Journals of Gerontology: Medical Sciences

cite as: J Gerontol A Biol Sci Med Sci, 2017, Vol. 72, No. 12, 1703–1709
doi:10.1093/gerona/glx041
Advance Access publication March 16, 2017

Research Article

Resveratrol Improves Vascular Function and Mitochondrial

Number but Not Glucose Metabolism in Older Adults
Rena M. Pollack,1 Nir Barzilai,1 Valentin Anghel,1 Ameya S. Kulkarni,1
Aaron Golden,1 Pilib O’Broin,2 David A. Sinclair,3 Michael S. Bonkowski,3
Alexander J. Coleville,4 Danielle Powell,1 Sharon Kim,1 Ruin Moaddel,5
Daniel Stein,1 Kehao Zhang,1 Meredith Hawkins,1 and Jill P. Crandall1
Albert Einstein College of Medicine, Bronx, New York. 2National University of Ireland Galway, Galway, Ireland. 3Harvard Medical School,
1

Boston, Massachusetts. 4Stanford University, Palo Alto, California. 5National Institute on Aging, Baltimore, Maryland.

Address correspondence to Jill P. Crandall, MD, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. E-mail: jill.
[email protected]

Received July 19, 2016; Editorial Decision Date February 17, 2017

Decision Editor: Stephen Kritchevsky, PhD

Abstract
Background: Resveratrol, a plant-derived polyphenol, has been reported to improve glucose metabolism and vascular function and to extend
life span in animal models, but studies in humans have been inconclusive.
Methods: In a randomized, double-blind crossover study, we treated older glucose-intolerant adults (n = 30) with resveratrol (2−3 g/daily)
or placebo, each for 6 weeks. A standard mixed-meal test was used to assess insulin sensitivity (Matsuda index) and secretion (C-peptide
deconvolution) and vascular function by reactive hyperemia peripheral arterial tonometry. Skeletal muscle samples were obtained for gene
expression using RNA-Seq analysis and to assess mitochondrial morphology.
Results: There were no changes in glucose tolerance, insulin sensitivity, weight, blood pressure, or lipid profile following resveratrol treatment.
Fasting reactive hyperemia index improved with resveratrol (2.02 ± 0.2 vs 1.76 ± 0.02, p = .002). RNA-Seq analysis yielded 140 differentially
expressed transcripts (corrected p-value ≤ .05), predominantly associated with mitochondrial genes and noncoding RNA. Ingenuity Pathway
Analysis confirmed that mitochondrial dysfunction (p = 2.77 × 10−12) and oxidative phosphorylation (p = 1.41 × 10−11) were the most
significantly perturbed pathways. Mitochondrial number, but not size, was increased.
Conclusions: Resveratrol treatment of older adults with impaired glucose regulation may have beneficial effects on vascular function, but not
glucose metabolism or insulin sensitivity. Changes in gene expression suggest effects similar to those observed with caloric restriction, which
has been shown to increase life and health span in animal models, although its significance for humans is uncertain. Future human studies
should address the appropriate dose range and low bioavailability of resveratrol.
Keywords: Prediabetes—Gene expression—Polyphenols—Aging

Aging in humans is a well-established risk factor for many disabling Resveratrol (trans-3, 5, 4′-trihydroxystilbene), a natural poly-
and chronic conditions, among them diabetes, cardiovascular dis- phenol found in fruits and medicinal plants, such as Japanese knot-
ease, Alzheimer’s disease, and cancer. In fact, the risk of death from weed (Polygonum cuspidatum), has emerged as an attractive agent
these causes is dramatically accelerated with increasing age, impos- to counteract age-related diseases. Interest in this compound has
ing a tremendous burden on health care systems across the world. accelerated in light of its association with the health benefits of red
For this reason, there is a need for effective interventions that have wine and its identification as a chemopreventive agent for cancer
the potential to delay or attenuate age-related chronic diseases and (1). Subsequent reports demonstrated resveratrol to be an activa-
to promote increased health span. tor of sirtuins, a family of NAD(+)-dependent deacetylase enzymes

