Effect of Resveratrol on Age-related Insulin Resistance and Inflammation in Humans
Phase: PHASE2
Conditions: Type 2 Diabetes Mellitus, Insulin Resistance
Sponsor: Albert Einstein College of Medicine

Hawkins, Meredith MD 
IND 103,034 
 
1 
Revised on 6/12/2014 
 
Effect of resveratrol on age-related insulin resistance and inflammation in humans 
 
OVERVIEW  
We hypothesize that resveratrol will improve insulin resistance, tissue lipid accumulation, 
inflammation, and mitochondrial function in middle aged and Type 2 Diabetes subjects. Using 
state of the art physiological tools for human investigation, we have shown that adipose tissue 
inflammation and tissue lipid accumulation are enhanced with aging in concert with systemic insulin 
resistance. 
We 
will 
administer 
resveratrol to overweight middle aged 
humans and study its effects on insulin 
action, 
tissue 
lipid 
accumulation, 
inflammation, 
and 
mitochondrial 
function. We will perform paired 
hyperinsulinemic clamps, the „gold 
standard‟ measure of insulin action, 
following both placebo and resveratrol 
treatment. We will perform adipose 
tissue and muscle biopsies to assess 
inflammatory response in adipocytes 
and adipose tissue macrophages, and 
to assess mitochondrial biology in 
muscle. Noninvasive 1H magnetic resonance spectroscopy (MRS) will be used to quantify the effects 
of resveratrol on tissue lipid accumulation.  
 
SPECIFIC AIMS: 
 
Human aging is associated with increased fat mass, insulin resistance and systemic inflammation. In 
addition to overall increases in fat mass, accumulation of lipid in such insulin-sensitive tissues as liver 
and skeletal muscle appears to contribute importantly to insulin resistance. Furthermore, age-related 
decreases in muscle mitochondrial ATP production rate and mitochondrial DNA copy number are likely 
to contribute to impaired insulin-mediated glucose disposal, as well as decreased muscular endurance 
and activity levels. Additionally, systemic inflammation is now believed to contribute to insulin 
resistance and thus ultimately to many age-related disorders, including diabetes mellitus, Alzheimer‟s 
disease, atherosclerosis and many cancers. Recently, it has been recognized that obesity and aging 
are both associated with increased macrophage infiltration into adipose tissue, and that these 
infiltrating macrophages are an important source of systemic cytokine production. We have previously 
reported that adipose macrophages from older humans display increased activation (and production of 
pro-inflammatory and pro-atherosclerotic peptides). This heightened reactivity of adipose 
macrophages in older individuals and in those with Type 2 diabetes is likely to contribute both to 
insulin resistance and to systemic inflammation.  
 
Mammalian Sirt1 seems to be particularly important in regulating glucose homeostasis, insulin action 
and nutrient sensing. Collectively, several studies have shown that activation of Sirt1 by 
pharmacological agents like resveratrol has intracellular effects that are strikingly similar to those 
observed with caloric restriction.  Specifically, resveratrol has been shown to improve mitochondrial 
function and appears to protect against metabolic disease by activating PGC-1alpha. Activation of 
PPAR-gamma in macrophages by resveratrol reduces activation of these inflammatory cells, which 
may be very pertinent to adipose tissue inflammation. It has been proposed that resveratrol may 
improve insulin resistance, although this has yet to be established in vivo in animals or humans. 
Preventing the of Sirt1 decline in human aging
Improves hepatic 
insulin resistance 
Improves 
Inflammation
Metabolic syndrome
Resveratrol
Prevention of: 
Improves
Hepatic steatosis 
Improves peripheral
insulin resistance
IRB NUMBER: 2007-534
IRB APPROVAL DATE: 04/30/2015

Hawkins, Meredith MD 
IND 103,034 
 
2 
Moreover, resveratrol has been shown to enhance cognitive function, reduce neural degeneration, and 
improve motor function in animal models. Thus, for this project we hypothesize that resveratrol 
will mediate important metabolic and anti-inflammatory effects that will improve insulin 
resistance in middle aged and older humans and in those with Type 2 diabetes. Specifically, we 
will determine whether and how Sirt1 activation with resveratrol will impact systemic glucose 
metabolism, lipotoxicity, mitochondrial dysfunction, inflammation, and motor and cognitive function in 
insulin resistant humans.  
 
Aim 1: To study the effects of resveratrol on age and diabetes related insulin resistance. We 
hypothesize that treatment of overweight, middle aged human subjects and those with Type 2 
diabetes with resveratrol will result in improved insulin sensitivity, as demonstrated by increased 
insulin-stimulated glucose uptake and enhanced insulin-mediated suppression of glucose production. 
Insulin action will be evaluated by stepped hyperinsulinemic euglycemic insulin „clamp‟ studies in 
middle aged subjects with normal glucose tolerance, with progressive increases in insulin levels 
designed to optimally examine both hepatic and peripheral insulin sensitivity.  
 
Aim 2: To examine the effects of resveratrol on systemic inflammation and/or on adipose 
tissue macrophages. We hypothesize that treatment of overweight, middle aged human subjects 
and in those with Type 2 diabetes with resveratrol will lead to reductions in systemic inflammatory 
markers and reduced content and/or activation of adipose tissue macrophages. Systemic 
inflammatory markers to be measured will include TNF-a, IL-6, IL-1b, MCP-1. Adipose tissue 
macrophages will be isolated from subcutaneous abdominal adipose tissue, and will be quantified by 
fluorescence activated cell sorting (FACS) analysis. Gene expression of a variety of pro-inflammatory 
and pro-atherosclerotic factors will be examined by „real-time‟ rt-PCR, to determine how resveratrol 
affects activation of adipose macrophages. 
 
Aim 3: To explore the effects of resveratrol on skeletal muscle metabolism and function. We 
hypothesize that resveratrol treatment will enhance mitochondrial metabolism and improve strength 
and contractility. In collaboration with the Mitochondrial Metabolic Core and Department of Neurology, 
we will examine the effects of systemic resveratrol on mitochondrial content, signaling molecules 
involved in mitochondrial biogenesis, DNA and protein oxidative damage, mitochondrial respiration in 
human skeletal muscle, and motor function and mobility 
 
Aim 4: Assess the effects of reveratrol on cognition. We hypothesize that resveratrol will enhance 
cognitive function in middle-aged individuals and in those with Type 2 diabetes. In collaboration with 
the Department of Neurology, we will examine cognition, including attention networks of alerting, 
orienting, and executive attention, as well as perform a clinical neuropsychology battery providing a 
comprehensive assessment of cognitive function, including measures of: a. intelligence, b. memory, 
c. language, d. executive function, e. visuospatial function and praxis, f. processing speed, and g. 
mental status.  
 
These proposed studies will address several unresolved questions about the role of 
Sirtuins/Sirt1 in humans: 
 
1. on insulin sensitivity in liver and in peripheral tissues (particularly skeletal muscle) 
2. on several aging-associated determinants of insulin resistance, including: 
 
tissue lipid accumulation  
 
adipose tissue and systemic inflammation  
 
mitochondrial and motor function  
 
cognitive function 
 
 
IRB NUMBER: 2007-534
IRB APPROVAL DATE: 04/30/2015

Ha w ki ns, Mere dit h M D 
I N D 1 0 3, 0 3 4 
 
3  
 
 
 
