
Spinach, raw 1 cup 30 7 1 7 35 0 0
Tomatoes, red, ripe, raw, year round average 1 medium whole 123 26 1 160 6 0 0
Corn, sweet, white, canned, whole kernel, regular 0.5 cup 128 82 1 291 9 0 0
Bread, pita, white, unenriched 1 pita, large (6-1/2" 60 165 1 307 14 0 0
Dinner Serving Size grams kcals Servings Short 6 Short 3 Long 6 Long 3
Cheese, gouda 1 oz 28 101 1 75 112 0 0
Salad dressing, mayonnaise, soybean oil, with salt 1 tbsp 14 49 0.5 2560 290 0 0
Pork, fresh, loin, tenderloin, separable lean only, 3 oz 85 159 1 417 9 34 0
Corn, sweet, yellow, frozen, kernels cut off cob, 0.5 cup 82 72 1 291 9 0 0
Crustaceans, shrimp, mixed species, cooked, 4 large 30 73 1 1353 80 18 80
Chickpeas (garbanzo beans, bengal gram), mature 1 cup 164 269 1 1825 71 0 0
Snacks Serving Size grams kcals Servings Short 6 Short 3 Long 6 Long 3
Apples, raw, with skin 1 medium (2-3/4" 138 81 1 120 25 0 0
Snacks, potato chips, plain, salted 1 oz 28 76 0.5 1698 27 0 0
Total Energy Choice = 2060 kcals total mg = 18661 1900 212 183
%Cal=8.15% 0.83% 0.09% 0.08%
These overall choices will give
KIM notes:
80% long 6 in your body's long total
Your energy allowance is 2250 kcals
Your Weight seems OK % long 6 in long total ==>>> 47% 58% 78%
Heart attack deaths/100,000 ===>>> 50 90 200
FIGURE 18.6 (continued) Two daily menu plans prepared with KIM-2. Each plan has 21
different familiar foods which lead to either 30% or 80% n-6 in HUFA.
The balance score summarizes in a single value the balance among 11 omega-3
and omega-6 essential fatty acids in a food [81]. It uses the same USDA Nutrient
Database information and expresses each essential fatty acid as mg/calorie for a
selected food. This allows the calorie-weighted average for foods eaten in a day
to equal the same en% value that is used in the rigorous overall planning software
noted earlier [80]. Whether or not a person chooses to calculate the calorie-weighted
average, the desirable or undesirable foods are quickly identified by a single omega
3–6 balance score value. Foods with more positive omega 3–6 balance food scores
will increase the predicted percent of omega-3 in tissue HUFA, whereas those with
more negative scores will increase the predicted percent of omega-6 in tissue HUFA
[81]. Obviously, a score of zero represents equal balance of the two types of com-
peting nutrients, and it is associated with a lower health risk assessment value than
occurs with typical Western diets.
Ways to “NIX the 6 and EAT the 3”

Omega 3–6 balance food scores for over 5000 food items are in searchable pdf files
readily available from a community learning site, fastlearner.org [82]. The scores
can be downloaded to home computers or mobile devices [83] for easy access when
choosing between desirable or undesirable foods or when discussing foods with
friends. A community wellness project to “NIX the 6 and EAT the 3” [83] led to
making a file of 270 most positive scores (EAT3) and a separate file of over 1000
most negative scores (NIX6). The lists can be rapidly scrolled to learn about foods
that fit personal preferences of taste and health risk. One can rapidly discover foods
with surprising and unexpected values.
Vegetables and Fruits

The USDA arranges foods into 25 groups [79] and updates the nutrient data
frequently. The categories of snacks, fast foods, and restaurant foods are continually
increasing and changing. A rank-ordered file of 3–6 balance scores for each of the
25 groups was made in 2012 with data from version SR24 (82; the USDA site now
has version SR25) and posted at the fastlearner.org site [82]. This allows one to scroll
through a very large variety of foods in each category to find those that fit personal
tastes. The scores for widely recommended fruits (286 items) and vegetables (669
items) are uniformly more positive (or less negative) than the average value near −6.5
for the current American diet [81]. Most fruits had scores near 0, although avocados
differed with its score of −10. It was reassuring to see positive scores for spinach
(+6), cauliflower (+5), broccoli (+3), turnips (+3), and squash (+2). However, bal-
anced values for potatoes [0], onions [0], and cabbage [0] become much more nega-
tive than the American average when served as potato salad (−21), sauteed onions
(−31), or cole slaw (−13).
The 3–6 balance scores quickly inform consumers of the effect that added food
oils have in the current US diet. Over the past decades, fats and oils commonly used
70 years ago, like butter (−1) and lard (−10), have been used less, and corn oil (−59)
and soybean oil (−50) are used more. Recent public interest in the healthy aspects
of a Mediterranean diet led to increased use of olive oil (−10) as a healthy lifestyle
change. This change lowers intakes of omega-6 fats, and a less-mentioned feature of
Mediterranean menus is seafood, which raises intake of omega-3 fats.
Food Oils and Fats

The n-3 and n-6 fats in food oils are often discussed in a fragmented manner as people
emphasize the presence of omega-3 nutrients while failing to note the competing
omega-6 nutrients that accompany them. The metabolic competition noted earlier is
an inescapable context whenever people eat the fats that maintain tissue HUFA and
their HRA values. A list of food oils at the efa education site [84] shows clearly that
the 925 mg of 18:3n-3 in a tablespoon of soybean oil (with a −50 score) is accompanied
by much more (6936 mg) of competing 18:2n-6. Similarly, the 1414 mg of 18:3n-3 in
a tablespoon of walnut oil (with a −5 score) is accompanied by 7194 mg of competing
18:2n-6. When these food oils are listed as a good source of omega-3, the consumer
may misinterpret the impact they will have on the HRA value. Omega 3–6 balance
scores include metabolic competition in the single assigned value for each food, and
they prevent omitting its consequence during discussion of healthy foods. The USDA
fats and oils group of 194 items has scores ranging from +263 to −84 with an average
value of −21. Consumers may find surprising values for oils they currently use, and
they may want to revise which they wish to continue using in their food repertoire.
Beans Are Legumes

Legumes are a good source of protein and fiber, and they include 100 items with
values near zero. They are useful foods to help make the average American diet
become more positive than its current −6.5 value. For example, a three-bean salad
with green beans (+1), pinto beans [0], and kidney beans [0] can be a very healthy
dish if it is not altered by a food oil with a very negative 3–6 balance score. Navy
beans [0], lima beans (−1), and black-eye peas (−2) all have scores more positive than
the US average foods.
However, eating soybean (−22) and peanut (−25) products makes the average
daily 3–6 balance value more negative than most people realize. For example, the
soy in a meatless hot dog (−20) makes it differ from a beef hot dog (−2). Even more
negative is meatless bacon (−39). People are likely unaware that many forms of
tofu have 3–6 balance values that range from −22 to −32. Plans that include tofu in
healthy meals merit more explicit review of how the food moves a consumer toward
the intended health goal.
Unexpected Values and Other Surprises

The 159 items in the cereal, grains, and pasta group have most scores ranging from 0
to −7 with an average of −3. One extreme item in the group, Chinese chow mein noo-
dles, has a value of −25, likely reflecting the presence of soy oil (−50). The dairy and
egg group (189 items) has many 3–6 balance scores between 0 and −3 and provides
a valuable source of protein, fat, and calcium. Most fish and seafoods (151 items)
have positive scores, except for the few that are served battered and fried (and of
course meatless fish sticks [−25] are not really seafood). Over 100 items in this group
have values ranging from +10 to +83, making them an obvious addition to diets
when a more positive average daily 3–6 balance is desired. Often discussed advice
to eat more nuts and seeds (133 items listed) merits careful logical thought to iden-
tify which nutrient feature weighs most highly in personal food choices. Although
very popular in snacks, nuts usually have many more calories per gram of fiber than
legumes, and more than half of them have 3–6 balance scores between −9 and −60.
Key Foods for Americans

The ease of using omega 3–6 balance scores was illustrated in a recent paper [81]
that reviewed top foods in a USDA Key Foods list [85]. The list contains 538
foods that were observed being consumed by Americans during 2007–2008 [86].
The unweighted average score of the top 100 items is about −6, equivalent to an
HRA value of 78% n-6 in HUFA. Removing ten food items with the most nega-
tive scores gave 90 remaining items with an unweighted average of about −3. This
simple step shifted the predicted HRA value from the typical American value near
78% to the Mediterranean value near 60% [81]. Not surprisingly, the items that were
removed are not common in traditional Mediterranean foods: soybean oil, −50; may-
onnaise, −46; tub margarine, −39; microwave popcorn, −37; Italian salad dressing,
−35; potato chips, −29; stick margarine, −28; vegetable shortening, −28; peanut but-
ter, −24; tortilla chip snacks, −24. Furthermore, Mediterranean menus include some
seafood items, and there were none in the United States’ top 100 foods. If some were
added to the 90 remaining items, the resulting overall average 3–6 balance would
have an even more positive value. Explicit information on the 3–6 balance of a food
gives an easy way to practice primary prevention. No prescriptions are needed.
The Seven Countries Study started long ago to examine CVD-associated risk
factors in different countries with an awareness that Mediterranean people had a
lower incidence of CVD than did Americans and that the CVD incidence was still
lower among people in Japan. The sense that some populations were unknowingly
practicing primary prevention triggered the large epidemiological study after World
War II. While clinical investigators put much attention on associated blood choles-
terol values, subsequent reports recognized that the wide range of ethnic food habits
of different populations causes a wide range of measured blood HUFA proportions
that is associated closely with CVD mortality [13,14]. Figure 18.5 reflects those val-
ues, and Figure 18.7 shows how the average % n-6 in HUFA associates with aver-
age omega 3–6 balance scores of different ethnic diets. Values of 30%–40% n-6
in HUFA associate with traditional Japanese foods (with an average balance score
near +1); however, HRA values in Japan are rising steadily as younger generations
eat more Western foods [49,87]. In addition, the traditional Mediterranean diet is
shifting under similar influences [88], and the average balance score seems likely to
become more negative than −3 (Figure 18.7).
The diet changes illustrate how a population’s food habits change under marketing
influences and how unintended consequences can follow food decisions uninformed
Jap
Am
M
Jap
an
ed
eri
an
Gr
tra
ite
Eu
can
mo
ee
rra
dit
rop
nla
/U
ion
ne
de
ean
SA
nd
a
rn
al
% omega-6 in blood HUFA

28% 33% 38% 43% 48% 53% 58% 63% 68% 73% 78% 83%
Average food 3–6 balance score
+3 +2 +1 0 –1 –2 –3 –4 –5 –6 –7
FIGURE 18.7 Relating blood HRA values with food balance scores. The horizontal bars
indicate the approximate HRA values reported for different populations.
about mediators of disease. Imprecise advice from health professionals to eat in a