© The Author 2017. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved.
1703
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1704 Journals of Gerontology: MEDICAL SCIENCES, 2017, Vol. 72, No. 12

thought to mediate the beneficial effects of caloric restriction (2). Montefiore Medical Center. Participants were instructed to abstain
In yeast, worms, and flies, resveratrol has been associated with sir- from nutritional supplements containing resveratrol and to main-
tuin-dependent increase in life span (3). Further study in vitro and tain their usual dietary and physical activity patterns. The compli-
in animal models has shown resveratrol to have beneficial effects on ance rate, defined as the proportion of tablets ingested relative to
glucose metabolism, vascular function, as well as anti-inflammatory the intended number, was calculated based on remaining tablets
and antioxidant properties (4). Resveratrol has also been shown returned at the end of the treatment period.
to promote mitochondrial biogenesis and improve mitochondrial
function (4,5). These potential benefits prompted the initiation of Study Visits and Interventions
human studies to test resveratrol’s role in the prevention and treat- Following a screening visit, the study consisted of two randomly
ment of chronic diseases such as diabetes and cardiovascular disease. assigned 6-week treatment periods (resveratrol and placebo). The
However, formal studies to examine its metabolic effects are limited duration of resveratrol treatment that may be required to demon-
and inconclusive (6–10). strate metabolic effects in humans has not been established. In an
Our group previously conducted a pilot study to examine the animal model, a single oral resveratrol dose acutely improved glu-
effects of resveratrol on glucose metabolism and vascular function in cose tolerance (12), whereas other studies have reported improved
older adults with impaired glucose tolerance (11). Promising effects glucose tolerance with chronic treatment (4) and our own prelimi-
were seen, including improvements in insulin sensitivity, postmeal nary studies suggest measurable effect after 4 weeks of treatment
plasma glucose, and vascular function. We therefore conducted this (11). We reasoned that some signal of efficacy should be apparent
randomized double-blind placebo controlled crossover study to fur- within 6 weeks, even if the full metabolic effects might take longer
ther characterize the effects of resveratrol treatment on metabolism, to emerge. Participants were instructed to begin study drug on the
vascular function, and mitochondrial biogenesis in a similar cohort. evening following the baseline measurements and continue through
the evening prior to metabolic testing at the end of each treatment
period. Following a 3-week washout period, the participants crossed
Methods
over to the other intervention for the second 6-week treatment
Participants period.
The study was approved by the Albert Einstein College of Medicine
Institutional Review Board, and written informed consent was Standard Mixed-Meal Test
obtained from all participants. Adults aged 50–80 years without a Participants were studied following an overnight fast and after a
prior diagnosis of diabetes were screened with a 75-g oral glucose standard breakfast consisting of 110-g carbohydrates, 20-g protein,
tolerance test, and those with fasting plasma glucose of <126 mg/dL and 20-g fat, at the beginning and end of each treatment period.
(or <140 if concurrent hemoglobin A1c [HbA1c] was <7%) and Participants were requested to maintain consistent nutrient intake
2-hour glucose of >170 mg/dL were eligible. Exclusions included on the days prior to each standard meal test. Blood sampling for
serious chronic or acute illness: active cancer (other than non-mel- glucose and insulin levels was performed fasting (Time 0) and 30, 60,
anoma skin cancer) or history of estrogen-dependent neoplasm (eg, 90, 120, 150, and 180 minutes following the mixed-meal through an
breast or endometrial cancer), symptomatic heart failure, chronic indwelling catheter. The assigned treatment (resveratrol or placebo)
obstructive pulmonary disease, inflammatory conditions, significant was administered with the meal at the test conducted at the end of
liver disease or renal disease, recent (within 3 months) cardiovascu- the 6-week treatment period. Insulin sensitivity was estimated using
lar event (myocardial infarction, revascularization, or stroke), and homeostasis model assessment (HOMA-IR) (13) and also from
prior bariatric or other gastric surgery or cigarette smoking. Also insulin and glucose levels obtained following the standard meal
exclusionary was use of drugs known to influence glucose metabo- challenge using the Matsuda index (14,15). Insulin secretion was
lism (eg, systemic glucocorticoids), antioxidant vitamins, warfarin, estimated using the C-peptide deconvolution method as described
or antiplatelet drugs (other than aspirin). Because of potential res- by Van Cauter and colleagues (16). β-Cell function was assessed
veratrol induced CYP450-related drug interactions, treatment with with the oral disposition index (DIo) calculated using the formula:
anti-epileptics, mexilitene, quinidine, cyclosporine, tacrolimus, HIV (ΔI0–30/ΔG0–30) × (1/I0) (17). Insulin and glucose area under the curve
protease inhibitors, and high-dose statin therapy was also exclusion- (AUC) were calculated using the trapezoidal method.
ary. Thirty-eight participants were enrolled in the study; eight par- Assays were performed in the core laboratories of the Einstein
ticipants dropped out (three for study-related adverse events) before Institute for Clinical and Translational Research: glucose, HbA1c,
completion of the second treatment period and are not included in lipoproteins, insulin (radioimmunoassay), C-peptide (radioimmu-
this analysis. noassay), high-sensitivity C-reactive protein (hs-CRP; latex-enhanced
turbidimetric assay), lipoprotein phospholipase A2 (Lp-PLA2;
Study Design ELISA, R&D Systems), and adiponectin (radioimmunoassay; Linco).
The study was a randomized double-blind placebo controlled cross- Blood chemistries, complete blood count, and urinalysis were per-
over study. Resveratrol capsules were obtained from RevGenetics formed in the clinical laboratories of Montefiore Medical Center.
Corporation, and independent verification of the resveratrol content Levels of resveratrol and metabolites were measured at 0, 30,
of the capsules used in this study was performed in the Proteomics 60, 120, and 180 minutes during the standard mixed-meal test
Facility, Laboratory for Macromolecular Analysis and Proteomics (SMMT) in the resveratrol treatment period. As a control, resvera-
at the Albert Einstein College of Medicine. A resveratrol dose of trol and metabolites were measured in samples obtained from three
1,500 mg twice daily was administered to the initial nine partici- randomly selected participants during a placebo period SMMT to
pants; however, because of gastrointenstinal side effects, subsequent confirm the sensitivity of the assay to distinguish between samples
participants received 1,000 mg twice daily. Randomization, blind- obtained during the resveratrol and placebo treatment periods.
ing, and dispensing were conducted in the research pharmacy of The concentrations of resveratrol and its metabolites in plasma
Journals of Gerontology: MEDICAL SCIENCES, 2017, Vol. 72, No. 12 1705