 
B A C K G R O U N D A N D SI G NI FI C A N C E  
T h e m et a b oli c s y n dr o m e of a gi n g a n d it s cli ni c al c o n s e q u e n c e s. T h e m et a b oli c s y n dr o m e of 
a gi n g d efi n e s a cl u st er of a b n or m aliti e s, i n cl u di n g i n s uli n r e si st a n c e, o b e sit y, a b n or m al gl u c o s e 
t ol er a n c e, d y sli pi d e mi a, a n d h y p ert e n si o n (1 ). O b e sit y a n d/ or c hr o ni c i n cr e a s e s i n n utri e nt i nt a k e ar e 
i n cr e a si n gl y r e c o g ni z e d t o pl a y c e ntr al r ol e s i n t h e p at h o g e n e si s of m a n y a g e-r el at e d di s e a s e s, 
i n cl u di n g di a b et e s m ellit u s, art eri o s cl er oti c a n d at h er o s cl er oti c v a s c ul ar di s e a s e s, 
m a n y t y p e s of 
c a n c er ( p arti c ul arl y br e a st, pr o st at e, c ol or e ct al a n d e n d o m etri al), a n d Al z h ei m er‟ s di s e a s e ( 2 ,3 ). 
T o g et h er, t h e s e c o n diti o n s c o ntri b ut e i m p ort a ntl y t o t h e o b e sit y-r el at e d i n cr e a s e i n all- c a u s e m ort alit y 
(4 ,5 ). F urt h er m or e, t h e pr e s e n c e of i n s uli n r e si st a n c e a n d t h e „ m et a b oli c s y n dr o m e‟ (i m p air e d gl u c o s e 
t ol er a n c e, d y sli pi d e mi a, h y p ert e n si o n, vi s c er al o b e sit y, d e cr e a s e d fi bri n ol y si s) i s hi g hl y c orr el at e d 
wit h all of t h e s e di s e a s e s ( 6 ,7 ,8 ,9 ,1 0 ,1 1 ,1 2 ). 
R ol e of a gi n g p er s e i n t h e p at h o g e n e si s of m et a b oli c c h a n g e s.  It i s p arti c ul arl y i m p ort a nt t o n ot e 
t h at a gi n g it s elf i s a s s o ci at e d wit h i n cr e a s e s i n i nfl a m m at or y a cti vit y (1 3 ). I n d e e d, c h a n g e s i n t h e 
i n n at e i m m u n e s y st e m h a v e b e e n n ot e d wit h a gi n g (1 4 ). I n p arti c ul ar, a gi n g i s a s s o ci at e d wit h 
i n cr e a s e d cir c ul ati n g l e v el s of c yt o ki n e s a n d a c ut e p h a s e pr ot ei n s i n vi v o, i n cl u di n g T N F- al p h a a n d 
I L- 6 (1 5 ,1 6 ,1 7 ,1 8 ). T h e c hr o ni c l o w- gr a d e i nfl a m m ati o n i n a gi n g a n d i s e pi d e mi ol o gi c all y r el at e d t o 
a g e- a s s o ci at e d di s or d er s ( e g, Al z h ei m er di s e a s e, at h er o s cl er o si s, t y p e 2 di a b et e s, et c.) a n d 
e n h a n c e d m ort alit y ri s k ( 1 9 ,2 0 ,2 1 ), a n d a d v er s el y i m p a ct s h y p ot h al a mi c p at h w a y s vit al t o e n er g y 
b al a n c e a n d gl u c o s e h o m e o st a si s ( 2 2 ). A m o n g t h e pr o p o s e d e x pl a n ati o n s f or t hi s p h e n o m e n o n, 
w hi c h h a s b e e n r ef err e d t o a s "i nfl a m m- a gi n g," ar e c o nti n u o u s e x p o s ur e t o a nti g e ni c l o a d s a n d str e s s 
(1 9 ).  
F urt h er m or e, t h er e i s a r e m ar k a bl e si mil arit y i n p at h ol o gi c pr o c e s s e s b et w e e n a gi n g a n d di a b et e s 
m ellit u s, ( 2 3 ). It i s e s p e ci all y n ot e w ort h y t h at n o n di a b eti c ol d er i n di vi d u al s c a n d e v el o p mi cr o v a s c ul ar 
c h a n g e s, e g. r eti n o p at h y ( 2 4 ,2 5 ) a n d n e ur o p at h y (2 6 ), t h at ar e p at h ol o gi c all y i n di sti n g ui s h a bl e fr o m 
t h o s e s e e n i n di a b et e s m ellit u s ( D M). I n d e e d, b ot h a gi n g a n d D M ar e a s s o ci at e d wit h i n cr e a s e d 
a v ail a bilit y of c ert ai n s u b str at e s s u c h a s F F A, a s w ell a s i n cr e a s e d i nfl a m m ati o n a n d alt er e d b o d y 
c o m p o siti o n.  
Eff e ct s of a gi n g o n 
mit o c h o n dri al f u n cti o n: Mit o c h o n dri a ar e k n o w n a s t h e “ p o w er pl a nt s” of 
e u k ar y oti c c ell s, g e n er ati n g m o st of t h eir a d e n o si n e tri p h o s p h at e ( A T P) s u p pl y t hr o u g h o xi d ati v e 
p h o s p h or yl ati o n a n d el e ctr o n tr a n sf er ( 2 7 ). T h e y ar e f urt h er m or e i n v ol v e d i n a n u m b er of ot h er 
i m p ort a nt pr o c e s s e s, s u c h a s a p o pt o si s, c ell si g n ali n g, a n d c o ntr ol of c ell gr o wt h a n d diff er e nti ati o n. 
T h e m a st er r e g ul at or of mit o c h o n dri al bi o g e n e si s a n d f u n cti o n h a s b e e n s h o w n t o b e p er o xi s o m e 
pr olif er at or- a cti v at e d r e c e pt or g- c o a cti v at or- 1 α ( P G C- 1 α). P G C- 1 α i n d u c e s mit o c h o n dri a s p e cifi c 
tr a n s cri pti o n f a ct or s, s u c h a s n u cl e ar r e s pir at or y f a ct or s ( N R F s). N R F s i n t ur n a cti v at e mit o c h o n dri al 
tr a n s cri pti o n f a ct or A ( T F A M) a n d t h er e b y i n d u c e mit o c h o n dri al ( mt D N A) r e pli c ati o n (2 8 ,2 9 ).  
 
Wit h i n cr e a si n g a g e t h er e a p p e ar s t o b e a d e cli n e i n mit o c h o n dri al f u n cti o n a n d n u m b er ( 3 0 ,3 1 ,3 2 ). I n 
t h e el d erl y, d e cli n e s i n mit o c h o n dri al tr a n s cri pti o n l e v el s (3 0 ), m u s cl e mit o c h o n dri al pr ot ei n s y nt h e si s 
(3 3 ) a n d, i m p ort a ntl y, d e cr e a s e s i n k e y mit o c h o n dri al e n z y m e s a n d m a xi m al A T P pr o d u cti o n c a p a cit y 
( M A P R) h a v e b e e n o b s er v e d (3 0 ). Si n c e t h e tr a n s cri pti o n of n u cl e ar e n c o d e d g e n e s of mit o c h o n dri al 
pr ot ei n s i s r e g ul at e d b y P G C- 1 α ( 2 8 ), it i s p o s si bl e t h at t h e f u n d a m e nt al pr o bl e m i s at t h e l e v el of 
n u cl e ar a n d mit o c h o n dri al g e n e e x pr e s si o n. R e c e nt st u di e s h a v e s u g g e st e d t h at t h e u n d erl yi n g 
c a u s e f or mit o c h o n dri al d y sf u n cti o n m a y b e a gi n g- a s s o ci at e d r e d u cti o n s i n A M P- a cti v at e d pr ot ei n 
ki n a s e ( A M P K) a cti vit y ( 3 4 ). A cti v at e d A M P K p h o s p h or yl at e s a n d t h er e b y i n d u c e s P G C- 1 α g e n e 
e x pr e s si o n i n s k el et al m u s cl e ( J a e g er et al. 2 0 0 7). L o w er a cti vit y l e v el s wit h i n cr e a si n g a g e c o ul d 
f urt h er m or e pr e s e nt a d e cr e a s e d sti m ul u s f or D N A r e pli c ati o n, r e s ulti n g i n r e d u c e d mt D N A c o p y 
I R B N U M B E R: 2 0 0 7- 5 3 4
I R B A P P R O V A L D A T E: 0 4/ 3 0/ 2 0 1 5