healthy way is not adequate to prevent the food-related problems noted in this chap-
ter. Rather, the professionals and the public need explicit information to identify and
prevent the preventable nutrient imbalances that cause the mediators of disease. The
explicit molecular processes described produce explicit mediator actions that fit in
the context of valid surrogates for harmful clinical outcomes. The explicit informa-
tion on the balance of the 11 n-3 and n-6 nutrients in thousands of foods gives readers
the tools to choose explicit foods and prevent eating an undesired balance of those
important nutrients.
CONCLUSIONS
Health-care professionals identify many signs and symptoms that predict risk of dis-
ease and death, and they use them as targets to treat. However, primary prevention
of the need to treat requires a sharp focus on identifying and preventing explicit pre-
ventable factors that cause those signs and symptoms. Evidence supports the hypoth-
esis that a preventable imbalance between n-3 and n-6 nutrients combines with an
imbalance in the intake and use of food energy to cause harmful health conditions
in America. We have tools that help consumers convert imprecise nutrition advice
into explicit actions that make voluntary changes in their daily life. Healthy people
do not need treatments.
ACKNOWLEDGMENT
Dr. N. Schoene provided helpful advice in preparing this chapter.
REFERENCES
1. Sadik CD, Luster AD. 2012. Lipid-cytokine-chemokine cascades orchestrate leukocyte
recruitment in inflammation. J Leukoc Biol 91(2):207–215.
2. Lee TH, Menica-Huerta JM, Shih C, Corey EJ, Lewis RA, Austen KF. 1984.
Characterization and biologic properties of 5,12-dihydroxy derivatives of eicosapentae-
noic acid, including leukotriene B5 and the double lipoxygenase product. J Biol Chem
259:2383–2389.
3. Lee TH, Sethi T, Crea AE et al. 1988. Characterization of leukotriene B3: Comparison of
its biological activities with leukotriene B4 and leukotriene B5 in complement receptor
enhancement, lysozyme release and chemotaxis of human neutrophils. Clin Sci (Lond)
74:467–475.
4. Terano T, Salmon JA, Moncada S. 1984. Biosynthesis and biological activity of leukot-
riene B5. Prostaglandins 27:217–232.
5. Mohrhauer H, Holman, RT. 1963. The effect of dose level of essential fatty acids upon
fatty acid composition of the rat liver. J Lipid Res 4:151–159.
6. Mohrhauer H, Holman RT. 1963. Effect of linolenic acid upon the metabolism of lin-
oleic acid. J Nutr 81:67–74.
7. Hansen AE, Wiese HF, Boelsche AN, Haggard ME, Adam DJD, Davis H. 1963. Role of
linoleic acid in infant nutrition. Clinical and chemical study of 428 infants fed on milk
mixtures varying in kind and amount of fat. Pediatrics 31:171–192.
8. Cuthbertson WFJ. 1976. Essential fatty acid requirements in infancy. Am J Clin Nutr
20:559–568.
9. Lands WEM, Morris AJ, Libelt B. 1990. Quantitative effects of dietary polyunsaturated
fats on the composition of fatty acids in rat tissues. Lipids 25:505–516.
10. Lands WEM, Libelt B, Morris A et al. 1992. Maintenance of lower proportions of n-6
eicosanoid precursors in phospholipids of human plasma in response to added dietary
n-3 fatty acids. Biochem Biophys Acta 1180:147–162.
11. Lands WEM. 2003. Functional foods in primary prevention or nutraceuticals in second-
ary prevention? Curr Topics Nutraceutical Res 1:113–120.
12. Strandjord SE, Lands B, Hibbeln JR. Validation of an equation predicting highly unsatu-
rated fatty acid compositions of human blood fractions from dietary intake. Nutr Metab
(submitted).
13. Lands WEM. 2003. Diets could prevent many diseases. Lipids 38:317–321.
14. Lands B. 2008. A critique of paradoxes in current advice on dietary lipids. Prog Lipid
Res 47:77–106.
15. Ameur A, Enroth S, Johansson A et al. 2012. Genetic adaptation of fatty-acid metabo-
lism: A human-specific haplotype increasing the biosynthesis of long-chain Omega-3
and Omega-6 fatty acids. Am J Hum Genet 90:809–820.
16. Sergeant S, Hugenschmidt CE, Rudock ME et al. 2012. Differences in arachidonic
acid levels and fatty acid desaturase (FADS) gene variants in African Americans
and European Americans with diabetes or the metabolic syndrome. Br J Nutr
107:547–555.
17. Bäck M, Dahlén SE, Drazen JM et al. 2011. International Union of Basic and Clinical
Pharmacology. LXXXIV: Leukotriene receptor nomenclature, distribution, and patho-
physiological functions. Pharmacol Rev 63:539–584.
18. Smith WL, Urade Y, Jakobsson PJ. 2011. Enzymes of the cyclooxygenase pathways of
prostanoid biosynthesis. Chem Rev 111:5821–5865.
19. Cook HW, Lands WEM. 1975. Further studies of the kinetics of oxygenation of arachi-
donic Acid by soybean lipoxygenase. Can J Biochem 53:1220–1231.
20. Hemler ME, Lands WEM. 1980. Evidence for a peroxide-initiated free radical mecha-
nism of prostaglandin biosynthesis. J Biol Chem 255:6253–6261.
21. Kulmacz RJ, Pendleton RB, Lands WEM. 1994. Interaction between peroxidase
and cyclooxygenase activities in prostaglandin-endoperoxide synthase. J Biol Chem
269:5527–5536.
22. Kulmacz RJ, Lands WEM. 1997. Peroxide tone in eicosanoid signaling. In Oxidative
Stress and Signal Transduction, eds. H.J. Forman and E. Cadenas. pp. 134–156. New
York: Chapman & Hall.
23. Wada M, DeLong CJ, Hong YH et al. 2007. Enzymes and receptors of prostaglandin
pathways with arachidonic acid-derived versus eicosapentaenoic acid derived substrates
and products. J Biol Chem 282:22254–22266.
24. Murphy RC, Pickett WC, Culp BR, Lands WEM. 1981. Tetraene and pentaene leukot-
rienes: Selective production from murine mastocytoma cells after dietary manipulation.
Prostaglandins 22:613–622.
25. Grimminger F, Wahn H, Mayer K, Kiss L, Walmrath D, Seeger W. 1997. Impact of
arachidonic versus eicosapentaenoic acid on exotonin-induced lung vascular leakage:
Relation to 4-series versus 5-series leukotriene generation. Am J Respir Crit Care Med
5:513–519.
26. Lands WEM, Culp BR, Hirai A, Gorman, R. 1985. Relationship of thromboxane gen-
eration to the aggregation of platelets from humans: Effects of eicosapentaenoic acid.
Prostaglandins 30:819–825.
27. Aoki T, Narumiya S. 2012. Prostaglandins and chronic inflammation. Trends Pharmacol
Sci 33(6):304–311.
28. Furuyashiki T, Narumiya S. 2011. Stress responses: The contribution of prostaglandin

E(2) and its receptors. Nat Rev Endocrinol 7:163–175.
29. Yuhki K, Kojima F, Kashiwagi H et al. 2011. Roles of prostanoids in the pathogenesis of
cardiovascular diseases: Novel insights from knockout mouse studies. Pharmacol Ther
129:195–205.
30. Spite M, Hellmann J, Tang Y et al. 2011. Deficiency of the leukotriene B4 receptor,
BLT-1, protects against systemic insulin resistance in diet-induced obesity. J Immunol
187:1942–1949.
31. Narumiya S, Furuyashiki T. 2011. Fever, inflammation, pain and beyond: Prostanoid
receptor research during these 25 years. FASEB J 25:813–818.
32. Tanaka K, Furuyashiki T, Kitaoka S et al. 2012. Prostaglandin E2-mediated attenuation
of mesocortical dopaminergic pathway is critical for susceptibility to repeated social
defeat stress in mice. J Neurosci 32:4319–4329.
33. Studer M, Briel M, Leimenstoll B, Glass TR, Bucher HC. 2005. Effect of different
antilipidemic agents and diets on mortality: A systematic review. Arch Intern Med
165:725–730.
34. Kolata G. 1987. Heart institute is major player in clinical trials. Science 237:851–853.
35. Dolecek TA, Granditis G. 1991. Dietary polyunsaturated fatty acids and mortality in the
Multiple Risk Factor Intervention Trial (MRFIT). World Rev Nutr Diet 66:205–216.
36. De Gruttola VG, Clax P, DeMets DL et al. 2001. Considerations in the evaluation of sur-
rogate endpoints in clinical trials: Summary of a national institutes of health workshop.
Contr Clin Trial 22:485–502.
37. Consensus conference. 1985. Lowering blood cholesterol to prevent heart disease.
JAMA 253:2080–2086.
38. Centers for Disease Control and Prevention: A Public Health Action Plan to Prevent
Heart Disease and Stroke. 2012. http://www.cdc.gov/dhdsp/action_plan/ (accessed on
February 25, 2013).
39. AHA Statistics Committee and Stroke Statistics Subcommittee. 2011. Heart disease
and stroke statistics—2011 update. http://circ.ahajournals.org/content/123/4/e18.extract
(accessed on February 25, 2013).
40. Austin MA. 1989. Plasma triglyceride as a risk factor for coronary heart disease. The
epidemiologic evidence and beyond. Am J Epidemiol 129:249–259.
41. National Research Council. 1989. Recommended Dietary Allowances, 10th edn.,
Washington, DC: National Academy Press.
42. Han CY, Umemoto T, Omer M et al. 2012. NADPH oxidase-derived reactive oxygen
species increases expression of monocyte chemotactic factor genes in cultured adipo-
cytes. J Biol Chem 287:10379–10393.
43. Lambertucci RH, Hirabara SM, Silveira Ldos R, Levada-Pires AC, Curi R, and Pithon-
Curi TC. 2008. Palmitate increases superoxide production through mitochondrial
electron transport chain and NADPH oxidase activity in skeletal muscle cells. J Cell
Physiol 216:796–804.
44. Ramsden CE, Ringel A, Feldstein AE et al. 2012. Lowering dietary linoleic acid reduces
bioactive oxidized linoleic acid metabolites in humans. Prostaglandins Leukot Essent
Fatty Acids 87:135–141.
45. Zhang X, Dong F, Ren J, Driscoll MJ, Culver B. 2005. High dietary fat induces NADPH
oxidase-associated oxidative stress and inflammation in rat cerebral cortex. Exp Neurol
191:318–325.
46. Homko CJ, Cheung P, Boden G. 2003. Effects of free fatty acids on glucose uptake and
utilization in healthy women. Diabetes 52:487–491.
47. Verschuren WM, Jacobs DR, Bloemberg BP et al. 1995. Serum total cholesterol and
long-term coronary heart disease mortality in different cultures. Twenty-five year
follow-up of the seven countries study. JAMA 274:131–136.
48. Ogushi Y, Hamazaki T, Kirihara Y. 2009. Blood cholesterol as a good marker of health
in Japan. World Rev Nutr Diet 100:63–70.
49. Lands WEM, Hamazaki T, Yamazaki K et al. 1990. Changing dietary patterns. Am J Clin
Nutr 51:991–993.
50. Ross R. 1999. Atherosclerosis is an inflammatory disease. Am Heart J 138(5 Pt 2):
S419–S420.
51. Kastelein JJ, Akdim F, Stroes ES et al. 2008. ENHANCE Investigators: Simvastatin with
or without ezetimibe in familial hypercholesterolemia. N Engl J Med 358:1431–1443.
52. Carey J. 2008. Do cholesterol drugs do any good? http://www.businessweek.com/
magazine/content/08_04/b4068052092994.htm (accessed on February 25, 2013).
53. Carey J. 2008. Heart disease: Not about cholesterol? http://www.businessweek.com/
bwdaily/dnflash/content/apr2008/db20080414_688906.htm (accessed on February 25,
2013).
54. Couzin J. 2008. Cholesterol veers off script. Science 322:220–223.
55. Ridker PM, Danielson E, Fonseca FA et al. 2008. JUPITER Study Group: Rosuvastatin
to prevent vascular events in men and women with elevated C-reactive protein. N Engl J
Med 359:2195–2207.
56. James M, Proudman S, Cleland L. 2010. Fish oil and rheumatoid arthritis: Past, present
and future. Proc Nutr Soc 69:316–323.
57. Trelle S, Reichenbach S, Wandel S et al. 2011. Cardiovascular safety of non-steroidal
anti-inflammatory drugs: Network meta-analysis. BMJ 342:c7086. doi: 10.1136/bmj.
c7086.
58. Collins FD, Sinclair AJ, Royle JP, Coats DA, Maynard AT, Leonard RF. 1971. Plasma
lipids in human linoleic acid deficiency. Nutr Metab 13:150–167.
59. Harris WS, Mozaffarian D, Rimm E et al. 2009. Omega-6 fatty acids and risk for car-
diovascular disease: A science advisory from the American Heart Association Nutrition
Subcommittee of the Council on Nutrition, Physical Activity, and Metabolism; Council
on Cardiovascular Nursing; and Council on Epidemiology and Prevention. Circulation
119:902–907.
60. Ramsden CE, Zamora D, Leelarthaepin B et al. 2013. Use of dietary linoleic acid for
secondary prevention of coronary heart disease and death: Evaluation of recovered data
from the Sydney Diet Heart Study and updated meta-analysis. BMJ 346:e8707. doi:
10.1136/bmj.e8707.
61. Ramsden CE, Hibbeln JR, Majchrzak SF, Davis JM. 2010. n-6 fatty acid-specific and
mixed polyunsaturate dietary interventions have different effects on CHD risk: A meta-
analysis of randomised controlled trials. Br J Nutr 104:1586–1600.
62. Blasbalg TL, Hibbeln JR, Ramsden CE, Majchrzak SF, Rawlings RR. 2011. Changes in
consumption of omega-3 and omega-6 fatty acids in the United States during the 20th
century. Am J Clin Nutr 93:950–962.
63. Lands B. 2009. Planning primary prevention of coronary disease. Curr Atheroscler Rep
11:272–280.
64. Lands WEM. 2005. Fish, Omega-3 and Human Health, 2nd edn., Champaign, IL: AOCS
Press.
65. Gu Y, Schupf N, Cosentino SA, Luchsinger JA, Scarmeas N. 2012. Nutrient intake and
plasma β-amyloid. Neurology 78:1832–1840.
66. Cockbain AJ, Toogood GJ, Hull MA. 2012. Omega-3 polyunsaturated fatty acids for the
treatment and prevention of colorectal cancer. Gut 61:135–149.
67. Alvheim AR, Malde MK, Osei-Hyiaman D et al. 2012. Dietary linoleic acid elevates
endogenous 2-AG and anandamide and induces obesity. Obesity 20:1984–1994.
68. Hibbeln JR. 2009. Depression, suicide and deficiencies of omega-3 essential fatty acids
in modern diets. World Rev Nutr Diet 99:17–30.
69. Hibbeln JR, Nieminen LR, Blasbalg TL, Riggs JA, Lands WEM. 2006. Healthy intakes
83:1483S–1493S.
70. Freeman MP, Hibbeln JR, Wisner KL et al. 2006. Omega-3 fatty acids: Evidence basis
for treatment and future research in psychiatry. J Clin Psychiatry 67:1954–1967.
71. Wei C, Hua J, Bin C, Klassen K. 2010. Impact of lipid emulsion containing fish oil on
outcomes of surgical patients: Systematic review of randomized controlled trials from
Europe and Asia. Nutrition 26:474–481.
72. Lands B. 2011. Prevent the cause, not just the symptoms. Prostaglandins Other Lipid
Mediat 96:90–93.
73. Lands B. 2009. Measuring blood fatty acids as a surrogate indicator for coronary heart
disease risk in population studies. World Rev Nutr Diet 100:22–34.
74. Lin YH, Salem N Jr, Wells EM et al. 2012. Automated high-throughput fatty acid
analysis of umbilical cord serum and application to an epidemiological study. Lipids
47:527–539.
75. Stark KD, Beblo S, Murthy M et al. 2005. Comparison of bloodstream fatty acid com-
position from African-American women at gestation, delivery, and postpartum. J Lipid
Res 46:516–525.
76. Stark KD. 2008. The percentage of n-3 highly unsaturated fatty acids in total HUFA as
a biomarker for omega-3 fatty acid status in tissues. Lipids 43:45–53.
77. Armstrong JM, Metherel AH, Stark KD. 2008. Direct microwave transesterification of
fingertip prick blood samples for fatty acid determinations. Lipids 43:187–196.
78. Diet Balance Spreadsheet. http://www.efaeducation.org/sig/dietbalance.html (accessed
on February 28, 2013).
79. USDA Nutrient Database. http://www.nal.usda.gov/fnic/foodcomp/search/ (accessed on
February 28, 2013).
80. Software to Choose Foods: A free interactive a computer-aided personal food choice
program called KIM-2 (Keep It Managed, ver.2). http://www.efaeducation.org/sig/kim.
html
81. Lands B, Lamoreaux E. 2012. Describing essential fatty acid balance as 3–6 differences
rather than 3/6 ratios. Nutr Metab 9:46–54.
82. Omega 3–6 Balance Scores. http://www.fastlearner.org/Omega3-6Balance.htm (accessed
on February 28, 2013).
83. An App for Omega 3–6 Balance Scores. http://www.fastlearner.org/Omega3-
6BalanceApp.htm (accessed on February 28, 2013).
84. Essential Fats In Food Oils. http://www.efaeducation.org/sig/esstable.html
85. Haytowitz DB. 2012. List of key foods based on NHANES 2007–08. http://www.
ars.usda.gov/SP2UserFiles/Place/12354500/Data/KeyFoods/Keyfoods_0708.xlsx
(accessed on February 28, 2013).
86. Birtwhistle R. 2008. What ever happened to the Mediterranean diet? QJM 101:741–742.
19 Spices and Dietary
Supplements with
Anti-Inflammatory Activity
Bharat B. Aggarwal and David Heber
CONTENTS
Introduction............................................................................................................. 317
History of Spices..................................................................................................... 318
Turmeric and Curcuminoids................................................................................... 319
Chili and Capsaicin................................................................................................. 321
Ginger and Gingerol............................................................................................... 321
Black Pepper and Piperine...................................................................................... 322
Cinnamon and Cinnamaldehyde............................................................................. 322
Fenugreek and Parthenolide.................................................................................... 323
Homologous Structures and In Vitro Antioxidant Function of Spices.................... 323
Spices and In Vivo Inhibition of Lipid Oxidation................................................... 324
Dietary Supplements Containing Spices................................................................. 325
Conclusion.............................................................................................................. 326
References............................................................................................................... 326
INTRODUCTION
Although the saying “Add spice to your life,” is highly Western, much of the Western
diet is spice-free. Similarly, some Western names of people such as Anise, Ginger,
Rosemary, Mace, Pepper, Basil, Tulsi, Sage, Jasmine, Angelica, Curry, or Chili are
also connected with spices. All this indicates Western countries have been always
fascinated by the spices. As mentioned, much of the Western diet is spice-free.
Ketchup and mustard are the primary American spices along with pepper and salt,
which may not even be a spice. In fact, while fat, salt, and sugar are used to flavor
processed foods, which are part of an obesogenic diet, it may be that spices can pro-
mote increased intakes of fruits and vegetables. There are already examples of spices
that have found their way onto the American palate. Oregano was virtually unknown
in America until American pizza was developed using oregano and tomato sauce.
The flavors and aromas of pizza are related almost exclusively to oregano. Spices can
have both direct antioxidant benefits for the diet but can also be used to substitute for
excess salt and to promote the intake of fruits and vegetables.
317
A spice is edible, aromatic, and dried. It comes from a plant’s root, bark, flower,
bud, leaves, or stem. Since herbs are dried, they concentrate antioxidants, such as
the polyphenols found in fruits and vegetables, so that their antioxidant potency on a
weight basis far outstrips most fruits and vegetables.
Phenolic compounds in these plant materials are closely associated with their
antioxidant activity, which is mainly due to their redox properties and their capacity
to block the production of reactive oxygen species. More recently, their ability to
interfere with signal transduction pathways involving various transcription factors,
protein kinases, phosphatases, and other metabolic enzymes has also been demon-
strated. Volatile or aromatic antioxidants are also important. While pomegranate
has one of the greatest antioxidant potencies among fruits based on its polyphenols,
its antioxidant potency on a weight basis is outstripped by clove. Spices such as
clove contain aromatic or volatile antioxidants, which impart the flavor and aromas
characteristic of spices and add additional antioxidant potency. However, spices are
consumed in much smaller amounts than fruits or vegetables.
Consumed in the nutritional range of 500 mg to 1 g, spices can contribute signifi-
cantly to antioxidant potency of the diet and may have additional biological effects.
In addition to antioxidant potency, many spice phytonutrients have additional prop-
erties including anti-inflammatory properties, which will be the focus of this chapter.
HISTORY OF SPICES
Throughout the ancient and medieval world, spices carried a high value and were
considered so special that they formed a vital part of international trade. Spices
have been used for centuries, serving a variety of purposes in a wide variety of
cultures. They have been used as flavor agents, as colorants to add special taste
to dishes, and also as preservatives to prevent the growth of bacteria. But today,
the importance of spices has become even more evident than at any other time
throughout history.
Alexander the Great’s campaigns in Central Asia around 330 BC are often
credited with the dissemination and adoption of herbs and spices among many
cultures because they introduced Asian, Persian, Indian, and Greek cultures and
ideas [1,2]. Early records indicate that herbs and spices were used as medicines in
ancient Egypt and Asia and as food preservatives in ancient Rome and Greece [3].
Herbs and spices continued to be used during the middle ages for flavoring, food
preservation, and/or medicinal purposes [4]. In countries such as India where pov-
erty and malnutrition are unbridled, knowledge of plant-derived antioxidants and
spices could reduce the cost of health care. India has a rich history of using various
herbs, spices, and herbal components for treating various diseases [5]. It has been
believed for some time that dietary factors play a key role in the development of
some human diseases, including cardiovascular disease. Several herbs and spices
of culinary origin were included in the approved monographs, such as caraway oil
and seed, cardamom seed, cinnamon bark, cloves, coriander seed, dill seed, fennel
oil and seed, garlic, ginger root, licorice root, mint oil, onion, paprika, parsley herb
and root, peppermint leaf and oil, rosemary, sage, thyme, turmeric root, and white
mustard seed [6].
Spices and Dietary Supplements with Anti-Inflammatory Activity 319
The use of herbal medicine has skyrocketed over the last 10 years, with out-
of-pocket costs estimated at more than $5 billion in the United States alone. Most
herbal medicinals have multiple effects modulating the cardiovascular system
[7]. In the traditional Indian systems of medicine Ayurveda and Siddha, various
spices and herbs are described to possess medicinal properties, such as being anti-
thrombotic, antiatherosclerotic, hypolipidemic, hypoglycemic, anti-inflammatory,
antiarthritic, etc. [8]. Because spices have very low calorie content and are relatively
inexpensive, they are reliable sources of antioxidants and other potential bioactive
compounds in the diet [9].
Spices that are primarily used in India and surrounding countries were sought for
centuries by such great explorers as Marco Polo, Vasco de Gama, and Christopher
Columbus. The spices are primarily herbs, including leaves (as in mint and cilantro),
seeds (e.g., fenugreek), barks (e.g., cinnamon), fruit (e.g., black pepper, red chili,
cardamom, mango, and pomegranate), and roots (e.g., turmeric and licorice) of
plants that have been used for centuries to preserve food, enhance its color, make it
more aromatic, and improve its taste; perhaps more importantly, the spices were used
to improve the digestive qualities and medicinal value of the food. Several of these
spices have been shown to modulate inflammatory pathways [10].
TURMERIC AND CURCUMINOIDS