were determined by high-performance liquid chromatography– Results

tandem mass spectrometry analysis, using a previously described
Baseline Characteristics
protocol (18).
Thirty participants (19 men, 11 women) with a mean age of 67 ± 7
completed the study. The participants were overweight to obese, with
Endothelial Function Testing
a mean body mass index of 31.5 ± 5.3 and waist circumference of
Endothelial function testing was performed fasting and 90 minutes
103 ± 12 cm, moderately insulin resistant, with baseline HOMA-IR
following the standard meal, using reactive hyperemia peripheral
of 4.08 ± 2.7 and HbA1c was 6.1% ± 0.4%. Mean fasting glucose
arterial tonometry (19), which measures arterial pulse wave ampli-
and 2-hour plasma glucose were 111 ± 14 and 173 ± 36 mg/dL,
tude in the finger before and after 5 minutes of blood flow occlusion
respectively. Hypertension (antihypertensive treatment or clinic
using a standard blood pressure cuff (EndoPAT; Itamar Medical).
blood pressure of >140/90) was present in 16/30, statin treat-
The reactive hyperemia index (RHI) is the ratio of the average pulse
ment in 7/30, and aspirin therapy in 8/30. Baseline characteristics
amplitude in the posthyperemic phase divided by the average base-
of the participants (n = 8) who dropped out prior to completion
line amplitude, with normalization to the signal in the control arm to
of the study did not differ significantly from these who completed
compensate for any systemic changes. Augmentation index (a meas-
(Supplementary Table 1).
ure of arterial stiffness) is also derived from the PAT signal.