Hawkins, Meredith MD 
IND 103,034 
 
4 
numbers (35).  In support of this, various studies have shown a positive effect of exercise on PGC-1α 
expression and mtDNA copy number (31,34,36,37). In particular, exercise is likely to significantly 
stimulate AMPK activity, and thus PGC-1α phosphorylation (34).  
Mitochondrial function and insulin action: There has been increasing evidence suggesting an 
association between muscle mitochondrial dysfunction and insulin resistance. Mitochondrial oxidative 
activity and ATP synthesis are both reduced in states of impaired glucose tolerance and insulin 
resistance. This was seen in a recent study by Petersen et al. (2003) comparing mitochondrial 
function between healthy, lean, elderly and healthy, lean, younger volunteers (38). Their results 
showed a ~40% reduction in mitochondrial activity and oxidative phosphorylation in addition to insulin 
resistance in the elderly subjects compared to the controls. Another study noted reduced 
mitochondrial activity and increased skeletal muscle triglyceride storage in young insulin-resistant 
offspring of diabetic parents compared to insulin sensitive controls (39). However, whether the 
mitochondrial dysfunction is primary or secondary to the pathogenesis of insulin resistance is 
currently unknown. A number of studies have proposed that muscle mitochondrial dysfunction may 
play an etiological role in insulin resistance (32,34,40). Reznick et al. reported a strong relationship 
between insulin resistance, dysregulated intracellular lipid metabolism and age-related reductions in 
AMPK-activity (34). They suggested that decreased activation of AMPK, and thus PGC-1α, as well as 
mitochondrial fatty acid oxidation with aging could lead to mitochondrial dysfunction and increased 
muscular triglyceride accumulation, thereby predisposing to insulin resistance and type 2 diabetes 
mellitus (T2DM). Increased intracellular triglyceride storage in liver and skeletal muscle results 
impairs insulin-stimulated glucose uptake in muscle (41).  
An alternative hypothesis is that insulin resistance itself leads to mitochondrial dysfunction with age 
since insulin resistant states, such as type 2 diabetes mellitus (42), decrease the transcription and 
translation of mitochondrial proteins (43, 44, 45) and mitochondrial biogenesis in muscle is enhanced 
by insulin (46). A study by Karakelides et al. recently demonstrated a reduced skeletal muscle 
mitochondrial ATP production rate (MAPR) and gene transcript levels following insulin deprivation 
(47). Reduced muscle mitochondrial function may also affect numerous other cellular functions 
including synthesis of proteins, such as contractile proteins (35,48).  Age-related declines in activity 
levels, ectopic accumulation of fat, and sarcopenia may be related to reduced contractile protein 
synthesis in combination with decreased ATP production in aging and could result in insulin 
resistance. 
SIRT1 effects on glucose metabolism and insulin action: Recent research has found one of the 
seven sirtuin genes, SIRT1, to impact mitochondrial function and biogenesis through positive 
regulation of PGC-1α activity (49,50). SIRT1 is the mammalian homologue of NAD-dependent histone 
deacetylase Sir2 (silencing information regulator 2) and belongs to the family of proteins collectively 
known as sirtuins. It is involved in processes such as cell survival, fat metabolism, and insulin and 
glucose production (51) and has been shown to control the hepatic gluconeogenic/glycolytic 
pathways in the fasted state through deacetylation of PGC-1α (50,52). One way in which lifespan can 
be extended in mammals is through caloric restriction.  Aging significantly reduced SIRT1 activity in 
heart of rats (53). Loss of SIRT1 with age was accelerated in mice with accelerated aging but was not 
observed in long-lived growth hormone-receptor knockout mice (54). Since Sirt1 expression is 
induced in multiple tissues including the liver, brain and muscle during periods of caloric restriction, it 
seems likely that Sirt1 may be a potential candidate for enhancing mammalian longevity.  
SIRT1-induced activation of PGC-1α in cultured liver cells has a differential effect on hepatic glucose 
production, repressing glycolysis but increasing gluconeogenesis (GNG) (52). By deacetylating 
forkhead transcription factor FoxO1 in hepatocytes SIRT1 increases the expression of gluconeogenic 
genes (55).  Moreover, the over-expression of pancreatic beta-cell specific SIRT1 in mice has been 
shown to improve glucose tolerance and increase glucose stimulated insulin secretion, whereas the 
expression of SIRT1 is decreased in insulin resistant cells and the inhibition of SIRT1 induces insulin 
IRB NUMBER: 2007-534
IRB APPROVAL DATE: 04/30/2015