Turmeric (Curcuma longa) has been described in the literature of Ayurveda (science
of long life), dating from about 3000 BC, for a wide variety of ailments including
obesity. The name turmeric derives from the French word terre-merite (meritori-
ous earth), referring to the color of ground turmeric, which resembles a mineral
pigment. It is known as Safaran des Indes in French and simply as yellow root
in many languages. In many cultures, its name is based on the Latin curcuma. In
Sanskrit, turmeric has at least 53 different names, including jawarantika, which
destroys fever; mehagni, killer of fat; rabhangavasa, which dissolves fat; and ratri-
manika, which glows in the night (referring to its fluorescent property). One of us
(DH) actually experienced this last property when taking a picture of an open sack
of turmeric outside a spice shop in Jaipur. On using the flash, the turmeric flashed
back brightly. When the flash was turned off, the turmeric was simply a bright yel-
low powder but did not flash.
Turmeric has been found to contain more than 100 different chemicals [11–13].
The main component is a volatile oil, containing turmerone, and other coloring
agents called curcuminoids. Curcumin is the yellow pigment present in the spice
turmeric and is a diferuloylmethane. Most research on turmeric has centered on
curcumin and curcuminoids. Curcuminoids consist of curcumin demethoxycur-
cumin, 5ʹ-methoxycurcumin, and dihydrocurcumin, which are natural antioxi-
dants [14]. In a standard preparation, turmeric contains moisture (>9%), curcumin
(5%–6.6%), extraneous matter (<0.5% by weight), mold (<3%), and volatile oils
(<3.5%). There are also reports of omega-3 fatty acid in turmeric. The volatile
oils include d-α-phellandrene, d-sabinene, cinol, borneol, zingiberene, and ses-
quiterpenes. Turmeric contains a variety of sesquiterpenes, including germacrone,
termerone, ar-(+)-, α- and β-termerones, β-bisabolene, a-curcumene, zingiberenel,
β-sesquiphellanderene, bisacurone, curcumenone, dehydrocurdione, procurcuma-

diol, bis-acumol, curcumenol, isoprocurcumenol, epiprocurcumenol, procurcum-
enol, zedoaronediol, and curlone, many of which are specific for species. The
responsible components for the aroma of turmeric are turmerone, arturmerone, and
zingiberene. The rhizomes are also reported to contain four new polysaccharides—
ukonans along with stigmasterole, β-sitosterole, cholesterole, and 2-hydroxymethyl
anthraquinone. Nutritional analysis showed that 100 g of turmeric contains 354
kcal, 10 g of total fat, 3 g of saturated fat, 38 mg of sodium, 2525 mg of potassium,
65 g of total carbohydrates, 21 g of dietary fiber, 3 g of sugars, and 8 g of protein.
Curcumin has been reported to modulate numerous targets in inflammatory
pathways that have been linked to obesity and insulin resistance. First, curcumin
has been shown to downregulate the expression of TNF in various tissues [15].
Second, curcumin suppresses NF-κB activation induced by a wide variety of
inflammatory agents through inhibition of degradation of IκBα [16]. Third, cur-
cumin can inhibit the activation of IKK linked to the activation of NF-κB, and
this leads to the suppression of expression of inflammatory enzymes and factors
including cyclooxygenase-2 (COX-2) and vascular endothelial growth factor [17].
Fourth, the spice has been shown to downregulate the expression of various NF-κB-
regulated proinflammatory adipocytokines including chemokines (such as MCP-1,
MCP-4, and eotaxin) [18] and interleukins (IL-1, IL-6, and IL-8) [19]. Curcumin
also suppresses the expression of plasminogen activator inhibitor type-1 through
the inhibition of the transcription factor early growth response (Egr)-1 gene prod-
uct [20] that has been closely linked with insulin resistance and obesity. Fifth,
curcumin has been reported to mimic most antidiabetic drugs in that it activates
PPAR-γ in hepatic stellate cells [21]. Sixth, it has been shown to downregulate
activation of c-Jun NH2 terminal kinase (JNK) [19]. Seventh, curcumin has been
shown to inhibit the Wnt/β-catenin pathway, which is closely linked to obesity [22].
Curcumin inhibits Wnt pathway signaling through downregulation of the tran-
scription coactivator p300 [23]. Another potential mechanism by which curcumin
may inhibit β-catenin signaling is through inhibition of glycogen synthase kinase
(GSK)-3β, which directly causes the phosphorylation of β-catenin. Curcumin was
found to inhibit GSK-3β with as little as 66 nM IC50 [24]. Eighth, curcumin has
been shown to induce the expression of hemeoxygenase (HO)-1 through the activa-
tion of Nrf2 in pancreatic cells and thus mediate the survival of these cells [25,26].
Ninth, curcumin downregulates the secretion of insulin-like growth factor-1 but
induces the expression of insulin-like growth factor binding protein-3 [27]. Tenth,
curcumin interrupts leptin signaling by reducing phosphorylation levels of the
leptin receptor (Ob-R) and its downstream targets [28]. In addition, curcumin sup-
presses gene expression of Ob-R in HSCs. Finally, curcumin has been reported to
increase the expression of adiponectin, which negatively controls obesity [29]. All
of these molecular mechanisms may be acting in the animal models and human
studies that have been conducted.
Since the report in 1972 that curcumin could lower blood glucose levels in
human diabetic subjects [30,31], more than 3000 reports have been published on
curcumin, with more than 300 on its effects on obesity and obesity-associated
diseases.
Several pilot studies have been done in human subjects with curcumin to exam-
ine its effect on obesity-related parameters. One of the first studies showed that cur-
cumin lowered blood sugar levels in diabetic patients [30]. Another study examined
the effect of curcumin in humans on the levels of HDL- and LDL-cholesterol [32].
Administration of 10 mg curcumin per day for 30 days to eight human subjects
increased HDL-cholesterol, decreased LDL-cholesterol, and increased APO A but
decreased APO B and APO A/B. The same group reported another study with cur-
cumin in human subjects with atherosclerosis [33]. In this study, 10 mg curcumin
was administered twice a day for 15 days to 16 men and 14 women. Curcumin
significantly lowered the levels of plasma fibrinogen in both men and women.
CHILI AND CAPSAICIN