Metabolic Parameters
Skeletal Muscle Samples
Cardiometabolic variables are shown in Table 1. There were no
Skeletal muscle biopsies were performed in the fasting state prior
significant differences in metabolic parameters observed during
to the SMMT in 16 participants. A muscle sample of ~50–100 mg
the resveratrol or placebo treatment periods. Fasting plasma glu-
was obtained with a spring-loaded biopsy needle (Bard Instruments)
cose (111 ± 13 vs 113 ± 13 mg/dL, p = .28), 3-hour glucose AUC
in the mid-thigh region (vastus lateralis) following local anesthesia
(508 ± 88 vs 513 ± 80, p = .73), and 3-hour insulin AUC (330 ± 156
extending into the muscle area. A ~1 mg muscle sample was pre-
vs 366 ± 228, p = .47) were observed during resveratrol and pla-
served in glutaraldehyde for electron microscopy (EM). The remain-
cebo treatment periods, respectively. Insulin sensitivity assessed by
ing sample was immediately homogenized in Trizol, frozen in liquid
the Matsuda index was unchanged (2.3 ± 1.7 vs 2.0 ± 0.99, p = .46).
nitrogen, and stored at −80°C for subsequent mRNA extraction and
Insulin secretion assessed by C-peptide deconvolution was slightly
analysis of gene expression. The RNA-Seq analysis was performed at
reduced (8.9 ± 8 vs 8.1 ± 3 pmol/kg/min, p = .03) after resvera-
Einstein’s Sequencing Core Facility, using multiplexed 100 bp single-
trol treatment, but β-cell function assessed by the disposition index
end sequencing on an Illumina HiSeq2500 (http://www.illumina.
(1.65 ± 0.8 vs 1.53 ± 0.7 mM−1, p = .45) showed no difference
com/technology/mrna_seq.ilmn).
EM was performed on skeletal muscle samples postfixed with
1% osmium tetroxide followed by 1% uranyl acetate. Sections
Table 1. Cardiometabolic Variables During Standard Meal
(80 nm) were prepared and stained with uranyl acetate followed by
Challenge Test; Placebo Versus Resveratrol (n = 30)
lead citrate. No fewer than 10 images per sample were analyzed at
8,000× magnification using a JEOL 1200EX transmission electron Variable Resveratrol Placebo p
microscope at 80 kV. EM was performed on five randomly selected
muscle sample pairs. Age, y 67 (7) —
Male gender, n (%) 19 (63) —
BMI (kg/m2) 32 (5) —
Statistical Analysis Fasting plasma glucose (mg/dL) 111 (12) 113 (13) .28
Data are presented as mean (± SD) for baseline values. Placebo versus Glucose AUC0-180 508 (88) 513 (80) .73
resveratrol variables (eg, peak and AUC glucose, insulin, Matsuda Peak postmeal glucose (mg/dL) 200 (34) 202 (34) .84
index, RHI, etc.) were compared using a paired t test. A nonpara- HbA1c (%) 6.4 (0.4) 6.3 (0.4) .15
Fasting insulin (µU/mL) 17.0 (6.6) 17.5 (7.4) .54
metric test (Wilcoxon’s test) was used if data were not normally
Insulin AUC0-180 330 (156) 366 (228) .47
distributed. The study was designed to have 90% power to detect
HOMA-IR 4.8 (1.9) 5.1 (2.3) .40
a 20 mg/dL difference in the primary study outcome, glucose AUC, Matsuda index 2.3 (1.7) 2.0 (1.0) .46
using data from our earlier pilot study (11), in which we observed Insulin secretion (pmol/kg/min) 8.9 (3.0) 8.1 (2.5) .03
glucose AUC of 469 ± 23 versus 428 ± 19 (p = .001), at baseline Disposition index (mM−1) 1.65 (0.8) 1.53 (0.7) .45
and after 4 weeks of resveratrol, respectively. The possibility of a Weight (kg) 90.3 (17.6) 89.7 (17.2) .13
carry-over effect between treatment periods was analyzed using a Percent body fat (BIA) 29.2 (10.6) 28.7 (11.2) .87
mixed effects model controlling for the baseline value at the begin- Systolic blood pressure (mmHg) 132 (11) 130 (13) .38
ning of each treatment period. No evidence of carry-over effect was Diastolic blood pressure (mmHg) 79 (8) 78 (7) .19
observed. Data analysis was performed using SAS version 9.4. HDL cholesterol (mg/dL) 44 (11) 43 (10) .71
LDL cholesterol (mg/dL) 116 (40) 109 (35) .21
The raw RNA-Seq reads were quality controlled and sub-
Triglycerides (mg/dL) 157 (118) 125 (50) .08
sequently aligned to the hg19 build of the human genome using
hs-CRP (mg/L) 2.4 (2.1) 2.6 (2.3) .60
GSNAP (20). The mapped reads were then processed using the Adiponectin (µg/mL) 10.1 (6.8) 11.4 (10.3) .20
edgeR package (21) within the R/Bioconductor environment to Lp-PLA2 (ng/mL) 185.0 (65.2) 177.5 (58.7) .36
identify differentially expressed transcripts between pre- and post-
treatment cohorts with a false discovery rate of <0.05 (21,22). Note: AUC = area under the curve; BIA = bioimpedance analysis;
These gene lists were subsequently imported into Ingenuity Pathway BMI = body mass index; HDL = high-density lipid; hs-CRP = high-sensitivity
Analysis (23) to identify enriched pathways, Gene Ontology terms, C-reactive protein; LDL = low-density lipid; Lp-PLA2 = lipoprotein phospho-
and disease-relevant associations. lipase A2. Results are mean (SD).
1706 Journals of Gerontology: MEDICAL SCIENCES, 2017, Vol. 72, No. 12