Ha w ki ns, Mere dit h M D 
I N D 1 0 3, 0 3 4 
 
5  
r e si st a n c e (5 6 ,5 7 ). I n Z u c k er f a/f a r at s, SI R T 1 a cti v at or s w er e r e c e ntl y s h o w n t o i m pr o v e w h ol e- b o d y 
gl u c o s e h o m e o st a si s a n d i n s uli n s e n siti vit y i n a di p o s e ti s s u e, s k el et al m u s cl e a n d li v er ( 5 8 ). 
SI R T 1 e x ert s n u m er o u s a d diti o n al eff e ct s o n e n er g y m et a b oli s m. It m o d ul at e s gl u c o s e a n d li pi d 
m et a b oli s m t hr o u g h it s r e g ul ati o n of t h e a di p o c yt e- d eri v e d h or m o n e a di p o n e cti n ( 5 1 ). A di p o n e cti n 
h a s b e e n s h o w n t o i m pr o v e i n s uli n s e n siti vit y a n d m a y i n hi bit t h e d e v el o p m e nt of i n s uli n r e si st a n c e 
(5 9 ). SI R T 1 i n cr e a s e s t h e c o n c e ntr ati o n of a di p o n e cti n t hr o u g h d e a c et yl ati o n of F o x O 1 
(6 0 ). I n 
w hit e a di p o s e ti s s u e, SI R T 1 i s t h o u g ht t o pl a y a r ol e i n a di p o g e n e si s b y pr o m oti n g f at m o bili z ati o n 
t hr o u g h d o w n-r e g ul ati o n of P G C- 1 α, r e s ulti n g i n d e cr e a s e d tri gl y c eri d e st or a g e a n d i n cr e a s e d 
s e cr eti o n of fr e e f att y a ci d s ( F F A) ( 6 1 ).  SI R T 1 m a y al s o c o ntri b ut e t o t h e pr o c e s s of i nfl a m m ati o n, 
c o n si d eri n g it i n hi bit s t h e tr a n s cri pti o n of n u cl e ar f a ct or- k a p p a B ( N F- k a p p a B), o n e of t h e k e y 
r e g ul at or s of i nfl a m m ati o n (5 1 ). T h e pr o-i nfl a m m at or y st at e i s o n e of t h e c h ar a ct eri sti c s of o b e sit y a n d 
t h e m et a b oli c s y n dr o m e (6 2 ). T h u s, a s SI R T 1 i s i n cr e a si n gl y b ei n g r e c o g ni z e d a s a n i m p ort a nt 
r e g ul at or of m et a b oli c h o m e o st a si s a n d mit o c h o n dri al f u n cti o n, t ar g eti n g SI R T 1 t o a m eli or at e a g e-
r el at e d mit o c h o n dri al d y sf u n cti o n a n d i n s uli n r e si st a n c e s h o w s gr e at pr o mi s e.  
R e s v er atr ol’ s 
eff e ct s 
o n i n s uli n 
a cti o n 
a n d 
gl u c o s e 
h o m e o st a si s : 
R e s v er atr ol ( 3, 5, 4‟-
tri h y dr o x y stil b e n e) i s a pl a nt d eri v e d p ol y p h e n oli c c o m p o u n d m ai nl y w ell k n o w n f or it s a nti o xi d a nt a n d 
p h yt o e str o g e ni c pr o p erti e s a n d i s m ai nl y f o u n d i n t h e s ki n of gr a p e s a n d i n r e d wi n e ( 6 3 ). Pr e vi o u s 
st u di e s h a v e al s o r e v e al e d it s a nti-t u m ori g e ni c a n d a nti-i nfl a m m at or y q u aliti e s ( 5 1 ,6 3 ).  It h a s b e e n 
s h o w n t o p o s s e s s a nti pl at el et ( 6 4 ,6 5 ) a n d c ar di o pr ot e cti v e, a nti- at h er o s cl er oti c eff e ct s (6 6 ,6 7 ). 
H o wit z et al. ( 2 0 0 3) d e m o n str at e d t h e a bilit y of r e s v er atr ol t o si g nifi c a ntl y i n cr e a s e t h e a cti vit y of 
SI R T 1 t hr o u g h all o st eri c i nt er a cti o n, r e s ulti n g i n i n cr e a s e d P G C- 1 α a cti vit y ( 6 8 ). R e s v er atr ol h a s b e e n 
s h o w n t o pr o d u c e SI R T 1- d e p e n d e nt eff e ct s i n m a m m ali a n c ell s t h at i m pr o v e or g a ni s m al h e alt h a n d 
c ell ul ar f u n cti o n, s u c h a s i n cr e a s e d i n s uli n s e n siti vit y, mit o c h o n dri al n u m b er, a n d i m pr o v e d m ot or 
f u n cti o n (4 9 ). I n a st u d y b y B a ur et al. ( 2 0 0 6) r e s v er atr ol w a s a bl e t o pr e v e nt t h e n e g ati v e eff e ct s of 
e x c e s s c al ori e i nt a k e i n mi c e o n a hi g h c al ori e di et ( 4 9 ). I n t h eir st u di e s, t h e p h y si ol o g y of mi d dl e-
a g e d mi c e o n a hi g h- c al ori e di et s hift e d t o w ar d s t h at of mi c e o n a st a n d ar d di et w h e n tr e at e d wit h 
r e s v er atr ol, si g nifi c a ntl y i n cr e a si n g t h eir s ur vi v al.  R e s v er atr ol h a s al s o b e e n s h o w n t o i n cr e a s e 
mit o c h o n dri al bi o g e n e si s a n d o xi d ati v e p h o s p h or yl ati o n, a s w ell a s r e d u c e di et-i n d u c e d o b e sit y a n d 
i n s uli n r e si st a n c e i n a r o d e nt m o d el (5 0 ).  F urt h er m or e, i n str e pt o z ot o ci n-i n d u c e d di a b eti c r at s, 
r e s v er atr ol tr e at m e nt i m pr o v e d h y p er gl y c e mi a, i n s uli n r e si st a n c e, h y p ertri gl y c eri d e mi a, a n d c o m m o n 
di a b eti c s y m pt o m s s u c h a s w ei g ht l o s s, p ol y di p si a a n d p ol y p h a gi a ( 6 9 ). I n c o n cl u si o n, gi v e n t h e 
p ot e nti al of r e s v er atr ol, t hr o u g h a cti v ati o n of SI R T 1, t o p o siti v el y i m p a ct mit o c h o n dri al f u n cti o n a n d 
m et a b oli c h o m e o st a si s, m a k e s it a n o pti m al i n str u m e nt of i n v e sti g ati o n i n o ur pr o p o s e d st u di e s. 
R e s v er atr ol’ s eff e ct s o n n e ur al f u n cti o n: 
R e c e ntl y, 
m u c h att e nti o n h a s b e e n p ai d t o t h e 
n e ur o pr ot e cti v e eff e ct s of r e s v er atr ol. A n u m b er of st u di e s h a v e d e m o n str at e d pr ot e cti v e eff e ct s of 
SI R T 1 a cti v ati o n a g ai n st a m yl oi d- β t o xi cit y i n Al z h ei m er‟ s di s e a s e ( 7 0 ,7 1 ). N e ur o d e g e n er ati o n i n t h e 
hi p p o c a m p u s w a s r e d u c e d a n d l e ar ni n g i m p air m e nt w a s pr e v e nt e d i n a m o u s e m o d el of Al z h ei m er‟ s 
di s e a s e w h e n i ntr a c er e br o v e ntri c ul ar r e s v ert ar ol w a s gi v e n ( 7 2 ). Of n ot e, Al z h ei m er‟ s di s e a s e i s 
a s s o ci at e d wit h i m p air e d mit o c h o n dri al  e n er g y m et a b oli s m ( 7 3 ), a n d mit o c h o n dri al d y sf u n cti o n i n 
Al z h ei m er‟ s di s e a s e h a s b e e n s h o w n t o b e r el at e d t o a g e- d e p e n d e nt i nt er a cti o n s of a m yl oi d- β wit h 
mit o c h o n dri al pr ot ei n s ( 7 4 ). SI R T 1 e x pr e s si o n m a y a cti v at e g e n e s r e s p o n si bl e f or n e ur o n al pr ot e cti o n 
a n d pr ot e ct n e ur ol o gi c al f u n cti o n i n di s or d er s s u c h a s s ei z ur e s a n d br ai n i s c h e mi a ( 7 5 ,7 6 ). 
R e s v er atr ol h a s al s o b e e n s h o w n t o pr e v e nt di a b et e s-i n d u c e d l e ar ni n g a n d m e m or y i m p air m e nt, 
m o d ul at e c h oli n er gi c n e ur otr a n s mi s si o n, a n d i m pr o v e c o g niti o n i n str e pt o z o ci n-i n d u c e d di a b eti c r at s 
(7 7 ). Gi v e n t h e n e ur o pr ot e cti v e pr o p erti e s of r e s v er atr ol i n r o d e nt m o d el s, it w o ul d b e i ntri g ui n g t o 
f urt h er i n v e sti g at e t h e s e eff e ct s i n t h e pr o p o s e d h u m a n st u di e s. 
 
 
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6 
 
Resveratrol in Human Subjects 
RATIONALE  
Since a causal link between Sirt1 and aging and diabetes related metabolic changes has not yet been 
established in humans, this protocol is indispensable to establishing the clinical relevance of this 
program. Sirt1 shows significant potential as a useful therapeutic target that can be used in 
ameliorating many of the conditions associated with the metabolic decline of aging and in Type 2 
diabetes  We are particularly interested in investigating the role of Sirt1 in the regulation of glucose 
homeostasis, insulin action and mitochondria function in humans.  Furthermore, demonstrating the 
ability of resveratrol to reverse the metabolic changes of aging and Type 2 diabetes would be of 
tremendous therapeutic importance, particularly in light of the feasibility and safety of longterm 
administration of this orally available agent. 
Our decision to study middle aged subjects is primarily due to the importance of timing this 
intervention to decrease aging-related metabolic changes prior to the presentation of serious age-
related diseases. An additional advantage is the feasibility of recruiting and studying subjects of this 
age group with somewhat arduous insulin clamp studies, biopsies and muscle function tests, as 
compared to a frailer group of elderly subjects. Our preliminary studies, shown below, indicate that 
individuals above 40 years of age already have substantial age-related declines in insulin sensitivity 
that would be appropriate targets of intervention. Additionally, since resveratrol appears to exert more 
beneficial effects in animal models of caloric excess, overweight subjects will be recruited for these 
studies. Similarly, subjects with Type 2 diabetes have established insulin resistance and nutrient 
excess as demonstrated by elevated free fatty acids and are likely to benefit from this intervention. 
The proposed in vivo studies will permit the study of effects on insulin action in concert with effects on 
adipose macrophage content and activation and muscle function, as well as circulating inflammatory 
mediators. Such a comprehensive analysis would be necessary to adequately connect the entire 
hypothesis in humans. 
 