Capsaicin is an active component of red chili. Numerous studies suggest that cap-
saicin has a potential against obesity and insulin resistance [34]. It has been shown
that capsaicin can suppress both NF-κB and signal transducer and activators of
transcription-3 pathways [35,36]. Capsaicin has been shown to induce apoptosis and
inhibit adipogenesis [37]. This nutraceutical was found to modulate adipokine gene
expression and protein release from adipocytes derived from obese mice [38]. These
investigators showed that capsaicin can also suppress the inflammatory response
of macrophages derived from adipose tissue. Capsaicin-desensitized rats exhibit a
long-term decrease in body fat and in brown adipose tissue [39]. Capsaicin mediates
its effects against adipogenesis and obesity through numerous mechanisms. One of
the mechanisms is the activation of its receptor, transient receptor potential vanil-
loid type 1 (TRPV-1) channels, in adipocytes [34,40]. In fact, TRPV-1-null mice are
protected from diet-induced obesity [41]. All these studies suggest the potential role
of red chili in preventing obesity.
A placebo-controlled, double-blind clinical trial of capsaicin in obese subjects has
been carried out. Body fat decreased in this eight-week intervention trial (P < 0.05)
[42]. A 12-week, placebo-controlled, double-blind, randomized study examined the
effect of a novel capsinoid on fatness and energy metabolism in humans [43]. These
investigators explored the safety and efficacy of capsinoids taken orally (6 mg/day)
for weight loss, fat loss, and change in metabolism. Treatment appeared to be safe
and was associated with abdominal fat loss. Capsinoid ingestion was associated with
an increase in fat oxidation. No significant difference in total change in adiposity
was noted, but abdominal adiposity decreased more in the capsinoid group than in
the placebo group, and this change correlated with the change in body weight. The
authors identified two common genetic variants that may be predictors of therapeutic
response. These included TRPV-1 Val585Ile and UCP2 -866 G/A, and they corre-
lated with change in abdominal adiposity.
GINGER AND GINGEROL

Gingerol is one of the most active compounds in ginger (Zingiber officinale).
Methanolic extracts of dried ginger have been shown to prevent fructose-induced
elevation of serum cholesterol, triglycerides, glucose, insulin, and gain in body
weight in rats [44]. Ginger extracts containing gingerol were found to enhance adi-
pocyte differentiation [45] and insulin-sensitive glucose uptake, thus suggesting its
potential for treating diabetes. Zingerone, another component of ginger, was found
to suppress the inflammatory responses of adipose tissue in obesity by suppressing
the inflammatory action of macrophages and release of MCP-1 from adipocytes [19].
Thus, these studies also suggest that ginger has potential in preventing obesity and
obesity-linked metabolic effects.
BLACK PEPPER AND PIPERINE

Piperine is an active component of black pepper that can effectively suppress lipid
peroxidation [46,47] and enhance the bioavailability of curcumin and other drugs
through the inhibition of drug-metabolizing enzymes in the liver [48]. The effect of
piperine was examined in Streptozotocin (STZ)-induced diabetic Sprague–Dawley
rats [49]. Treatment with piperine reversed the diabetic effects on glutathione disul-
fide (GSSG) concentration in the brain, on renal glutathione peroxidase (GPO) and
superoxide dismutase (SOD) activities, and on cardiac glutathione reductase activity
and lipid peroxidation. Piperine treatment did not, however, reverse the effects of
diabetes on hepatic glutathione (GSH) concentrations, lipid peroxidation, or GPO or
catalase activities; on renal SOD activity; or on cardiac GPO or catalase activities.
These data indicate that subacute treatment with piperine is only partially effective
as an antioxidant therapy in diabetes.
CINNAMON AND CINNAMALDEHYDE

Most of the studies in humans involving the effects of spices on obesity and diabetes
have tested cinnamon [50–60]. All these studies suggested that cinnamon modulated
the levels of various biomarkers linked with insulin resistance and obesity favor-
ably. For instance, Crawford [50] showed that taking cinnamon could lower serum
HbA1C in type 2 diabetes with HbA1C > 7. The difference between the groups was
found to be statistically significant. In another study, cinnamon was also found to
affect postprandial blood glucose levels, gastric emptying, and satiety in human
subjects [52].
Cinnamaldehyde is one of the active components of cinnamon. The essential oil of
cinnamon bark is about 90% cinnamaldehyde. There are numerous reports about the
role of cinnamon in obesity and diabetic conditions [61–65]. Antidiabetic effects of
cinnamon extracts have been demonstrated in db/db mice [66]. In vitro studies have
shown that cinnamon can increase the expression of PPAR-γ/α and their target genes
such as LPL, CD 36, GLUT 4, and ACO in 3T3-L1 adipocytes [66]. The transactivi-
ties of both full-length and ligand-binding domain of PPAR-γ/α were activated by cin-
namon. Furthermore, this spice in vivo was found to activate PPARγ and α, resulting
in improved insulin resistance and reduced fasting glucose, FFA, LDL-cholesterol,
and aspartate aminotransferase levels in high-caloric-diet-induced obesity and db/db
mice in its water extract form [67]. Another study showed that water extracts of cin-
namon reverse TNF-induced overproduction of intestinal apoB48-containing lipo-
protein in vivo by modulation of expression of inflammatory cytokines, insulin, and
lipoprotein-signaling pathways [68]. Similar to insulin, cinnamon has been shown to

regulate protein phosphorylation and dephosphorylation of insulin receptor through
inhibition of PTP-1 [69]. Cao et al. [70] showed that cinnamon-derived nutraceuticals
can increase the insulin receptor β levels and that water-soluble extracts and these
nutraceuticals together can increase the glucose-transporting protein 4 and tristetra-
prolin levels in adipocytes. Tristetraprolin mRNA levels in the adipocytes increased
by almost sixfold. The antidiabetic activity of cinnamon appears to be due to mul-
tiple constituents including cinnamaldehyde, hydrocinnamic acid, polyphenol type
A polymer, dihydroxyhydrocinnamic acid, and proanthocyanidines [55,63,66,71].
FENUGREEK AND PARTHENOLIDE

Fenugreek (Trigonella foenum-graecum) seed, used as a condiment, is documented
for amelioration of abnormalities in lipid homeostasis due to its hypolipidemic prop-
erties. The hypolipidemic effect of a novel thermostable extract of fenugreek seeds
has been examined on differentiating and differentiated 3T3-L1 cells and HepG2
cells cultured in normal or sterol-enriched conditions [72]. These extracts inhibited
accumulation of fat in differentiating and differentiated 3T3-L1 cells via decreased
expression of adipogenic factors such a PPAR-γ, sterol SREBP-1, and C/EBP-α.
Cellular triglycerides and cholesterol concentrations were decreased in HepG2
cells via reduced expression of SREBP-1. These extracts also upregulated LDL-
receptor expression, resulting in enhanced LDL uptake. Treating fat-supplement-fed
C57BL6/J mice with extracts for 15 days resulted in decrease of serum triglyceride,
LDL-cholesterol, and body weight. Thus, these studies suggest that fenugreek has
potential application in the management of dyslipidemia and its associated metabolic
disorders.
HOMOLOGOUS STRUCTURES AND IN VITRO

ANTIOXIDANT FUNCTION OF SPICES
A significant structural homology exists between curcumin, capsaicin derived
from red chili, piperine derived from black pepper, eugenol derived from cloves,
and gingerol derived from ginger. In addition to turmeric, several other spices, for
example, garlic, onions, red pepper, and fenugreek, have been shown to have benefi-
cial hypolipidemic or hypocholesterolemic effects [73–75]. In another study, dietary
supplementation with capsicum pigment but not rosemary suppressed Fe/ascorbic
acid–induced lipid peroxidation in the liver [76]. Several spice-derived nutraceuti-
cals have been identified that can inhibit oxidation of LDL in vitro, including cur-
cumin, capsaicin, eugenol, piperine, zingerone (ginger), and cuminaldehyde (cumin)
[77,78]. Manjunatha and Srinivasan [79] showed that dietary curcumin and capsaicin
inhibited the oxidation of LDL in rats.
Most of these spices also exhibit potent antioxidant activity [80,81]. For instance,
various spice-derived nutraceuticals suppress arachidonic acid–induced plate-
let aggregation in vitro in the following order of potency: eugenol > capsaicin >
curcumin > cinnamaldehyde > piperine [82]. The anti-inflammatory activity of
these spices is also indicated by their ability to directly inhibit 5-lipooxygenase, an
enzyme responsible for leukotriene production [83]. Based on IC50, their ability to
suppress 5-lipooxygenase was found to be eugenol > curcumin > cinnamaldehyde >
piperine > capsaicin.
SPICES AND IN VIVO INHIBITION OF LIPID OXIDATION

Over the past 30 years, there has been accumulating evidence that lipid oxidation can
play an important role in the processes of atherogenesis and carcinogenesis. Specific
proinflammatory oxidized phospholipids that result from the oxidation of LDL phos-
pholipids containing arachidonic acid are recognized by the innate immune system
in animals and humans and lead to inflammation, which can promote atherogenesis
and carcinogenesis. Fogelman et al. [84] reported that malondialdehyde, an obligate
product of the oxidation of arachidonic acid by lipoxygenase pathways, could cause
Schiff’s base formation with the ε amino groups of apolipoprotein B lysine residues
in LDL. Altered lipoproteins bind to macrophage scavenger receptors, resulting in
cholesteryl ester accumulation and the formation of foam cells.
Oxidatively modified LDL is present in the arterial walls of animals and humans
with atherosclerosis and leads to destabilization of atherosclerotic plaques [85–87].
Malondialdehyde can also react with deoxyadenosine and deoxyguanosine in DNA
and form DNA adducts that are mutagenic. Thus, the formation of malondialdehyde
has implications for atherogenesis and carcinogenesis [88]. Inhibition of the forma-
tion of malondialdehyde by antioxidants during the cooking of hamburger meat may
result in reduced concentrations of malondialdehyde in plasma and urine as the result
of inhibition of malondialdehyde formation ex vivo or the inhibition of its formation
or absorption from the gastrointestinal tract in vivo [89,90]. Such a reduction would
suggest that the processes of lipid peroxidation and DNA adduct formation could be
reduced [91,92].
The in vivo effects of spices on lipid peroxidation were demonstrated in humans
[93]. The effect of an antioxidant spice mixture on malondialdehyde formation
while cooking hamburger meat and its effects on plasma and urinary malondi-
aldehyde concentrations were studied. Eleven healthy volunteers consumed two
kinds of hamburger patties in a randomized order: one patty was seasoned with
a spice blend, and one patty was not seasoned with the spice blend. The produc-
tion of malondialdehyde in the meat and malondialdehyde concentrations in plasma
and urine after ingestion were measured by high-performance liquid chromatog-
raphy (HPLC). Rosmarinic acid from oregano was monitored to assess the effect
of cooking on spice antioxidant content. Forty percent of the added rosmarinic
acid remained in the spiced meat after cooking. There was a 71% reduction in the
malondialdehyde formed during cooking in the meat of the spiced burger patties
compared with the malondialdehyde concentration in the meat of the control pat-
ties. When these two types of patties were fed to volunteers, the plasma malondial-
dehyde concentration increased significantly in the control group as a change from
baseline (P = 0.026). There was a significant time-trend difference between the two
groups. Urinary malondialdehyde concentrations decreased by 49% in subjects
consuming the spiced meat patties compared with subjects consuming the control
patties. The overall effect of adding the spice mixture to hamburger meat before
cooking was a reduction in malondialdehyde concentrations in the meat, plasma,

and urine after ingestion. Gorelik et al. [90] have shown a similar effect adding red
wine to turkey meat before cooking. This effect was likely due to the polyphenols in
red wine but may extend to spices containing polyphenols as well. Therefore, cook-
ing hamburgers or a comparable meat product with a polyphenol-rich spice mixture
can significantly decrease the concentration of malondialdehyde, which suggests
potential health benefits for atherogenesis and carcinogenesis.
DIETARY SUPPLEMENTS CONTAINING SPICES