between resveratrol or placebo treatment. Weight, blood pressure, randomly selected participants during a placebo period SMMT and
HbA1c, low-density lipid cholesterol, hs-CRP, Lp-PLA2, and adi- none was detected.
ponectin were similarly unchanged.
Mitochondrial Function
Endothelial Function RNA extracted from skeletal muscle samples underwent deep tran-
Fasting RHI was significantly better following resveratrol treatment scriptomic study using RNA-Seq and yielded 140 differentially
(2.02 ± 0.2 vs 1.76 ± 0.02, for resveratrol and placebo, respectively, expressed transcripts (corrected p-value ≤ .05), with the dominant
p = .002; Figure 1). No difference in postprandial RHI was seen ones being associated with mitochondrial genes and noncoding RNA
following resveratrol treatment as compared to placebo (1.79 ± 0.4 (Supplementary Table 2). Ingenuity Pathway Analysis confirmed
vs 1.69 ± 0.38, p = .38). Augmentation index did not differ between that mitochondrial dysfunction (p = 2.77 × 10−12) and oxidative
resveratrol and placebo treatment periods, in either the fasting phosphorylation (p = 1.41 × 10−11) were the most significantly per-
(16.1 ± 15.4 vs 17.3 ± 13.4, p = .9) or postmeal conditions (6.2 ± 9.5 turbed (downregulated) pathways following resveratrol treatment
vs 5.7 ± 8.9, p = .8). (Table 2). The significantly differentially expressed genes included
ENDOG, IGFBP7, and QDPR, all of which have been previously
Resveratrol Levels implicated in modulating mitochondrial function in aging/longevity
Levels of resveratrol and metabolites were measured at 0, 30, 60, contexts (24–26). Pathways involving immune function (regulation
120, and 180 minutes during the SMMT in the resveratrol treatment of IL-2 expression and IL-1 signaling) were also significantly affected
period (Figure 2). No unmodified resveratrol was detectable at Time by resveratrol. EM performed in five pairs (resveratrol and placebo)
0, but became detectable at low levels at subsequent time points. of muscle samples showed mitochondrial number was increased
In contrast, levels of resveratrol metabolites (resveratrol-3-O-sulfate, with resveratrol treatment in all cases, but mitochondrial area and
resveratrol-3-O-glucuronide, and resveratrol-4′-O-glucuronide) morphology did not change (Supplementary Figure 1).
were detectable at Time 0 and increased substantially over the 180
minutes following resveratrol administration. As a control, resvera- Safety and Adherence
trol and metabolites were measured in samples obtained from three The first nine participants enrolled in the study were treated with
3 g of resveratrol daily. Three of these participants experienced
severe gastrointestinal symptoms, one requiring hospitalization
(Supplementary Table 3). Subsequently, the resveratrol dose was
lowered to 2 g/d for remaining participants, and no further gas-
trointestinal symptoms were reported. There were no other serious
adverse events or changes in laboratory safety parameters.
Adherence to study drug (assessed by pill count) was excellent,
with 94% and 92% of expected drug consumed during the resvera-
trol and placebo periods, respectively.