EXPERIMENTAL APPROACH AND METHODS: 
STUDY POPULATION AND RECRUITMENT 
Overweight, middle aged and type 2 diabetes subjects will be recruited by a variety of approaches, as 
described below. Subjects will range in age from 21 to 65 years, will be overweight (BMI 25-30 
kg/m2) with and without diabetes mellitus. . All subjects with a history of liver disease or liver 
abnormalities, hyperlipidemia, hypertension, heart disease, cerebrovascular disease, seizures, 
bleeding disorders, muscle disease, a known history of ovarian, uterine, breast cancer or smoking will 
be excluded.  The following subjects will also be specifically excluded: mentally disabled persons, 
prisoners, pregnant women, subjects with a history of ethanol or drug or toxin exposure which could 
be associated with neuropathy, any subject deemed incapable of giving voluntary informed consent, 
and subjects with a history of a bleeding disorder or with a prolonged PT or PTT. Furthermore, 
subjects allergic to Novocain, Benzocaine and Lidocaine will also be excluded. Given potential 
concerns (based on in vitro observations) that resveratrol might inhibit various cytochrome P450 
enzymes involved in drug metabolism, use of the following drugs will be exclusionary for this study: 
anti-coagulant and anti-platelet drugs, anti-epileptics, mexilitene, quinidine, cyclorpsorine, tacrolimus, 
and HIV protease inhibitors (see below under Protection of Human Subjects).  NSAIDS should be 
avoided as far as possible during the study period. Additionally, given confounding effects of extreme 
exercise on muscle triglyceride content, highly trained or professional athletes will be excluded from 
the studies. 
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7 
Women in the child-bearing age-group will be allowed to participate in the studies provided that they 
have negative pregnancy tests within a week of the studies and that they are not breast-feeding. 
Children below the age of 18 will be excluded from the current protocol, given the use of labeled 
isotopes. Prior to their enrollment in the study the purpose, nature, risks and benefits of the study will 
be explained to all subjects and their voluntary, informed, written consent will be obtained. All 
subjects will have a screening visit to allow for clinical evaluation, including history, physical 
examination and consent procedures. 
Recruitment and Screening Strategies for Clamp Studies 
Recruitment of middle aged subjects will be predominantly by local advertising. This will include 
posting flyers within Einstein and around the neighborhood and posting advertisements in area 
newspapers. Dr. Kishores‟ research group has had considerable success with these approaches in 
the recruitment of many middle aged, nondiabetic subjects to date.  
Screening: This visit will include a full history and physical examination, an oral glucose tolerance 
test and additional laboratory tests to determine eligibility, including serum electrolytes, BUN and 
creatinine, PT/PTT, liver function tests, CPK, and lipid profile, as well as a screening urinalysis, urine 
toxicology screening, and blood pressure measurements.  Safety laboratories will be repeated during 
the first and second pancreatic clamp studies and include: CBC, chemistry, liver function tests, CPK, 
and urinalysis. 
All subjects will present to the CRC fasting; consequently, visits will be scheduled in the morning. The 
oral glucose tolerance tests (only non-diabetic subjects) will consist of a 75g oral glucose challenge in 
the form of a beverage, with fasting (immediately prior to drinking beverage) and 2 hour venous blood 
samples for plasma glucose and insulin measurements. Physical examination will include a careful 
assessment of upper extremity veins, as well as measurement of height and waist circumference and 
percent body fat (by body impedence absorptiometry). Since public transportation in our local area is 
limited and many area residents do not drive, we will make arrangements to provide transportation to 
and from clinic visits, including screening visits, for all participants who require it. 
 
General Experimental Conditions:  
 
On the day of the first pancreatic clamp study the patients will receive a 28 days‟ supply of resveratrol 
capsules. The pilot study will be an open-labeled study. The study design requires that all 55 subjects 
participate in all of the proposed studies, as outlined below. Consequently, additional subjects will be 
recruited to compensate for those subjects who cannot complete all of the required studies. 
 
Prior to each study, subjects will meet with study personnel to discuss dietary recommendations. 
Each subject will be instructed to follow a standardized diet plan for three days prior to each of the 
clamp studies (40% fat: 20% saturated, 10% polyunsaturated, 10% monounsaturated; 35% 
carbohydrate; 25% protein). Subjects will be asked to refrain from vigorous exercise for three days 
prior to each clamp study. 
 
Aim 1: To study the effects of resveratrol on age-related insulin resistance 
 
We hypothesize that treatment of overweight, middle aged or Type 2 diabetes subjects with 
resveratrol will result in improved insulin sensitivity, as demonstrated by increased insulin-stimulated 
glucose uptake and enhanced insulin-mediated suppression of glucose production. 
We will study the following subject group: 
 
Overweight and either young or middle aged (BMI 25-30 kg/m2, age 21-65) nondiabetic 
subjects, n=40 diabetic subjects (BMI=25-35)  n=15 
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IND 103,034 
 
8 
Each subject will receive a 28 day course of resveratrol (n=55). RevGenetics will supply the 
resveratrol (500 mg capsules; 2 capsules bid) for our proposed studies. A certificate of analysis has 
been provided by the manufacturer.  
A comprehensive study of whole-body insulin action will be undertaken with specially designed 
„stepped‟ euglycemic (~90 mg/dl) -hyperinsulinemic clamp studies, both before and after 
administration of resveratrol.  
Thus, we will compare the following parameters under paired conditions in each subject, on day 0 
and day 28 of resveratrol treatment: 
a. hepatic insulin action: this will be determined as the ability of low infusion rates of insulin (20 
mU/m2.min above basal requirements) to suppress glucose production during the “low insulin 
phase” of the clamp studies. 
b. peripheral insulin action: this will be determined as the ability of high infusion rates of insulin 
(80 mU/m2.min above basal requirements) to stimulate glucose uptake during the “high insulin 
phase” of the clamp studies. 
 
In vivo clamp studies – General Conditions 
Subjects will be admitted on the day of the study to the General Clinical Research Center (GCRC) 
study room after an overnight fast. Subjects with type 2 diabetes will be admitted to the Jack Weiler 
Hospital of the Albert Einstein College of Medicine the night before the study. All anti-diabetic 
medications, including insulin, will be held from one day to one week prior to the clamp study date 
depending on the drug they are taking. They will not eat after 10 PM the night before the study (but 
can drink water). An intravenous catheter will be inserted into their left arm and insulin will be 
administered overnight to maintain their glucose at normal levels. Blood samples will be collected 
every hour for glucose values. At 8:00 am 2 intravenous cannulae will be established, one for 
infusions and a second one to be inserted in a retrograde fashion in a dorsal vein of the opposite arm 
for blood sampling. To obtain arterialized venous blood samples, this hand will be maintained at 65°C 
in a thermoregulated plexiglass box. 
A primed continuous infusion of D2G-glucose will be initiated at t=-120 min (2 hours prior to the onset 
of the „clamp‟ study). A bolus of 200 mg/m2 D2G for the duration of 3 min will be followed by a 
continuous infusion of 2 mg/min/m2 D2G for the entire study in all subjects (for total duration of 6 
hours) to quantify glucose turnover, specifically rates of peripheral glucose disposal and hepatic 
glucose production.  
These „pancreatic clamp‟ experiments will consist of 360 min insulin/somatostatin (250 µg/hr) or 
insulin/octreotide (30 mg/kg/min) infusions with replacement of glucoregulatory hormones (glucagon 1 
ng/kgmin; growth hormone 3 ng/kgmin). Throughout the entire 360 min the plasma glucose 
concentration will be maintained at basal levels (~90 mg/dl). 
Basal phase: From t=0 min to t=120, optimal insulin infusion rates will be selected in each individual 
by making frequent (~ every 25 mins) adjustments to the insulin infusion rates in order to establish 
those insulin infusion rates required to maintain euglycemia (90 mg/dl) without the requirement for 
exogenous glucose infusion. 
Low phase: Following establishment of basal insulin requirements during the basal phase, at t=120 
the insulin infusion rate will be increased by a fixed increment of 20 mU/m2/min, and will be 
maintained at this rate for 2 hours (T=120-240). Plasma glucose will be maintained at euglycemic 
concentrations (~90mg/dl) by a variable infusion of 20% dextrose for the entire study (78).  
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IND 103,034 
 