Within the nutritional range, spices provided within capsules or tablets when taken
with food should have similar effects to spices added to foods. However, there is
a limited amount of data on such dietary supplement bioavailability. Moreover,
for some supplements that have been tested, there is evidence of a lack of bio-
availability when bioavailability is defined by the active substances or marker
compounds of the spice entering the bloodstream. A key example of this issue
is curcumin, which is poorly absorbed from the intestine. Various approaches
including lipid emulsification, processing that results in micron-sized particles,
and simply giving very large amounts of curcumin in the gram range have all been
tried in clinical trials.
When tested in clinical trials with a disease endpoint, dietary supplements
have sometimes been termed nutraceuticals. The term was first coined by Stephen
DeFelice in 1989 from nutrition and pharmaceutical. According to DeFelice, a
nutraceutical can be defined as “a food (or part of a food) that provides medical or
health benefits, including the prevention and/or treatment of a disease” [94,95]. In
stark contrast to this classification, dietary supplements regulated under the Dietary
Supplement and Health Education Act are prohibited from preventing, mitigating, or
treating any disease condition. Therefore, nutraceuticals would have to fall under the
botanical drug classification at the Food and Drug Administration and would require
formal approval prior to marketing.
Beyond the regulatory issues, there has been a great deal of investigation in cell
culture systems looking at mechanisms of action and in animals looking at various
disease models as reviewed in this chapter, but there is a critical lack of human stud-
ies of spices sold as dietary supplements. Intake of any single biochemical or com-
bination of biochemicals believed to represent the action of the corresponding spice
should have at least as strong a biological effect as does the whole spice. The synergy
of the multiple substances found in spices is difficult to reproduce in supplements,
which purify extracts to enrich them in a particular ingredient, often called the active
ingredient. This drug-like approach can cause an imbalance in the spice mixture due
either to the absence of some compounds or excessive amounts of others.
Although over 100 pilot clinical trials have been done with spices and their com-
ponents in human subjects, more research is still needed to demonstrate their poten-
tial to help reduce health risks or aid in weight management. The possibility for
spices to exhibit multiple synergistic effects and the possibility of interacting with
existing drug treatment regimens exists just as it does for drugs. Unlike conventional
drugs, spices have been consumed by healthy people for centuries. Nonetheless,
much more research is needed on dietary supplements in the setting of a healthy

diet that optimizes the potential to see beneficial effects of spices in the nutritional
range. Studies of spices in much higher doses must be done under highly controlled
conditions. More information is needed on bioavailability, metabolism, and chemical
constituents, and these elements should be standard in nutritional research on dietary
supplements based on spices as they are for other phytochemical formulas.
CONCLUSION
The potentials of spices to improve human health and the tastes of healthy foods and
to counteract inflammation are all significant. Consuming spices in gram amounts
has significant effects on biological processes, and these amounts can be incorpo-
rated in the diet. Scientists in the food industry, the government, and academia must
interact to realize the full potential benefits of spices for human health.
REFERENCES
1. USC-UCLA Joint East Asian Studies Center. 1993. Along the silk road, people,
interaction & cultural exchange. http://www.ess.uci.edu/~oliver/silk3.html (accessed on
November 7, 2013).
2. Silk Road Study Group. 2000. Silk road. http://gallery.sjsu.edu/silkroad/intro.htm
(accessed on November 7, 2013).
3. Parry JW. 1953. The Story of Spices. New York: Chemical Publishing Co.
4. McCormick Science Institute. 2008. The history of spices. http://www.mccormickscien-
ceinstitute.com/Spice-Landing/History-of-Spices.aspx (accessed on July 17, 2013).
5. Ali SS, Kasoju N, Luthra A et al. 2008. Indian medicinal herbs as sources of antioxi-
dants. Food Intern. J. 41:1–15.
6. Blumenthal M. 1998. The Complete German Commission E Monographs, Therapeutic
Guide to Herbal Medicines. Austin, TX: American Botanical Council.
7. Frishman HW, Sinatra TS, Moizuddin M. 2004. The use of herbs for treating cardiovas-
cular disease. Semin. Integr. Med. 2:23–35.
8. Srivastava KC, Bordia A, Verma SK. 1995. Curcumin, a major component of food spice
turmeric (curcuma longa) inhibits aggregation and alters eicosanoid metabolism in
human blood platelets. Prostaglandins Leukot. Essent. Fatty Acids 52:223–227.
9. Muthulakshmi V, Vijayakumar V, Vasanthkumar M, Vasanthi HR. 2009. Food is medicine
and medicine is food: A siddha perspective. In: Functional Foods for Chronic Diseases,
Vol. 4, edn. DM. Martirosyan, pp. 274–317. Richardson, TX: D & A Inc/FF Publishing.
10. Aggarwal BB, Van Kuiken ME, Iyer LH, Harikumar KB, Sung B. 2009. Molecular
targets of nutraceuticals derived from dietary spices: Potential role in suppression of
inflammation and tumorigenesis. Exp. Biol. Med. (Maywood) 234:825–849.
11. Nakayama R, Tamura Y, Yamanaka H, Kikuzaki H, Nakatani N. 1993. Curcuminoid pig-
ments from Curcuma domestica. Phytochemistry 33:501–502.
12. Ohshiro M, Kuroyanagi M, Ueno A. 1990. Structures of sesquiterpenes from Curcuma
longa. Phytochemistry 29:2201–2205.
13. Ravindranath V, Satyanarayana MN. 1980. An unsymmetrical diarylheptanoid from
Curcuma longa. Phytochemistry 19:2031–2032.
14. Masuda T, Hidaka K, Shinohara A, Maekawa T, Takeda Y, Yamaguchi H. 1999. Chemical
studies on antioxidant mechanism of curcuminoid: Analysis of radical reaction products
from curcumin. J. Agric. Food Chem. 47:71–77.
15. Chan MM. 1995. Inhibition of tumor necrosis factor by curcumin, a phytochemical.
Biochem. Pharmacol. 49:1551–1556.
16. Singh S, Aggarwal BB. 1995. Activation of transcription factor NF-kappa B is sup-
pressed by curcumin (diferuloylmethane) [corrected]. J. Biol. Chem. 270:24995–25000.
17. Aggarwal BB, Shishodia S, Sandur SK, Pandey MK, Sethi G. 2006. Inflammation and
cancer: How hot is the link? Biochem. Pharmacol. 72:1605–1621.
18. Wang SL, Li Y, Wen Y et al. 2009. Curcumin, a potential inhibitor of up-regulation of
TNF-alpha and IL-6 induced by palmitate in 3T3-L1 adipocytes through NF-kappaB
and JNK pathway. Biomed. Environ. Sci. 22:32–39.
19. Woo HM, Kang JH, Kawada T, Yoo H, Sung MK, Yu R. 2007. Active spice-derived
components can inhibit inflammatory responses of adipose tissue in obesity by sup-
pressing inflammatory actions of macrophages and release of monocyte chemoattractant
protein-1 from adipocytes. Life Sci. 80:926–931.
20. Pendurthi UR, Rao LV. 2000. Suppression of transcription factor Egr-1 by curcumin.
Thromb. Res. 97:179–189.
21. Xu J, Fu Y, Chen A. 2003. Activation of peroxisome proliferator-activated receptor-
gamma contributes to the inhibitory effects of curcumin on rat hepatic stellate cell
growth. Am. J. Physiol. Gastrointest. Liver Physiol. 285:G20–G30.
22. Jaiswal AS, Marlow BP, Gupta N, Narayan S. 2002. Beta-catenin-mediated transacti-
vation and cell-cell adhesion pathways are important in curcumin (diferuylmethane)-
induced growth arrest and apoptosis in colon cancer cells. Oncogene 21:8414–8427.
23. Ryu MJ, Cho M, Song JY et al. 2008. Natural derivatives of curcumin attenuate the Wnt/
beta-catenin pathway through down-regulation of the transcriptional coactivator p300.
Biochem. Biophys. Res. Commun. 377:1304–1308.
24. Bustanji Y, Taha MO, Almasri IM, Al-Ghussein MA, Mohammad MK, Alkhatib HS.
2008. Inhibition of glycogen synthase kinase by curcumin: Investigation by simulated
molecular docking and subsequent in vitro/in vivo evaluation. J. Enzyme Inhib. Med.
Chem. 24:771–778.
25. Andreadi CK, Howells LM, Atherfold PA, Manson MM. 2006. Involvement of Nrf2,
p38, B-Raf, and nuclear factor-kappaB, but not phosphatidylinositol 3-kinase, in induc-
tion of hemeoxygenase-1 by dietary polyphenols. Mol. Pharmacol. 69:1033–1040.
26. Motterlini R, Foresti R, Bassi R, Green CJ. 2000. Curcumin, an antioxidant and anti-
inflammatory agent, induces heme oxygenase-1 and protects endothelial cells against
oxidative stress. Free Radic. Biol. Med. 28:1303–1312.
27. Xia Y, Jin L, Zhang B, Xue H, Li Q, Xu Y. 2007. The potentiation of curcumin on
insulin-like growth factor-1 action in MCF-7 human breast carcinoma cells. Life Sci.
80:2161–2169.
28. Tang Y, Zheng S, Chen A. 2009. Curcumin eliminates leptin’s effects on hepatic stellate
cell activation via interrupting leptin signaling. Endocrinology 150:3011–3020.
29. Weisberg SP, Leibel R, Tortoriello DV. 2008. Dietary curcumin significantly
improves obesity associated inflammation and diabetes in mouse models of diabesity.
Endocrinology 149:3549–3558.
30. Srinivasan M. 1972. Effect of curcumin on blood sugar as seen in a diabetic subject.
Indian J. Med. Sci. 26:269–270.
31. Srinivasan SR, Berenson GS, Radhakrishnamurthy B. 1970. Glycoprotein changes in
diabetic kidneys. Diabetes 19:171–175.
32. Ramirez Bosca A, Soler A, Carrion-Gutierrez MA et al. 2000. An hydroalcoholic extract
of Curcuma longa lowers the abnormally high values of human-plasma fibrinogen.
Mech. Ageing Dev. 114:207–210.
33. Ramirez-Bosca A, Soler A, Carrion MA et al. 2000. An hydroalcoholic extract of
Curcuma longa lowers the apo B/apo A ratio. Implications for atherogenesis prevention.
Mech. Ageing Dev. 119:41–47.
34. Suri A, Szallasi A. 2008. The emerging role of TRPV1 in diabetes and obesity. Trends
Pharmacol. Sci. 29:29–36.
35. Bhutani M, Pathak AK, Nair AS et al. 2007. Capsaicin is a novel blocker of constitutive
and interleukin-6-inducible STAT3 activation. Clin. Cancer Res. 13:3024–3032.
36. Singh S, Natarajan K, Aggarwal BB. 1996. Capsaicin (8-methyl-N-vanillyl-6-
nonenamide) is a potent inhibitor of nuclear transcription factor-kappa B activation by
diverse agents. J. Immunol. 157:4412–4420.
37. Hsu CL, Yen GC. 2007. Effects of capsaicin on induction of apoptosis and inhibition of
adipogenesis in 3T3-L1 cells. J. Agric. Food Chem. 55:1730–1736.
38. Kang JH, Kim CS, Han IS, Kawada T, Yu R. 2007. Capsaicin, a spicy component of hot
peppers, modulates adipokine gene expression and protein release from obese-mouse
adipose tissues and isolated adipocytes, and suppresses the inflammatory responses of
adipose tissue macrophages. FEBS Lett. 581:4389–4396.
39. Cui J, Himms-Hagen J. 1992. Rapid but transient atrophy of brown adipose tissue in
capsaicin-desensitized rats. Am. J. Physiol. 262:R562–R567.
40. Zhang LL, Yan Liu D, Ma LQ et al. 2007. Activation of transient receptor potential
vanilloid type-1 channel prevents adipogenesis and obesity. Circ. Res. 100:1063–1070.
41. Motter AL, Ahern GP. 2008. TRPV1-null mice are protected from diet-induced obesity.
FEBS Lett. 582:2257–2262.
42. Belza A, Frandsen E, Kondrup J. 2007. Body fat loss achieved by stimulation of thermo-
genesis by a combination of bioactive food ingredients: A placebo-controlled, double-
blind 8-week intervention in obese subjects. Int. J. Obes. (Lond.). 31:121–130.
43. Snitker S, Fujishima Y, Shen H et al. 2009. Effects of novel capsinoid treatment on fat-
ness and energy metabolism in humans: Possible pharmacogenetic implications. Am. J.
Clin. Nutr. 89:45–50.
44. Kadnur SV, Goyal RK. 2005. Beneficial effects of Zingiber officinale Roscoe on
fructose induced hyperlipidemia and hyperinsulinemia in rats. Indian J. Exp. Biol.
43:1161–1164.
45. Sekiya K, Ohtani A, Kusano S. 2004. Enhancement of insulin sensitivity in adipocytes
by ginger. Biofactors 22:153–156.
46. Gulcin I, Beydemir S, Hisar O. 2005. Effect of alpha-tocopherol on antioxidant enzyme
activities and lipid peroxidation in rainbow trout (Oncorhynchus mykiss). Acta Vet.
Hung. 53:425–433.
47. Srinivasan K. 2007. Black pepper and its pungent principle—Piperine: A review of
diverse physiological effects. Crit. Rev. Food Sci. Nutr. 47:735–748.
48. Shoba G, Joy D, Joseph T, Majeed M, Rajendran R, Srinivas PS. 1998. Influence of
piperine on the pharmacokinetics of curcumin in animals and human volunteers. Planta
Med. 64:353–356.
49. Rauscher FM, Sanders RA, Watkins JB 3rd. 2000. Effects of piperine on antioxidant
pathways in tissues from normal and streptozotocin-induced diabetic rats. J. Biochem.
Mol. Toxicol. 14:329–334.
50. Crawford P. 2009. Effectiveness of cinnamon for lowering hemoglobin A1C in
patients with type 2 diabetes: A randomized, controlled trial. J. Am. Board Fam. Med.
22:507–512.
51. Dugoua JJ, Seely D, Perri D et al. 2007. From type 2 diabetes to antioxidant activity: A
systematic review of the safety and efficacy of common and cassia cinnamon bark. Can.
J. Physiol. Pharmacol. 85:837–847.
52. Hlebowicz J, Darwiche G, Bjorgell O, Almer LO. 2007. Effect of cinnamon on post-
prandial blood glucose, gastric emptying, and satiety in healthy subjects. Am. J. Clin.
Nutr. 85:1552–1556.
53. Jitomir J, Willoughby DS. 2009. Cassia cinnamon for the attenuation of glucose
intolerance and insulin resistance resulting from sleep loss. J. Med. Food. 12:467–472.
54. Khan A, Safdar M, Ali Khan MM, Khattak KN, Anderson RA. 2003. Cinnamon improves
glucose and lipids of people with type 2 diabetes. Diabetes Care 26:3215–3218.
55. Mang B, Wolters M, Schmitt B et al. 2006. Effects of a cinnamon extract on plasma
glucose, HbA, and serum lipids in diabetes mellitus type 2. Eur. J. Clin. Invest.
36:340–341.
56. Roussel AM, Hininger I, Benaraba R, Ziegenfuss TN, Anderson RA. 2009. Antioxidant
effects of a cinnamon extract in people with impaired fasting glucose that are over-
weight or obese. J. Am. Coll. Nutr. 28:16–21.
57. Saraswat M, Reddy PY, Muthenna P, Reddy GB. 2009. Prevention of nonenzymic
glycation of proteins by dietary agents: Prospects for alleviating diabetic complications.
Br. J. Nutr. 101:1714–1721.
58. Solomon TP, Blannin AK. 2007. Effects of short-term cinnamon ingestion on in vivo
glucose tolerance. Diabetes Obes. Metab. 9:895–901.
59. Solomon TP, Blannin AK. 2009. Changes in glucose tolerance and insulin sensitivity
following 2 weeks of daily cinnamon ingestion in healthy humans. Eur. J. Appl. Physiol.
105:969–976.
60. Ziegenfuss TN, Hofheins JE, Mendel RW, Landis J, Anderson RA. 2006. Effects of
a water-soluble cinnamon extract on body composition and features of the metabolic
syndrome in prediabetic men and women. J. Int. Soc. Sports Nutr. 3:45–53.
61. Anderson RA, Broadhurst CL, Polansky MM et al. 2004. Isolation and characteriza-
tion of polyphenol type-A polymers from cinnamon with insulin-like biological activity.
J. Agric. Food Chem. 52:65–70.
62. Jia Q, Liu X, Wu X et al. 2009. Hypoglycemic activity of a polyphenolic oligomer-
rich extract of Cinnamomum parthenoxylon bark in normal and streptozotocin-induced
diabetic rats. Phytomedicine 16:744–750.
63. Onderoglu S, Sozer S, Erbil KM, Ortac R, Lermioglu F. 1999. The evaluation of long-
term effects of cinnamon bark and olive leaf on toxicity induced by streptozotocin
administration to rats. J. Pharm. Pharmacol. 51:1305–1312.
64. Subash Babu P, Prabuseenivasan S, Ignacimuthu S. 2007. Cinnamaldehyde—A poten-
tial antidiabetic agent. Phytomedicine 14:15–22.
65. Verspohl EJ, Bauer K, Neddermann E. 2005. Antidiabetic effect of Cinnamomum cassia
and Cinnamomum zeylanicum in vivo and in vitro. Phytother. Res. 19:203–206.
66. Kim SH, Hyun SH, Choung SY. 2006. Anti-diabetic effect of cinnamon extract on blood
glucose in db/db mice. J. Ethnopharmacol. 104:119–123.
67. Sheng X, Zhang Y, Gong Z, Huang C, Zang YQ. 2008. Improved insulin resistance and
lipid metabolism by cinnamon extract through activation of peroxisome proliferator-
activated receptors. PPAR Res. 2008:541348.
68. Qin B, Dawson H, Polansky MM, Anderson RA. 2009. Cinnamon extract attenuates
TNF-alphainduced intestinal lipoprotein ApoB48 overproduction by regulating inflam-
matory, insulin, and lipoprotein pathways in enterocytes. Horm. Metab. Res. 41:516–522.
69. Imparl-Radosevich J, Deas S, Polansky MM et al. 1998. Regulation of PTP-1 and insu-
lin receptor kinase by fractions from cinnamon: Implications for cinnamon regulation of
insulin signaling. Horm. Res. 50:177–182.
70. Cao H, Polansky MM, Anderson RA. 2007. Cinnamon extract and polyphenols affect
the expression of tristetraprolin, insulin receptor, and glucose transporter 4 in mouse
3T3-L1 adipocytes. Arch. Biochem. Biophys. 459:214–222.
71. Peng X, Cheng KW, Ma J et al. 2008. Cinnamon bark proanthocyanidins as reactive car-
bonyl scavengers to prevent the formation of advanced glycation endproducts. J. Agric.
Food Chem. 56:1907–1911.
72. Vijayakumar MV, Pandey V, Mishra GC, Bhat MK. 2010. Hypolipidemic effect of fenu-
greek seeds is mediated through inhibition of fat accumulation and upregulation of LDL
receptor. Obesity (Silver Spring). 18:667–674.
73. Kawada T, Hagihara K, Iwai K. 1986. Effects of capsaicin on lipid metabolism in rats
fed a high fat diet. J. Nutr. 116:1272–1278.
74. Sharma RD, Rukmini C.1986. Rice bran oil and hypocholesterolemia in rats. Lipids
21:715–717.
75. Srinivasan K, Patole PS, Kaul CL, Ramarao P. 2004. Reversal of glucose intoler-
ance by pioglitazone in high fat diet-fed rats. Methods Find. Exp. Clin. Pharmacol.
26:327–333.
76. Asai A, Nakagawa K, Miyazawa T. 1999. Antioxidative effects of turmeric, rosemary
and capsicum extracts on membrane phospholipid peroxidation and liver lipid metabo-
lism in mice. Biosci. Biotechnol. Biochem. 63:2118–2122.
77. Naidu KA, Thippeswamy NB. 2002. Inhibition of human low density lipoprotein oxida-
tion by active principles from spices. Mol. Cell Biochem. 229:19–23.
78. Reddy AC, Lokesh BR. 1994. Studies on the inhibitory effects of curcumin and eugenol
on the formation of reactive oxygen species and the oxidation of ferrous iron. Mol. Cell
Biochem. 137:1–8.
79. Manjunatha H, Srinivasan K. 2006. Protective effect of dietary curcumin and capsa-
icin on induced oxidation of low-density lipoprotein, iron-induced hepatotoxicity and
carrageenan-induced inflammation in experimental rats. FEBS J. 273:4528–4537.
80. Agbor GA, Oben JE, Ngogang JY, Xinxing C, Vinson JA. 2005. Antioxidant capacity of
some herbs/spices from Cameroon: A comparative study of two methods. J. Agric. Food
Chem. 53:6819–6824.
81. Shan B, Cai YZ, Sun M, Corke H. 2005. Antioxidant capacity of 26 spice extracts and
characterization of their phenolic constituents. J. Agric. Food Chem. 53:7749–7759.
82. Raghavendra RH, Naidu KA. 2009. Spice active principles as the inhibitors of human
platelet aggregation and thromboxane biosynthesis. Prostaglandins Leukot. Essent.
Fatty Acids. 81:73–78.
83. Prasad NS, Raghavendra R, Lokesh BR, Naidu KA. 2004. Spice phenolics inhibit
human PMNL 5-lipoxygenase. Prostaglandins Leukot. Essent. Fatty Acids
70:521–528.
84. Fogelman AM, Shechter I, Seager J, Hokom M, Child JS, Edwards PA. 1980.
Malondialdehyde alteration of low density lipoproteins leads to cholesteryl ester accu-
mulation in human monocyte-macrophages. Proc. Natl. Acad. Sci. USA 77:2214–2218.
85. Haberland ME, Fong D, Cheng L. 1988. Malondialdehyde-altered protein occurs in
atheroma of Watanabe heritable hyperlipidemic rabbits. Science 241:215–218.
86. Palinski W, Rosenfeld ME, Yla-Herttuala S et al. 1989. Low density lipoprotein under-
goes oxidative modification in vivo. Proc. Natl. Acad. Sci. USA 86:1372–1376.
87. Yla-Herttuala S, Palinski W, Rosenfeld ME et al. 1989. Evidence for the presence of
oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and
man. J. Clin. Invest. 84:1086–1095.
88. Marnett LJ. 1999. Lipid peroxidation-DNA damage by malondialdehyde. Mutat. Res.
424:83–95.
89. Gorelik S, Ligumsky M, Kohen R, Kanner J. 2008. The stomach as a “bioreactor”:
When red meat meets red wine. J. Agric. Food Chem. 56:5002–5007.
90. Gorelik S, Ligumsky M, Kohen R, Kanner J. 2008. A novel function of red wine poly-
phenols in humans: Prevention of absorption of cytotoxic lipid peroxidation products.
FASEB J. 22:41–46.
91. Gorelik S, Lapidot T, Shaham I et al. 2005. Lipid peroxidation and coupled vitamin
oxidation in simulated and human gastric fluid inhibited by dietary polyphenols: Health
implications. J. Agric. Food Chem. 53:3397–3402.
92. Gorelik S, Kohen R, Ligumsky M, Kanner J. 2007. Saliva plays a dual role in oxidation
process in stomach medium. Arch. Biochem. Biophys. 458:236–243.
93. Li Z, Henning SM, Zhang Y et al. 2010. Antioxidant-rich spice added to hamburger
meat during cooking results in reduced meat, plasma, and urine malondialdehyde con-
centrations. Am. J. Clin. Nutr. 91:1180–1184.
94. Brower V. 1998. Nutraceuticals: Poised for a healthy slice of the healthcare market?
Nat Biotechnol. 16:728–731.
95. Zeisel SH. 1999. Regulation of “nutraceuticals”. Science 285:1853–1855.
Level of oxidative stress
Low Intermediate High
Nrf-2 NF-κB Mitochondrial
Signaling pathway AP-1 PT pore
Outcome Antioxidant Inflammation Apoptosis