Discussion
In this cohort of older adults with impaired glucose tolerance, we
found no evidence of resveratrol effect on weight, carbohydrate tol-
erance, insulin sensitivity, or β-cell function. Beneficial effects were
seen on endothelial function, although other cardiovascular risk
Figure 1. Reactive hyperemia index (RHI) in resveratrol versus placebo factors (eg, lipids, blood pressure, hs-CRP) were largely unaffected.
treatment periods. RHI is the ratio of posthyperemia pulse amplitude divided
Importantly, we show evidence that resveratrol may increase mito-
by baseline amplitude (see Methods for details).
chondrial number and modulate pathways involved in oxidative
phosphorylation and inflammation in humans.
Studies in animal models have demonstrated positive effects of
resveratrol on glucose metabolism, including enhanced insulin sen-
sitivity and improvements in insulin secretion and glucose tolerance
(4,5). Human studies have mostly been small and of short duration,
used widely varying resveratrol doses and have shown mixed results
(27). The largest study (n = 214) of resveratrol in patients with
established diabetes reported improvement in fasting and postchal-
lenge glucose with doses up to 5 g/d, but no change in HbA1c or
insulin levels, making the results difficult to interpret (28). Among
the more rigorous studies, Timmers and colleagues demonstrated
improved metabolic profile (lower HOMA, triglyceride, and leptin
levels), reduced intrahepatic lipid content, and improved mitochon-
drial function in skeletal muscle in obese men using 150 mg/d of
Figure 2. Levels of RSV and its metabolites at 0, 30, 60, 120, and 180 minutes
during the SMMT in the resveratrol treatment period. RSV = resveratrol; resveratrol (9). Subsequently, exhaustive studies in healthy obese
RSV-G1 = resveratrol glucuronide 1; RSV-G2 = resveratrol glucuronide 2; men (1,500 mg/d) and healthy nonobese postmenopausal women
RSV-S = resveratrol sulfate; SMMT = standard mixed-meal test. (75 mg/d) failed to demonstrate any evidence of resveratrol effects
Journals of Gerontology: MEDICAL SCIENCES, 2017, Vol. 72, No. 12 1707

Table 2. Predominant Canonical Pathways Identified by Ingenuity Pathways Analysis of Skeletal Muscle Gene Expression

Canonical Pathway p Value Ratio Molecules

Mitochondrial dysfunction 2.5 × 10−12 12/71 MT-CO1, MT-ATP6, MT-ND5, MT-ND6, NDUFS7, MT-CYB, MT-
ND4, MT-CO3, MAPK9, SDHC, MT-Co2, MT-ND3
Oxidative phosphorylation 1.6 × 10−11 10/109 MT-Co1, MT-ATP6, MT-ND5, NDUFS7, MT-CYB, MT-ND4, MT-
CO3, SDHC, MT-CO2, MT-ND3
Regulation of IL-2 expression in activated and 3.7 × 10−3 3/79 RAC1, MAPK9, TOB1
anergic T lymphocytes
IL-1 signaling 5.5 × 10−3 3/91 TAB2, MAPK9, GNG7
SAPK/JNK signaling 6.0 × 10−3 3/94 RAC1, MAPK9, GNG7