9 
High phase: At t=240 min, insulin infusion rate will be increased to 80 mU/m2/min above basal 
requirements, and will be maintained at that 
rate for the final 2 hours of the study (t=240-
360). 
Plasma measurements: From t=0 to t=360 
min., blood samples will be obtained at hourly 
intervals for determinations of plasma glucose, 
insulin, glucagon, C-peptide, growth hormone, 
free fatty acids (FFA), glycerol, and lactate. 
Additional samples for D2G determinations will 
also be obtained every 15 min. Plasma 
glucose will be measured every 5-10 min in 
duplicate by a Beckman glucose analyzer. 
Plasma 
lipids 
(LDL, 
HDL, 
VLDL, 
total 
Cholesterol and Triglyerides) will be measured 
at t=0. 
Biopsies: From t=225-240 min, fat and muscle biopsies will be obtained. Participants will have fat 
biopsies performed in the periumbilical region. A small 0.5 cm cutaneous incision will be performed 
under local anaesthesia (Lidocaine 1% with Epinephrine) and 1-2 g of adipose tissue will be obtained 
by aspiration technique (79). A skelatl muscle biopsy will be obtained from vastus lateralis muscle 
using a cadence muscle biopsy needle following local anaesthesia extending into the muscle area. 
The total amount of muscle obtained will be ~300 mg. The biopsy specimens will be immediately 
homogenized in Trizol at the bedside, to inhibit any RNAase activity, and will subsequently be stored 
at –80 ˚C.  Pressure will be applied at the biopsy site for ~10 min following the biopsy. A pressure 
bandage will be applied and kept on 5-6 hours after the biopsy. Since the biopsies are performed in 4 
hours into the clamp and the subject will under direct observation for an additional 3-4 hours. The 
biopsy site will be examined prior to discharge and a pressure bandage will be reapplied for an 
additional two hours. Subjects will be able to remove the bandage once they get home. 
All infusions will be stopped at t=360 min. The patient will be given a standard meal and plasma 
glucose levels will be monitored at 15-30 min intervals for the next one hour. Dextrose infusion will be 
continued for approximately 45 minutes after the study in order to avoid hypoglycemia. The patient 
will be discharged after about one hour, provided that his or her condition remains stable.  
Indirect Calorimetry.  A PARVO MEDICS, (TrueOne® 2400 Metabolic Measurements 
Systems). will be used to estimate rates of carbohydrate and lipid oxidation from T=260-280 in all 
studies. Twenty minute averaged values for VO2 and VCO2 will be corrected for nitrogen excretion 
as urinary urea nitrogen measured enzymatically by the method of Kjeldahl (80). Net glucose and 
lipid oxidation rates will be calculated from indirect calorimetry. The equations used to calculate 
glucose and lipid oxidation from gas exchange and urinary urea nitrogen excretion have been 
published (81). Under conditions in which RQ> 1.0, the overestimation of glucose oxidation will be 
corrected as described (81). 
Analytical procedures – Plasma glucose will be measured with a Beckman glucose analyzer 
(Fullerton, CA) by use of the glucose oxidase method. Measurements of plasma insulin, C-peptide, 
glucagon, and growth hormone are undertaken throughout the studies in order to evaluate the 
adequacy of the “pancreatic clamp” technique, ie. inhibitory effects of somatostatin on hormone 
secretion, and uniformity of hormone replacement. All samples will be kept on ice until centrifuged 
(within 30 min). Plasma samples will then be sorted and handled by the Special Assay Core, and will 
be stored frozen at -20°C for later assays. Plasma hormone determinations will be performed by 
radioimmunoassay as previously reported (82), and these determinations will be provided by the 
Radioimmunoassay Core of the Diabetes Research and Training Center. 
Plasma insulin(µU/mL)
Plasma glucose (mg/dl)
0
60
120
180
240
300
360
Time (min)
Variable Glucose Infusion
50
100
150
200
Basal Insulin
Glucose Tracer
Somatostatin, GH, Glucagon
100
200
300
400
0
Low Insulin
High Insulin
Plasma insulin(µU/mL)
Plasma glucose (mg/dl)
0
60
120
180
240
300
360
Time (min)
Variable Glucose Infusion
50
100
150
200
Basal Insulin
Glucose Tracer
Somatostatin, GH, Glucagon
100
200
300
400
0
Low Insulin
High Insulin
Plasma glucose (mg/dl)
0
60
120
180
240
300
360
Time (min)
0
60
120
180
240
300
360
Time (min)
Variable Glucose Infusion
50
100
150
200
50
100
150
200
Basal Insulin
Glucose Tracer
Somatostatin, GH, Glucagon
100
200
300
400
0
100
200
300
400
0
Low Insulin
High Insulin
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Substrate determination- Plasma FFA levels will be determined by using an acyl-CoA oxidase 
based colorimetric kit (Wako, Osaka, Japan) and glycerol will be measured using colorimetric 
enzymatic methods (83,84). Plasma lactate will be measured by fluorometric enzyme techniques 
(85). 
Glucose Kinetics- Plasma for labeled glucose radioactivity will be treated as previously described 
(86). Rates of glucose appearance (Ra) and disappearance (Rd) and other indices of glucose 
turnover will be estimated by using the steady-state equation of Steele (87). Endogenous glucose 
production (EGP) will be determined by subtracting the rates of glucose infusion from the tracer-
derived rate of appearance of glucose. Data for glucose turnover represent the mean values during 
the final 60 min of the low insulin period (t=60-120 min), and during the final 60 min of the 
hyperglycemic period (t=180-240 min). 
These techniques are approved by the CCI under Protocols 2000-200 and 2006-414. 
 
Aim 2: To examine the effects of resveratrol on systemic inflammation and/or on adipose 
tissue macrophages. 
This aim will make use of the adipose biopsy specimens from Aim 1. We will therefore examine 
the effects of resveratrol on macrophage content and activation in adipose tissue, and on systemic 
markers of inflammation, in the same n=55 overweight, middle aged (age 45-65) nondiabetic and 
diabetic subjects. We will be utilizing the younger 21-44 year old population to assess whether 
resveratrol has the same beneficial effects in younger subjects.  
We hypothesize that treatment of insulin resistant subjects with resveratrol will lead to reductions in 
systemic inflammatory markers and reduced content and/or activation of adipose tissue 
macrophages. Systemic inflammatory markers to be measured will include TNF-a, IL-6, IL-1b, MCP-
1. Adipose tissue macrophages will be isolated from subcutaneous abdominal adipose tissue, and will 
be quantified by fluorescence activated cell sorting (FACS) analysis. Gene expression of a variety of 
pro-inflammatory and pro-atherosclerotic factors will be examined by „real-time‟ rt-PCR, to determine 
how resveratrol affects activation of adipose macrophages. 
Following administration of resveratrol (n=55) for 28 days, we will examine: 
1. Adipose macrophage content, by fluorescence activated cell sorting (FACS) analysis. 
These data will additionally be confirmed by immunohistochemistry 
2. Activation of adipose macrophages, as assessed by expression and production of a 
variety of pro-inflammatory and pro-atherosclerotic factors, using „real-time‟ rt-PCR and 
immunohistochemistry 
3. Plasma levels of cytokines and acute phase reactants 
Adipose biopsy specimens- Adipose biopsy specimens will be obtained as described above. 
Suctioned adipose tissue samples will be suspended in PBS and washed with a 40 m cell strainer. 
Approximately 1 gram of tissue will be immediately homogenized in Trizol (Life Technologies, 
Bethesda, MD) with a Tissumizer homogenizer. This technique will ensure optimized yields of mRNA 
by the inhibition of RNAses. The homogenate will be frozen on dry ice, transported to the laboratory 
and stored at –800C. Subsequent mRNA extraction and analysis of gene expression will be 
performed in the Gene Expression Core lab. Remaining fresh adipose biopsy specimens will be 
immediately processed as described below. 
Collagenase digestion of adipose tissue and cell separation: The adipose tissue will be digested 
with collagenase type 1 (Worthington Biochemical, Lakewood, NJ; 0.05gm per 30ml of Hanks 
Balanced Salt solution with 4% BSA) for 30 minutes at 37°C with intermittent shaking. Adipocytes will 
be subsequently separated from stromal cells by centrifugation at 2500 rpm for 10 minutes followed 
by filtration. Macrophages will be separated from stromal cells by CD14+ coated antibody beads 
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(Dynabeads, Dynal Biotech, Lake Success, NY) by the manufacturer‟s method. The separated 
adipocytes and macrophages will be stored in TRIzol.  
Macrophage quantification: It is possible that the 28 day duration of therapy and effects of 
resveratrol on adipocyte chemoattractant factors could also impact numbers of macrophages in fat 
(see below). We will therefore quantify adipose tissue macrophages with immunohistochemistry 
and/or 
FACS 
analysis. 
To 
measure 
macrophage 
content 
in 
adipose 
tissue 
by 
immunohistochemistry, samples will be fixed and embedded in paraffin (21). Sections of tissue will 
be stained for expression of F4/80 (macrophage-specific) with a monoclonal antibody. The total 
number of nuclei and the number of nuclei of F4/80 expressing cells will be counted for each field. We 
have also developed a technique to count macrophages by Fluorescence Activated Cell Sorting 
(FACS) analysis. After digestion with collagenase, the pellet of stromal cells will be treated with red 
blood cell lysing buffer for 5 min followed by incubation with saturating amounts of FITC-labeled 
human CD14+ antibody (BD Pharmingen) in staining buffer (PBS with 1 mg/ml BSA and 12 mM 
NaN3, pH 7.2) on ice for 20 min, washed and analyzed immediately by using a FACScalibur flow 
cytometer (Becton Dickinson, San Jose, CA). 
Quantitative „Real Time‟ Reverse Transcriptase Polymerase Chain Reaction (rt-PCR): From the 
whole fat samples and isolated cells, total RNA will be extracted with TRIzol. cDNA will be made 
using Superscript First Strand Synthesis System for rt-PCR (Invitrogen Technologies). Gene 
expression will be quantified using the protocol for the LightCycler instrument (Roche Diagnostics, 
Indianapolis, IN), using SYBRGreen (Roche Diagnostics, Indianapolis, IN) for fluorescent detection of 
double-stranded DNA. Plasmid standard curves will be generated to calculate mRNA copy numbers. 
Gene product specificity will be confirmed by melting curves and by running the PCR products on 
agarose gels. All reactions will be performed at least three times. The genes of interest will include 
PAI-1, TNF-, TGF-, and interleukins 1- and 6 both in whole fat and in macrophages. Expression of 
these inflammatory factors will thus provide a measure of macrophage activation. In skeletal muscle 
samples we will quantify Socs-1/2/3 gene expression, as a documented target of cytokine signaling 
(44). We will also perform immunohistochemistry and Western blotting to measure protein levels of 
these factors.  
Measurement of circulating cytokines and acute phase reactants: Plasma samples will be 
aliquoted into Eppendorff tubes and stored at –200C. Subsequent measurements of plasma leptin, 
TNF-a, PAI-1, angiotensinogen, IL-1-b, IL-6, resistin, Acrp30 and SAA will be performed by the 
Special Assays Core. 
Aim 3: To explore the effects of resveratrol on skeletal muscle metabolism and function.  
 