enzymes proteins proteins
FIGURE 2.1 Hierarchical oxidative stress model. A low oxidative stress induces Nrf2, a
transcription factor implicated in the transactivation of gene coding for antioxidant enzymes.
An intermediate amount of ROS triggers an inflammatory response through the activation of
NF-κB and AP-1, and a high amount of oxidative stress induces perturbation of the mitochon-
drial PT pore and disruption of the electron transfer, thereby resulting in apoptosis or necro-
sis. (Adapted from Williams, M.S. and Kwon, J., J Free Radic. Biol. Med., 37, 1144, 2004.)
TNF-α
TNFR1
TRADD TRAF2
RING
RIP Ubc13
γ γ Uev1A
P P TRAF2
IKK
complex α β β α
MEKK3 Ub 53
P P
Ub 53
Ub 63
Ub
P
P IκBα
p50 p65
us
le
uc
N
p50 p65 Target gene
FIGURE 2.2 TNFR1 signaling. In this model of TNFR1 signaling, the IKK complex is
activated while associated with the receptor. The IKK complex is recruited to the receptor in a
TNF-α-dependent manner. This recruitment requires TRAF2 and may also involve the interac-
tion between IKKγ and RIP. TRAF2 is thought to activate the IKK complex via a ubiquitin-
dependent signaling pathway. The TRAF2/ubiquitin signaling complex may lead to the
activation of MEKK3, although this has yet to be demonstrated. RIP is also likely to play a role
in activation of the IKK complex, possibly by interacting with MEKK3 (From Yang, J. et al.,
Nat. Immunol. 2, 620, 2001.) Once activated, the IKK complex phosphorylates IκBα on serines
32 and 36, leading to its proteasome-mediated degradation. (Reprinted with permission from
Silverman, N. and Maniatis, T., Genes Dev., 15, 2321. Copyright 2001 by Cold Spring Harbor
Laboratory Press, www.genesdev.org 2321.)
TNFR1, IL-1R1, TLRs, BCR, TCR LTβR, BAFFR, CD40, HTLV, EBV
Classical pathway Alternative pathway
NIK
NEMO NEMO
Ub
Ub
Ub P P
IKKα IKKβ
Ub
P P
Ub
IκBα P IKKα IKKα
P
p50 p65
Ub
P P Ub
IκBα Ub
Ub P P
p50 p65 Ub p100
RelB p52 RelB

p50 p65
Inflammatory proteins Lymphoid organ development and

p50 p65 p52 RelB homeostasis proteins
FIGURE 2.3 Classical and alternative pathways of NF-κB activation. Ligation of TNFR1, IL-1/TLR, TCR, and BCR induces IKK-dependent IkBα
phosphorylation on S32 and 36, which induces ubiquitination and degradation of the inhibitory protein, thus allowing NF-κB to migrate into the nucleus
and transactivate inflammatory genes (classical pathway). Upon ligation of LTbR, BAFFR or CD40 or infection by HTLV or EBV, the alternate path-
way is induced. It enhances NF-κB inducing kinase (NIK)- and IKKα-dependent processing of p100 into p52, which binds DNA in association with its
partners and stimulates genes implicated in lymphoid organ development and organogenesis. These stimuli also activate the classical pathway.
LPS TLR4
CD14 MD-2
TAB2
MyD88
DD
TIR
IRAK
IRAK
Ubc13
TRAF6 Uev1A
RING
63 Ub TRAF6
63 Ub
63 Ub TAB2 TAK1
Ub
TAB1
γ γ
P P
IKK
complex α β β α
P P
β-T r C P
P U biq u iti n
P IκBα
p50 p65 Prote a s o m e
s
leu
c
Nu
p50 p65 Target gene
FIGURE 2.4 LPS signaling pathway in mammals. In this model, LPS is recognized by a
complex of three proteins: CD14, MD-2, and TLR4. TLR4 activates the intracellular signaling
cascade by recruiting MyD88 and IRAK to the membrane. IRAK associates with the recep-
tor complex transiently; once released IRAK can associate with and activate TRAF6. The
TRAF6 RING finger, in combination with Ubc13 and Uev1A, mediates the K63-extended
polyubiquitination of TRAF6 itself. The TAK1/TAB1/TAB2 complex is activated by its asso-
ciation with ubiquitinated TRAF6. Interestingly, the TAK1-associated protein TAB2 translo-
cates from the membrane fraction to the cytoplasmic fraction upon treatment with IL-1. Once
activated, the TAK1 complex phosphorylates and activates the IKK complex. The activated
IKK complex then phosphorylates IκBα, leading to its ubiquitination and degradation by the
proteasome.
High-fat diet High-fat diet
Low-fat diet Low ω-3 fatty acids High ω-3 fatty acids
Interleukin-10
Interleukin-10 Polyunsaturated Arginase
Arginase Saturated ω-3 fatty acids
fatty acids TNFα
DHA, EPA
Insulin sensitive MCP1