on insulin sensitivity, body composition, or energy expenditure resveratrol’s reported ability to increase activity of endothelial nitric
(8,10). It has been suggested that resveratrol’s effect may be lim- oxide synthase and inhibit the vasoconstrictor, endothelin (43).
ited to individuals with metabolic compromise (29), as is the case However, when administered with the standard meal, no additional
in rodent models (30). Our cohort, selected to have obesity, insu- effects of acute administration were apparent. No effect of resvera-
lin resistance, and impaired glucose tolerance would appear ideal trol on augmentation index was demonstrable in our study. However,
to test resveratrol’s metabolic effects, while avoiding the potential others have reported that resveratrol improves arterial pulse wave
confounding of established diabetes and its treatment. Our failure, velocity (a measure of arterial stiffness) in nonhuman primates (44),
then, to demonstrate any detectable effect on glucose metabolism and there is an ongoing study in humans to explore this (45).
suggests other study design issues—for example, dose or duration The gene expression studies showed evidence for a pronounced
of treatment, may be critical. Some evidence suggests that high-dose perturbation of mitochondrial activity in the muscle samples taken
resveratrol (as used in our study) may act via a SIRT1-independent with resveratrol versus placebo. The most notably perturbed path-
pathway and could actually impair insulin action via excess acti- ways identified by Ingenuity Pathway Analysis are defined as “mito-
vation of PGC1α (31). Possibly, longer treatment with lower dose chondrial dysfunction” and “oxidative phosphorylation,” with the
resveratrol may be required to modulate metabolism, as was the case majority of mitochondrial transcripts involved in both pathways
in the rodent studies (30). Furthermore, although the Matsuda index being downregulated. Furthermore, the relevant transcripts are all
appears to have better predictive value as a surrogate index of insu- associated with the functioning of the four mt-DNA encoded mito-
lin sensitivity relative to HOMA-IR, both measures are recognized chondrial complexes (I, III, IV, and V) involved in the electron trans-
to have limitations as longitudinal measures of insulin sensitivity in port chain. Interestingly, metformin—an antidiabetic drug known to
response to therapeutic interventions (32). However, it may also be target a number of aging-related mechanisms—is shown to inhibit
the case that resveratrol simply does not have important effects on mitochondrial complex I and to influence reactive oxygen spe-
glucose metabolism in humans. cies production and mitochondrial bioenergetics, suggesting some
The rapid metabolism of resveratrol, and thus very low res- similarity to the action of resveratrol (46,47). Downregulation of
veratrol levels following oral dosing, has been a conundrum in the genes associated with mitochondrial bioenergetics has been previ-
field. Unmodified resveratrol was present at very low levels follow- ously reported in caloric restriction studies in rhesus monkeys (48),
ing administration during the SMMT, but metabolites showed a where it was suggested that the caloric restriction group was effec-
robust increase. Furthermore, resveratrol metabolites were detect- tively in a hypometabolic state associated with reduced activity of
able at Time 0, prior to supervised dose administration, providing the mitochondrial electron transport system. Analysis of adenosine
evidence of adherence to chronic dosing during the study. Others triphosphate production and mitochondrial energy metabolism in
have demonstrated that specific resveratrol metabolites retain bio- resveratrol-treated participants can provide further insights into the
logical activity in some tissues (33) and that intracellular reservoirs role of resveratrol on mitochondrial activity and its role in targeting
of resveratrol metabolites may undergo deconjugation and result in aging. Pathways involved in immune regulation were also perturbed
significant intracellular levels of resveratrol (34). Although “thera- with resveratrol, although two circulating inflammatory markers,
peutic” resveratrol levels have not been defined, we feel confident hs-CRP and Lp-PLA2, were unchanged.
that our participants had substantial resveratrol exposure during EM, performed in a subset of samples, demonstrated an increase
the 6-week treatment period, including during the SMMT. Levels of in mitochondrial number, but not size. Resveratrol is known to
resveratrol and metabolites were not measured in every participant increase the activity of PGC1α (4), an important regulator of mito-
during placebo treatment. However, detectable levels resulting from chondrial mass and function. PGC1α’s stimulation of mitochondrial
dietary intake—estimated to rarely exceed 6–8 mg/d (35)—appear number appears to be mediated by ENDOG (which encodes the
unlikely. mitochondrial enzyme endonuclease G), the expression of which
Resveratrol treatment has been reported to improve a variety of was increased in our participants following resveratrol treatment.
vascular biomarkers, including expression of adhesion molecules Furthermore, because ENDOG expression has been reported to
(VCAM, ICAM) and inflammatory cytokines (IL-6, TNF-α, PAI-1) decline with aging (25) and is stimulated by resveratrol, this could
(7,9,36–38), as well as reduction in oxidative stress (39). Endothelial provide a potential mechanism for some of resveratrol’s putative
function, assessed by flow-mediated vasodilation, has been reported antiaging effects. The pathway analysis of our samples showing per-
to improve following acute (40) and chronic (41) resveratrol dos- turbation of pathways involved in mitochondrial dysfunction and
ing of as little as 10 mg. We observed a modest, but significant oxidative phosphorylation provides indirect support for the signifi-
improvement in RHI (a nitric oxide–dependent phenomenon) (42) cance of the change in mitochondrial number. Expression of several
in response to chronic resveratrol treatment. This is consistent with mitochondrial genes was downregulated with resveratrol (shown in
1708 Journals of Gerontology: MEDICAL SCIENCES, 2017, Vol. 72, No. 12