We hypothesize that resveratrol treatment will enhance mitochondrial metabolism and improve 
strength and contractility. We will examine the effects of resveratrol on mitochondrial content, 
signaling molecules involved in mitochondrial biogenesis, DNA and protein oxidative damage, and 
mitochondrial respiration in human skeletal muscle. It is anticipated that mitochondrial ATP production 
and aerobic muscular function will improve in concert with enhanced insulin sensitivity. 
This aim will make use of the muscle biopsy specimens from Aim 1. We will therefore examine the 
effects of resveratrol on muscle mitochondrial function and aerobic muscular function in the same 
n=25 overweight, middle aged (age 45-65) nondiabetic and diabetic subjects. Again,We will be 
utilizing the younger 21-44 year old population to assess whether resveratrol has the same beneficial 
effects in younger subjects. 
Following administration of resveratrol (n=55) for 28 days, we will examine: 
1. Muscle mitochondrial content  
2. Muscle mitochondrial function 
3. Muscle strength and mobility 
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Muscle biopsies: human skeletal muscle biopsies will be obtained as described above, and stored at 
–80 °C for analysis of the following: 
 
mitochondrial content  
 
signaling molecules involved in mitochondrial biogenesis 
 
DNA and protein oxidative damage, and  
 
mitochondrial respiration  
 
skeletal muscle mitochondrial function (citrate synthase and succinate dehydrogenase 
activity), content, and morphology by electron microscopy. 
Muscle strength and mobility tests: will be conducted by our collaborators from the Department of 
Neurology and assess the following: 
 
Mobility measures: Quantitative gait studies will be conducted using a 28-foot (20 feet 
recording surface) computerized walkway with embedded pressure sensors (GAITRite, CIR 
systems).  Subjects will walk on the mat at their „normal pace‟ for two trials in a quiet well-lit 
room wearing comfortable footwear and without any attached monitoring devices. The 
walkway includes four unmarked feet at either end to account for initial acceleration and 
terminal deceleration. Based on footfalls recorded on the walkway, the software automatically 
computes various gait parameters as the mean of two trials.  The GAITRite system is widely 
used in clinical and research settings, with excellent inter-rater and test-retest reliability (kappa 
>0.9) (88,89). Our collaborators in the Department of Neurology have experience using this 
system for the past 8 years in community based studies. 
 
 
 
Walking while talking test (WWT): Subjects are asked to walk the course for two trials while 
reciting alternate letters of the alphabet using protocols developed and validated in our 
subjects. Number of errors while reciting letters will also be recorded. Subjects are given 
practice trials as required. The order of the initial letter on WWT is also randomly varied 
between „A‟ and „B‟ to minimize practice effects. The WWT performance has been related to 
increased attention demands (90) and falls (91). 
 
Chair rise: Time taken to get up from a chair five times unassisted and without pausing is 
recorded (seconds). This test assesses lower extremity strength and balance, and is a 
component of the widely used Short Physical Performance Battery (92). 
 
Unipedal Stance. The unipedal stance measures ability to stand on one foot (maximum 30 
sec), and is considered a sensitive measure of balance (93).  
 
Stair climbing: Difficulty with climbing up or down stairs is associated with functional 
limitations. 
 
Muscle strength: Tests of grip and leg strength is commonly used to assess power, and 
define frailty. 
 
Aim 4: To assess the effects of resveratrol on cognition.  
We hypothesize that resveratrol will enhance cognitive function in middle-aged individuals. In 
collaboration with the Department of Neurology we will therefore examine the effects of resveratrol on 
cognition in the same n=25 overweight, middle aged (age 45-65) nondiabetic and diabetic subjects. 
We will be utilizing the younger 21-44 year old population to assess whether resveratrol has the same 
beneficial effects in younger subjects. 
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Following administration of resveratrol (n=25) for 28 days, we will examine: 
 
Executive control: The Attention Network Test is a combination of the cued reaction time 
(RT) and the flanker task (94,95). The ANT provides estimates of three separate attention 
networks of alerting, orienting, and executive attention (95,96). Efficiency of the three 
attentional networks is assessed by measuring how response times are influenced by alerting 
cues, spatial cues, and flankers (i.e., congruent or incongruent conditions). The task is 
relatively short and simple so that reliable estimates of the three attention networks can be 
obtained within 35 minutes for various healthy and pathological populations.  
 
Clinical neuropsychology battery: provides comprehensive assessment of cognitive 
function (97,98,99).  This battery consists of traditional, standardized measures of: a. 
intelligence, b. memory, c. language, d. executive function, e. visuospatial function and praxis, 
f. processing speed, and g. mental status. These tests were used in our previous research to 
demonstrate informative associations between cognitive and motor function (100,101,102).  
 
Schedule of proposed clinical studies: 
Day -1: Subjects visit the Ferkauf School of Psychology at 8 am for motor and cognitive function 
tests. Subject with type 2 diabetes will present at 2 pm for cognitive and motor testing then be 
admitted to Weiler Hospital at 5 pm the night before the clamp study.  
Day 0: Subjects admitted to GCRC at 8 am following a 10 hour fast,  
 
first stepped insulin clamp study  
 
fat and muscle biopsies 
Days 1-28: Subjects receive oral resveratrol (last dose on day of final clamp study) 
Day 27: Subjects visit the Ferkauf School of Psychology at 8 am for motor and cognitive function 
tests. Subject with type 2 diabetes will present at 2 pm for cognitive and motor testing then be 
admitted to Weiler Hospital at 5 pm the night beofer the study.  
Day 28: Subjects admitted to GCRC at 8 am following a 10 hour fast 
 
second stepped insulin clamp study  
 
fat and muscle biopsies 
STATISTICAL ANALYSIS 
Data collected from each subject will be edited, entered, verified and entered into a FoxPro data base 
system in the GCRC. Data quality checking will be performed by computer programs designed to 
detect errors using accepted ranges, logic checks, and missing values. Outliers will be checked 
against the original record and correct values will be entered into a final data file. Data from FoxPro 
files will be transferred into SAS System Version 6.12 (SAS Institute, Cary, NC) for analysis. We shall 
utilize SAS for all data management and statistical analysis. Descriptive statistics of all the variables 
at different time points will be derived and reported. 
For the specific aims of this study, repeated measurements of several parameters including glucose 
metabolism and gene expression will be measured in each subject, with the principal endpoint being 
the rate of whole-body glucose uptake during the final hour of the high insulin clamp. The null 
hypothesis is that glucose uptake will be equal in each pair of experiments (ie. prior to and following 
45 days‟ treatment with resveratrol or placebo). To determine the changes in glucose uptake between 
the paired experimental protocols for each subject group, paired Student‟s t-tests will be performed. 
For between-group comparisons (ie. between the two subject groups randomized to resveratrol or 
placebo), the principal endpoint will be the difference in whole-body glucose uptake (GU) between 
the 2 experimental conditions (eg. Resveratrol vs. placebo), with null hypothesis that the response to 
the experimental intervention will be the same in all subject groups. To determine the changes in 
glucose uptake between the paired experimental protocols for each subject group, analysis of 
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variance (ANOVA) will be performed by using PROC GLM in the SAS statistical package. Within 
group effects will be derived from the analysis.  
All values will be presented as the mean +/- SE. 
 