Insulin resistant Insulin sensitive
ω-3 M1 macrophage
M2 macrophage Saturated
GPR120 fatty acids
(ani-inflammatory) fatty acids
M1 macrophage NFκB
(proinflammatory) JNK
Adipose cell TLR4 β-Arrestin Cytokines

TNFα
FIGURE 3.2 A high-fat diet with a disproportionate ratio of saturated fatty acids to ω-3
fatty acids triggers activation of Toll-like receptor 4 (TLR4) in adipocytes and circulat-
ing immune cells. This launches an inflammatory cascade that results in the recruitment
of proinflammatory M1 macrophages, increased secretion of TNFα, and insulin resistance
in adipocytes. The addition of ω-3 fatty acids to the diet activates the G protein-coupled
receptor GPR120 on proinflammatory M1 macrophages (Oh et al., 2010), which in turn atten-
uates the inflammatory response and recruits anti-inflammatory M2 macrophages to adipose
tissue. Eventually, these M2 macrophages restore secretion of interleukin-10 and improve
insulin sensitivity. (Courtesy of A.R. Saltiel, Life Sciences Institute, Departments of Internal
Medicine and Molecular and Integrative Physiology, University of Michigan Medical School,
Ann Arbor, MI. With permission.)
MRI
Food intake
Weeks: 0 8 10 12 14 16
End study
HF/HS feeding
Weeks: 0 8
(a)
Before HF/HS feeding After HF/HS feeding
AXB19b/PgnJ
AXB19/PgnJ
BxH20/K J
BXA14/Pgn
ccJ
BXD 20/TyJ
BX A12/P J
CX A13 gnJ
Bx 11/Ty
SJL B12 /PgnJ

iAJ
C58/J
/H
BXD
BX A1/ K/H J
D 87/ yJ J
n
0
BX K Rww yJ
g
D3 Pg IJ
J
BXxH2 2/P
/T ww
BX BX
PL/ J
9/T nJ
A /J
Bx D T
D4 D
B xB1
12 R
BX
yJ
J
y
J wJ
4/ 9/T
D 10
Bx 24 Ty w
Body fat percentage
A D b 1/ 1/R iAJ
BX XB1 19 /Ty D 6
D6 0/ /Ty J 20 Bx XD 13/H nJ J
BX 0/R Pgn J B XB /Pg ww
w J C xA4 5/R J
BX A2/P wJ B D7 hiLt
BX D32/ gnJ 30 BX D/S yJ
D
BXD 21/ yJ T NO D15/T
T BX BL/6J nJ
BXD 43/Rw yJ
BXD 85/Rww J
w 40 C57 19a/Pg
66/R J AxB 5/RwwJ
ww BXD4 /TyJ
RIIIS/ J 50 BXH19
BXD16/T J
yJ CxB3/ByJ
SEA/GnJ BXD8/TyJ
BTBRTtf/J CXB6/ByJ
LG/J BXD34/TyJ
129X1/SvJ BXD49/RwwJ
BUB/BnJ
BxA11/PgnJ SWR/J
CBA/J
BXD6
AKR/J AxB8 8/RwwJ
2J
DBA/ yJ BXD /PgnJ
D 3 1/T J BX 13/Ty
Bx Rww BX D55/R J
62/ iLtJ Cx A24/ wwJ
BXD N/SH gnJ C B7/ PgnJ
P
NO xA7/ /MyJ J BX 57L/J ByJ
B A c C H
M 2/Kc acJ B 3H 6/T
H 2 /L wJ Ax XD /He yJ
Bx ZW /Rw A/J B6 40/ J
N 51 /P Ty
BX XD 86/ gnJ J
BX BxH 8/T nJ
D gn J
g
B D /P
CX D3 9/T yJ
D 74/ Rw
BX
D3 /P
J
BX A16 /Rw wJ
BX B11 6/T yJ
84 R wJ
Bx xA8
Bx D50 4/Rw J
D7 /H yJ
/R ww
BX D6
w J
9/R iA
ww J
B
BX D71/R wwJ
FVB wJ
BX D48/R wJ
J
BxH /N
CXB 4/TyJ
BX D56/Rw
J
BX 8/TyJ
yJ
AxB15 M/J
BXH /TyJ
BXD6
CE/J
C57BL gnJ
BXD70/Rw /J
4/B
BxD5/TyJ
BALB/cJ
AXB2/PgnJ
BXD14/TyJ
wJ
BXD73/RwwJ
KS
S
/P
w
ww
(b)
FIGURE 6.1 Natural variation in gene-by-diet interactions. (a) Schematic of study design
with indicated time points for HF/HS feeding, magnetic resonance imaging (MRI), food
intake monitoring, and end of study. (b) Body fat percentage in male mice (108 strains) before
and after 8 weeks of HF/HS feeding. Error bars represent SEM.
(continued )
0%–50%
400 50%–100%
100%–150%
150%–200%
Body fat percentage growth
200%–250%
300 250%–300%
>300%
200
100
0 2 4 6 8
(c) Weeks on diet
5.0 5.0
4.5 4.5
Food intake (g/day)
Food intake (g/day)
4.0 4.0
3.5 3.5
3.0 3.0
2.5 r = 0.45 2.5 r = 0.52
p = 4.18e–33 2.0 p = 1.49e–45
2.0
20 30 40 50 60 15 20 25 30 35 40
(d) Body weight—4 weeks on diet (g) (e) Lean mass—4 weeks on diet (g)
5.0 5.0
4.5 4.5
Food intake (g/day)
Food intake (g/day)
4.0 4.0
3.5 3.5
3.0 3.0
2.5 2.5 r = 0.01
r = 0.18 p = 0.807
2.0 p = 4.33e–06 2.0
10 20 30 40 0 100 200 300 400
Body fat percentage— Body fat percentage
(f) 4 weeks on diet (g) growth—0–4 weeks
FIGURE 6.1 (continued) Natural variation in gene-by-diet interactions. (c) Biweekly

percent body fat percentage increase in male mice with indicated body fat percentage increase
after 8 weeks of HF/HS feeding. (d–g) Correlation of food intake (g/day/mouse) with body
weight (d), lean mass (e), body fat percentage—4 weeks on HF/HS diet (f), and body fat
percentage growth—0–4 weeks (g), regression line. r, bi-weight mid-correlation; p, p value.
(From Parks, B.W. et al., Cell Metab., 17, 141, 2013. With permission.)
Chow diet HF/HS diet PC2 (4%)
HF/HS
Chow
Verrucomicrobia
Actinobacteria
Proteobacteria
Firmicutes
Other Bacteroidetes
Tenericutes
Actinobacteria Firmicutes Proteobacteria

PC3 (3.3%)
Bacteroidetes Other Tenericutes
Verrucomicrobia
PC1 (9.5%)
(a) (b)
Chow HF/HS
Akkermansia
Lachnospiraceae_unclassified
Ruminococcaceae_unclassified
Clostridium
Bifidobacterium
Turicibacter
Clostridiaceae_unclassified
Dorea
Roseburia
Hydrogenoanaerobacterium
Erysipelotrichaceae_unclassified
Lactococcus
Butyricicoccus
Anaeroplasma
Oscillibacter
Barnesiella
Porphyromonadaceae_unclassified
–6.0 –4.8 –3.6 –2.4 –1.2 0.0 1.2 2.4 3.6 4.8 6.0
(c) LDA score (log 10)
FIGURE 6.2 Robust shifts in gut microbiota composition after HF/HS feeding. (a) Relative
abundances of the different phyla after chow diet and HF/HS feeding (average among 52
matched strains). (b) Principal coordinates analysis (PCoA) plot of the unweighted UniFrac
distances. Each circle representing a different mice strain is colored according to the dietary
conditions. PC1, PC2, and PC3 values for each mouse sample are plotted; percent variation
explained by each PC is shown in parentheses. (c) Linear discriminant analysis (LDA) cou-
pled with effect size measurements identifies the most differentially abundant taxons between
chow and HF/HS diets. HF/HS-diet-enriched taxa are indicated with a positive LDA score
and taxa enriched in normal chow diet have a negative score. Only taxa meeting an LDA
significant threshold >2 are shown. (From Parks, B.W. et al., Cell Metab., 17, 141, 2013. With
permission.)
3
Immunosuppression
PGE2, IL-10, TGF-B1
Basement Hypoxic areas

membrane MMPs, uPA,
1
Invasion cathepsins
? VEGF, bFGF,PDGF, MMPs,

?
Stroma IL-8, Ang1
2
Angiogenesis
EGF EGF
4
Metastasis
FIGURE 7.1 The roles of different subpopulations of TAMs in tumor progression.

(1) Invasion: TAMs secrete a variety of proteases to break down the basement membrane
around areas of proliferating tumor cells (e.g., ductal carcinoma in situ in the breast), thereby
prompting their escape into the surrounding stroma where they show deregulated growth.
(2) Angiogenesis: In areas of transient (avascular) and chronic (perinecrotic) tumor hypoxia,
macrophages cooperate with tumor cells to induce a vascular supply for the area by upregu-
lating a number of angiogenic growth factors and enzymes. These diffuse away from the
hypoxic area and, together with other proangiogenic stimuli in the tumor microenviron-
ment, stimulate endothelial cells in neighboring, vascularized areas to migrate, proliferate,
and differentiate into new vessels. (3) Immunosuppression: Macrophages in hypoxic areas
secrete factors that suppress the antitumor functions of immune effectors within the tumor.
(4) Metastasis: A subpopulation of TAMs associated with tumor vessels secretes factors like
EGF to guide tumor cells in the stroma toward blood vessels where they then escape into the
circulation. In the stromal compartment (both acellular regions and others where they are in
close contact with tumor cells), TAMs secrete growth factors to stimulate tumor cell division
and/or undefined factors that promote tumor cell motility. (From Lewis, C.E. and Pollard,
J.W., Cancer Res., 66, 605, 2006.)
Adipose tissue Insulin sensitivity
Insulin clearance
FFA, SAA adn Liver
leptin, IL-6 Hepatic glucose output
VLDL-TG
CRP
FFA, adn
leptin, IL-6
SAA, IL-8, Muscle
Leptin, IL-6
IL-6, TSP-1
FFA Insulin sensitivity
Brain Blood vessels

Pancreas
Insulin secretion
Food intake
Energy Inflammation
expenditure Atherogenesis
FIGURE 8.3 Adipose signals influence systemic metabolism and appetite. Dysfunctional adipose tissue in obesity produces more proinflammatory
factors (e.g., FFA, SAA, IL-6) and less anti-inflammatory factors (e.g., adiponectin). These exacerbate inflammation and hence risk for metabolic dis-
eases by affecting liver, skeletal muscle, beta cells, as well as blood vessels. Insulin–glucose homeostasis becomes impaired as a result of increased
hepatic glucose output and muscle insulin resistance, and basal insulin secretion from pancreas is increased, most likely by FAs. Leptin normally regu-
lates food intake and energy expenditure through its effects on the central nervous system. Besides leptin levels are commonly elevated in the obese
state, most obese persons are resistant to the weight-reducing effects of leptin. (Reprinted from Mol Aspects Med, 34(1), Lee, M.J., Wu, Y., and Fried,
S.K., Adipose tissue heterogeneity: Implication of depot differences in adipose tissue for obesity complications, 1–11, Copyright 2013, with permission
from Elsevier.)
Fatty liver
Retroperitoneal Preperitoneal
Pancreas
Stomach
Retroperitoneal
Abdominal sc
perinephric
(superficial)
Intestine
sc deep
Mesenteric
Omental
Gluteal sc
Thigh
(femoral) sc
FIGURE 8.4 Major adipose depots in humans. Subcutaneous adipose tissues include
abdominal, femoral, and gluteal. Intraperitoneal (visceral) adipose tissues are associated
with digestive organs. Omental is attached to the stomach and mesenteric and epiploic are
associated with the intestine and colon, respectively. Retroperitoneal fat is located in the ret-
roperitoneal compartment. (Reprinted from Mol Aspects Med, 34(1), Lee, M.J., Wu, Y., and
Fried, S.K., Adipose tissue heterogeneity: Implication of depot differences in adipose tissue
for obesity complications, 1–11, Copyright 2013, with permission from Elsevier.)
Normal adiposity
Energy-dense food Lack of physical

( fat + sugar content) activity/exercise
Positive
energy balance
Smoking
Unfavorable genotype
Maladaptive response
to stress
Subcutaneous obesity Visceral obesity

Healthy adipose tissue Dysfunctional adipose tissue
Altered FFA Altered release

metabolism of adipokines
No ectopic fat Lipid overflow–ectopic fat
Muscle fat
Low muscle fat ( intracellular lipid)
Low epicardial fat Epicardial fat
Low liver fat and

normal function Liver fat and
altered function
Normal metabolic profile Altered metabolic profile
Absence of clinical criteria