Supplemental Table 2), but the specific functional consequences of 7. Ghanim H, Sia CL, Abuaysheh S, et al. An antiinflammatory and reac-
these changes and the relationship to changes in mitochondrial num- tive oxygen species suppressive effects of an extract of Polygonum cuspi-
ber cannot be directly addressed from our data. datum containing resveratrol. J Clin Endocrinol Metab. 2010;95:E1–E8.
doi:10.1210/jc.2010-0482
Formal safety testing of nutritional supplements such as res-
8. Poulsen MM, Vestergaard PF, Clasen BF, et al. High-dose resveratrol
veratrol is generally not required by regulatory agencies, including
supplementation in obese men: an investigator-initiated, randomized,
the U.S. Food and Drug Administration, so safety data are limited.
placebo-controlled clinical trial of substrate metabolism, insulin sensitiv-
The gastrointestinal side effects of resveratrol observed in our study ity, and body composition. Diabetes. 2013;62:1186–1195. doi:10.2337/
have been reported previously (49) and appear to be dose related. db12-0975
Although widely used and presumed safe, the potential adverse 9. Timmers S, Konings E, Bilet L, et al. Calorie restriction-like effects of
effects of long-term pharmacologic doses of resveratrol have not 30 days of resveratrol supplementation on energy metabolism and
been studied. metabolic profile in obese humans. Cell Metab. 2011;14:612–622.
doi:10.1016/j.cmet.2011.10.002
10. Yoshino J, Conte C, Fontana L, et al. Resveratrol supplementation does
Conclusions not improve metabolic function in nonobese women with normal glucose
tolerance. Cell Metab. 2012;16:658–664. doi:10.1016/j.cmet.2012.09.015
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effects similar to those observed with caloric restriction, which has 12. Chi TC, Chen WP, Chi TL, et al. Phosphatidylinositol-3-kinase is involved in
been shown to increase life and health span in many animal models; the antihyperglycemic effect induced by resveratrol in streptozotocin-induced
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Funding
Metabolism. 2006;55:676–690. doi:10.1016/j.metabol.2006.01.002
This study was supported by the American Diabetes Association (1-11-CT- 16. Van Cauter E, Mestrez F, Sturis J, Polonsky KS. Estimation of insulin secre-
12), the Glenn Foundation for Medical Research, the NIH/National Center tion rates from C-peptide levels. Comparison of individual and standard
for Advancing Translational Science (NCATS) Einstein-Montefiore CTSA kinetic parameters for C-peptide clearance. Diabetes. 1992;41:368–377.
Grant Number UL1TR001073, the Einstein-Sinai Diabetes Research Center doi:10.2337/diab.41.3.368
(NIH-5P60 DK20541, RO1 AG028730 to D.A.S.), and the Intramural 17. Utzschneider KM, Prigeon RL, Faulenbach MV, et al. Oral disposition
Research Program at the National Institute on Aging (R.M.). index predicts the development of future diabetes above and beyond fast-
ing and 2-h glucose levels. Diabetes Care. 2009;32:335–341. doi:10.2337/
dc08-1478
Acknowledgments 18. Fiori JL, Shin YK, Kim W, et al. Resveratrol prevents β-cell dedifferen-
The authors thank the staff of the Einstein Clinical Research Center, Dr. Hillel tiation in nonhuman primates given a high-fat/high-sugar diet. Diabetes.
Cohen for assistance with study design, K.S.S. Doussou for analysis of plasma 2013;62:3500–3513. doi:10.2337/db13-0266
resveratrol levels, and the study volunteers. 19. Bonetti PO, Pumper GM, Higano ST, Holmes DR Jr, Kuvin JT, Lerman
A. Noninvasive identification of patients with early coronary athero-
sclerosis by assessment of digital reactive hyperemia. J Am Coll Cardiol.
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