Presence of clinical criteria
for metabolic syndrome
for metabolic syndrome
(including hypertrigly-
ceridemic waist)
FIGURE 8.5 The lipid overflow–ectopic fat model. Excess visceral fat accumulation might
be causally related to the features of insulin resistance, but might also be a marker of a dys-
functional adipose tissue being unable to appropriately store the energy excess. According
to this model, the body’s ability to cope with the surplus of calories (resulting from excess
caloric consumption, a sedentary lifestyle, or a combination of both factors) might, ultimately,
determine the individual’s susceptibility to developing metabolic syndrome. There is evi-
dence suggesting that if the extra energy is channeled into insulin-sensitive subcutaneous
adipose tissue, the individual, although in positive energy balance, will be protected against
the development of the metabolic syndrome. However, in cases in which adipose tissue is
absent, deficient, or insulin resistant with a limited ability to store the energy excess, the
triacylglycerol surplus will be deposited at undesirable sites such as the liver, the heart, the
skeletal muscle and in VAT—a phenomenon described as ectopic fat deposition. (Reprinted
by permission from Macmillan Publishers Ltd. Nature, Després, J.P. and Lemieux, I.,
Abdominal obesity and metabolic syndrome, 444(14), 881–887, Copyright 2006.)
Genetically modified environmental factors
Decreased physical activity, inadequate nutrition, obesity, and infection
Signal (ROS, fatty acids, AGES, etc.)
Cells—macrophages,
PRRs
endothelium, adipocytes
NF-κβ
Chronic complications of Nucleus

type 2 diabetes
(atherosclerosis and Pathogenesis of
dyslipidemia) type 2 diabetes
Skeletal muscle—
Blood—clotting insulin resistance
CRP, fibrinogen IL-1β, TNF-α
Endothelium—permeability Cytokines Apoptosis of pancreatic β-cells—
VCAM-1, ICAM-1 impaired insulin secretion
IL-1β, TNF-α
Liver—Apps, glucose
output, free fatty acids Adipose tissue—
IL-6, IL-1β insulin resistance
IL-6, TNF-α
FIGURE 9.1 Innate immunity and T2DM. Cell components of the innate immune system,
such as macrophages, endothelial cells, and adipocytes detect, through pattern-recognition
receptors (PRRs), potential environmental threats to the host, which are represented by sig-
nals such as reactive oxygen species (ROS), fatty acids, and advanced glycation end products
(AGES). This process activates nuclear transcription factors, such as nuclear factor-kappa B
(NF-κB), which induce immune inflammatory genes, which in turn cause the release of cyto-
kines. These cytokines act in many cells in the body to produce the clinical and biochemical
features of type 2 diabetes and its chronic complications. APPs, acute-phase proteins; CRP,
C-reactive protein; IL, interleukin; TNF-α, tissue necrosis factor alpha; VCAM-1, vascular
cell adhesion molecule 1; ICAM-1, vascular endothelial growth factor expression of intercel-
lular adhesion molecule 1. (From Santos-Tunes, R. et al., J. Can. Dent. Assoc., 76, a35, 2010.
With permission.)
Periodontitis Diabetes mellitus/obesity
IL-1β, TNF-α, IL-6, IL-8, PGE2, LPS Fatty acids, lipids, AGES
PRRs
Cell
PKCs JNK IKKβ ROS

PS302
IRS-1 IκB
PS307 NF-κB
Nucleus NF-κB
Inflammatory markers
and mediators
Insulin resistance
Endothelium cells Immune cells
Adipocytes
Hepatocytes Skeletal muscle cells
FIGURE 9.2 Proposed mechanism by which periodontal inflammatory mediators may con-
tribute to the development of insulin resistance in individuals with both type 2 diabetes and
periodontitis. The inflammatory mediators originating from periodontal sources can inter-
act systemically with lipids, free fatty acids, and advanced glycation end products (AGES),
all of which are characteristic of diabetes. This interaction induces or perpetuates activa-
tion of the intracellular pathways, such as the I-kappa-B (IκB), I-kappa-B kinase-β (IKKβ),
nuclear factor-kappa B (NF-κβ), and the protein c-Jun N-terminal kinase (JNK) axes, all of
which are associated with insulin resistance. The activation of these inflammatory pathways
in immune cells (monocytes or macrophages), endothelium cells, adipocytes, hepatocytes,
and muscle cells promotes and contributes to an increase in the overall insulin resistance,
which makes it difficult to achieve metabolic control in patients with both type 2 diabetes and
periodontitis. IL, interleukin; IRS-1, insulin receptor substrate-1; LPS, lipopolysaccharide;
PGE2, prostaglandin E2; PKCs, protein kinases C; PRRs, pattern-recognition receptors;
pS302 (serine-302) and pS307 (serine-307), examples of serine sites; ROS, reactive oxygen
species; TNF-α, tumor necrosis factor alpha. (From Santos-Tunes, R. et al., J. Can. Dent.
Assoc., 76, a35, 2010. With permission.)
UFP
Redox NP
chemistry
Mito Endosome
NADPH
oxidase
Fe2+
Lysosome
PAHs Fenton ROS
Quinones reaction Mito
Ca2+
ROS ROS Ca2+
ROS
ATP ?
Cyt C
Nrf2 Cyt C Nrf2 Ca2+
JNK, NF-κB ATP
Caspases
Caspases
HO-1, Phase II
enzymes
Cytokines
Tier 1: Cell defense

Tier 2: Proinflammation
Tier 3: Apoptosis/necrosis
FIGURE 14.2 Comparison of the mechanisms of ROS generation induced by UFP and NM
outside or inside of cells. Ambient UFP usually contains large amount of organic chemical
such as polycyclic aromatic hydrocarbons (PAHs) and quinines and transition metals such
as Fe and Cu, which can generate ROS through redox chemistry both outside and inside
of cells. UFPs have also been found to lodge in mitochondria, causing damage to mito-
chondrial function and structure, which can also produce more ROS. Cells under oxidative
stress will have tiered responses, including cell defense (tier 1), proinflammation (tier 2),
and mitochondria-mediated cell death (tier 3). Nanomaterial (NM) are uniform in size and
can also generate ROS via crystal structural defects or under UV conditions. NM are taken
up into cells via endocytosis, which includes phagocytosis, clathrin-dependent endocytosis,
caveolae-mediated endocytosis, or macropinocytosis depending on specific cell types. After
cells take up NM, endosomes are formed, and ROS can be produced via the formation of
NADPH oxidase. After a series of fusion and fission processes, endosomes will fuse with
lysosomes. NM can break loose from lysosomes and interact with other organelles such as
mitochondria, which can produce more ROS. The cells under oxidative stress will go through
tiered oxidative stress responses as described previously. (From Li, N. et al., Free Radic. Biol.
Med., 44(9), 1689, 2008.)
Proinflammatory Anti-inflammatory
IL-6
TNF
IL-1ra
TNF-R IL-10
Sepsis
Anti-inflammatory
IL-6
IL-1ra
IL-10
Exercise
FIGURE 15.1 During sepsis, there is a marked and rapid increase in circulating TNF-α,
which is followed by an increase in IL-6. In contrast, during exercise, the marked increase in
IL-6 is not preceded by elevated TNF-α. (From Pedersen, B.K. and Febbraio, M.A., Physiol.
Rev., 88, 1379, 2008. With permission from the American Physiological Society.)
IL-6
IL-6Rα/gp130Rβ
P13-K p-STAT3
p-Akt p-AMPK Blood vessel
Glucose uptake Fat oxidation IL-6

Liver
IL-6
Increased hepatic
Contraction glucose production
during exercise
IL-6 IL-6
IL-6 IL-6
IL-6
IL-6
Adipose tissue
IL-6
Increased lipolysis
FIGURE 15.2 Skeletal muscle expresses and releases myokines into the circulation. In
response to muscle contractions, both type I and type II muscle fibers express the myokine
IL-6, which subsequently exerts its effects both locally within the muscle (e.g., through
activation of AMPK) and—when released into the circulation—peripherally in several organs
in a hormone-like fashion. Specifically, in skeletal muscle, IL-6 acts in an autocrine or para-
crine manner to signal through a gp130Rβ/IL-6Rα homodimer, resulting in the activation
of AMP kinase and/or PI3 kinase to increase glucose uptake and fat oxidation. IL-6 is also
known to increase hepatic glucose production during exercise or lipolysis in adipose tissue.
(Modified from Pedersen, B.K. and Febbraio, M.A., Physiol. Rev., 88, 1379, 2008. With per-
mission from the American Physiological Society; Reprinted from Curr. Opin. Clin. Nutr.
Metab. Care., 10(3), Pedersen, B.K. and Fischer, C.P., Physiological roles of muscle-derived
interleukin-6 in response to exercise, 265–271, Copyright 2007, with permission from Elsevier.)
NUTRITION
Immunonutrition
Interactions of Diet, Genetics, and Inflammation
The interaction of immune function and nutrition underlies the low-grade
chronic inflammation involved in the etiology of many common obesity-
associated and age-related chronic disease conditions. This close interaction
is the genesis of the term immunonutrition, which represents a new
interdisciplinary field of nutritional and medical research. Immunonutrition:
Interactions of Diet, Genetics, and Inflammation introduces the breadth
of this field, which implicates nutrition in both immune function and in the
etiology, prevention, and treatment of common diseases influenced by
inflammation and immune imbalance, including obesity, diabetes, heart
disease, asthma, autoimmune diseases, and common forms of cancer .
The book begins by reviewing the basic mechanisms of immunity and cellular
mechanisms of cytokine activation. It discusses the effects of dietary fat
intake and changes in Western diet and lifestyle linked to inflammation. It
also describes the interaction of genetics and environment in the modulation
of immune function and inflammation and addresses exercise and skeletal
muscle as an endocrine and immune organ. The book reviews the entire
spectrum of inflammation and cancer from causation to its role in tumor
therapy. It examines abdominal obesity and metabolic diseases, interactions
between nutrition and autoimmunity in systemic lupus erythematosus and
rheumatoid arthritis, and inflammation associated with type 2 diabetes,
heart disease, kidney disease, Alzheimer’s disease, and asthma.
Considering potential nutrition-based treatments, the book explores approaches

for reducing abdominal obesity, anti-inflammatory effects of phytochemicals,
practical strategies for increasing fruit and vegetable intake, and anti-
inflammatory properties of spice phytonutrients. In addition, it explores how
uninformed food choices related to fats and oils create a balance of tissue-
selective signals that produce harmful health outcomes and how to restore a
healthy balance.
K14496
6000 Broken Sound Parkway, NW

Suite 300, Boca Raton, FL 33487
711 Third Avenue
New York, NY 10017
an informa business
2 Park Square, Milton Park
w w w. c r c p r e s s . c o m Abingdon, Oxon OX14 4RN, UK w w w. c rc p r e s s . c o m

Immunonutrition Interactions of

Uploaded by

Document Informationclick to expand document information

Document Informationclick to expand document information

Copyright:

Available Formats

Immunonutrition Interactions of

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Immunonutrition Interactions of

Uploaded by

Copyright:

Available Formats

Immunonutrition

Boca Raton London New York

CRC Press is an imprint of the

No claim to original U.S. Government works

International Standard Book Number-13: 978-1-4665-0386-1 (eBook - PDF)

Chapter 1 Evolution of Innate and Adaptive Immunity.........................................1

Chapter 2 Cellular Mechanisms of Cytokine Activation..................................... 19

Chapter 3 Cellular Lipids and Inflammation....................................................... 39

Chapter 4 Biomarkers of Inflammation and the Western Diet............................ 53

Chapter 5 Phytochemicals and Immune Function............................................... 67

Chapter 6 Genetic and Environmental Modifiers of Immune Function.............. 85

Chapter 7 Cancer and Inflammation.................................................................. 101

Chapter 8 Abdominal Obesity: Pathophysiology and Related Metabolic

Chapter 9 Type 2 Diabetes and Inflammation................................................... 141

Chapter 10 Heart Disease and Inflammation....................................................... 149

Chapter 11 Chronic Kidney Disease and Inflammation...................................... 167

Chapter 12 Alzheimer’s Disease and Inflammation............................................ 181

Chapter 13 Nutrition in Autoimmunity: A Focus on Systemic Lupus

Chapter 14 Asthma and Inflammation................................................................. 229

Chapter 15 Muscle and Immune Function........................................................... 245

Chapter 16 Approaches to Reducing Abdominal Obesity................................... 259

Chapter 17 Barriers to Fruit and Vegetable Consumption and Practical

Chapter 18 Healthy Fats and Oils: Balancing Omega-3 and

Chapter 19 Spices and Dietary Supplements with

David Heber MD, PhD, FACP, FACN

Bharat B. Aggarwal, PhD

Dr. Bharat B. Aggarwal is a Ransom Horne,

• ARTOI Award, Association for Research Integrated Oncology Therapies,

Caroline M. Apovian Ana F.T.A. Junqueria

David Heber Zhaoping Li

Maureen McMahon Gary W. Small

FIGURE 1.1 Adaptive immune function is a late evolutionary development in vertebrates

of signaling pathways modulating NFκB activation and inflammation discussed

The mammalian The Drosophila

IRAK TRAF6 Pelle

EVOLUTION OF INNATE IMMUNITY

Innate Immune System in Plants

Innate Immune System in Humans

EVOLUTION OF CELLULAR IMMUNITY

receptors on amoebas discriminate between food to be engulfed and digested from

Immunity and Inflammation

products, foreign materials, or invading pathogens. Most leukocytes cannot divide or

ADAPTIVE IMMUNE SYSTEM

MALNUTRITION AND IMMUNE FUNCTION

with histological evidence of atrophy being greatest in the T-lymphocyte areas of

IMMUNE FUNCTION IN OBESITY

MACROPHAGE RECEPTORS FOR OMEGA-3 FATTY ACIDS

IMMUNE FUNCTION AND VITAMIN AND MINERAL BALANCE

pathogens. Retinoic acid is required also for production of mature phagocytes in

group, the blood concentrations of IgE, CD4+, CD4+/CD8+, WBC, lymphocyte

PRACTICAL CONSIDERATIONS FOR

TRANSCRIPTION FACTORS AND MITOGEN-ACTIVATED

Nuclear Factor-K appa B

asbestos, dietary agents, environmental pollutants, obesity, and various infectious

Nuclear Factor of Activated T-Cells

Extracellular Signal–Regulated Kinases

c-Jun N-Terminal Kinases