HUMAN SUBJECTS RESEARCH SECTION 
 
Protection of Human Subjects 
 
1. 
RISKS TO THE SUBJECTS 
 
 
 
Human Subjects Involvement  
Subject involvement will include a preliminary screening visit for history, physical exam and lab 
testing to determine eligibility. Subsequently, each subject will participate in two infusion studies with 
frequent blood glucose monitoring.  
Human Subject Characteristics 
The study population will consist of at least 40 nondiabetic subjects and 15 diabetic subjects with the 
following characteristics: ages 21-65 years, with BMI between 25 and 30 kg/m2. The following 
subjects will be specifically excluded: children, mentally disabled persons, prisoners, pregnant 
women, fetuses, subjects with a history of ethanol or drug or toxin exposure which could be 
associated with neuropathy, any subject deemed incapable of giving voluntary informed consent, and 
subjects with a history of a bleeding disorder or with a prolonged PT or PTT; a history of uterine, 
breast, or ovarian cancer, renal disease, and liver disease or impairment. Women in the child-bearing 
age-group will be allowed to participate, provided they have negative pregnancy tests. The research 
topic to be studied is not relevant to the involvement of special classes or groups of subjects and 
therefore no criteria for their involvement are specified.  
 
 
Table I  DEMOGRAPHY OF RECRUITMENT AREA 
*Data from United States Census 2000  (http://www.census.gov/) 
 
 
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Bronx 
 
Manhattan 
 
Westchester County 
(Borders the Bronx) 
Population 
1, 332,650 
1,537,195 
923,459 
Racial Distribution 
 
 
 
  White (Non-Hispanic) 
15% 
50% 
65% 
  Hispanic 
48% 
24% 
16% 
  African American 
36% 
14% 
14% 
  Asian 
3% 
10% 
5% 
Age Distribution 
 
 
 
  >25 years 
40% 
27% 
32% 
  25-44 years 
31% 
38% 
31% 
  45-64 years 
19% 
23% 
23% 
  65+ years 
10% 
12% 
14% 
Table II  TARGETED/PLANNED ENROLLMENT:  Number of Subjects 
 
ETHNIC CATEGORIES 
Sex/Gender 
Females 
Males 
Total 
Hispanic or Latino 
8 
8 
16 
Not Hispanic or Latino 
12 
12 
24 
Ethnic Category: Total of All Subjects 
20 
20 
40 
 
RACIAL CATEGORIES 
American Indian/Alaska Native 
0 
0 
0 
Asian 
0 
0 
0 
Native Hawaiian or Other Pacific Islander 
0 
0 
0 
Black or African American 
8 
8 
16 
White 
12 
12 
24 
Racial Categories: Total of All Subjects 
20 
20 
40 
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Sources of Materials/Use of Data: 
All data and records obtained in this study will be used for research purposes only. The protocol will 
be submitted for review and approval by the Institutional Review Board known as the Committee on 
Clinical Investigations of the Albert Einstein College of Medicine of Yeshiva University (CCI). The CCI 
is responsible to ensure human subject protections in compliance with institutional policies and 
federal regulations including the HIPAA privacy law. 
Potential Risks:  
Potential risks to the subjects include the following: 
 
1. Blood withdrawal: The total amount of blood sampled will not exceed 190 ml per study, less than 
half of that donated by a blood donor.  
2. 20% dextrose: The infusion of 20% dextrose may be associated with local venous irritation. This 
will be minimized by use of a large-bore antecubital vein in which the glucose will be infused.  
3. D2G: is a non-radioactive glucose tracer and is not associated with any known side effects at the 
doses used.  
4. Glucagon infusion in high doses can cause gastrointestinal symptoms such as cramps and 
nausea, and produce hyperglycemia in persons with glucose intolerance. The doses to be employed 
here are intended to maintain plasma glucagon at basal levels.  
5. Growth Hormone: At higher doses and for prolonged periods of exposure, growth hormone (rhGH) 
has either insulin-like or insulin-antagonistic effects on carbohydrate and lipid metabolism, however 
the proposed infusion is for physiological replacement during SRIF infusion and is unlikely to have 
adverse effects.  
6. Hypoglycemia: is unlikely to develop in these subjects because of the desired plasma glucose 
targets (90 mg/dl during the normoglycemic studies), the low rates of insulin infusion, and the 
constant blood glucose monitoring.  
7. Resveratrol: Resveratrol is present in significant amounts in a variety of foods, including grapes, 
blueberries, peanuts and red wine and over-the-counter resveratrol supplements are widely used.  
Formal safety testing is not required for marketing of nutritional supplements, but to date, there have 
been no published reports of side effects at the commonly used doses (up to 1000 mg /day).  A 
number of in vitro studies have demonstrated that resveratrol can be an inhibitor of various 
cytochrome P450 enzymes that are involved in drug metabolism. The relevance of these in vitro 
observations to in vivo use is unclear primarily because oral resveratrol is rapidly metabolized by the 
liver, free resveratrol levels are quite low and resveratrol metabolites have not been shown to be 
inducers or inhibitors of CYP enzyme activity.  Nonetheless, caution is warranted, especially for drugs 
with a narrow therapeutic window (anti-epileptics, mexilitene, quinidine, cyclorpsorine, tacrolimus, HIV 
protease inhibitors) and use of these drugs will be exclusionary for this study.  Furthermore, due to 
the potential estrogenic effects of resveratrol, a medical history of ovarian, uterine, and breast cancer 
will be exclusionary for the study. 
8. Somatostatin: At the proposed physiological replacement doses has no known side effects.  
9. Ocreotide: At the proposed physiological replacement doses has no known side effects. 
 
2.  ADEQUACY OF PROTECTION AGAINST RISKS 
Recruitment and Informed Consent Procedures: 
We will recruit at least 40 overweight, middle aged, nondiabetic subjects and 45 subjects with Type 2 
diabetes by local advertising. Consent will be initially obtained during telephone screening performed 
by a research fellow or the study coordinator.  Formal consent procedures which adhere to the 
Committee on Clinical Investigations of the Albert Einstein College of Medicine will be followed. 
Specifically, each subject will be verbally informed in layman's language of the purpose, benefits and 
possible risks of the studies.  They will then read the written consent form in the presence of a 
member of the research team and a physician who will answer any further questions. The subject and 
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witness shall be asked to sign the consent form which will be kept in the patient‟s chart. Each 
subject's potential participation in related experiments (for example, when each subject is asked to 
perform repeat studies) will be explicitly stated.  
All clinical data on the subjects will be confidential. Data generated in these studies will be considered 
only relevant for research purposes and will not be included in any clinical databases or the patient‟s 
chart. Since the GCRC databases are coded, the study data will not be available on any clinical 
database system. 
Protection Against Risk: 
Procedures employed to protect subjects from undue risks include the following:  
 
1) 
Blood withdrawal: Blood withdrawal during any single study will be limited to less than 190cc 
and subsequent studies will be separated by at least, a four week interval.  The infusion and 
withdrawal catheters may produce infection or local hematoma, but strict aseptic technique will be 
observed by the experienced physician performing the procedure.  
2) 
20% glucose: Vein irritation with 20% glucose infusion will be prevented by use of a large-bore 
antecubital vein. 
3) 
Glucagon: Symptoms of mild nausea will be treated with antiemetic medications, if needed. 
4) 
Hypoglycemia: All of these studies will be performed by a physician, hypoglycemia will be 
prevented by blood glucose sampling every 5-10 minutes and if inadvertent hypoglycemia develops 
can be rapidly treated by glucose infusion.  
5) 
Resveratrol: Given data from a number of in vitro studies demonstrating that resveratrol can 
be an inhibitor of various cytochrome P450 enzymes involved in drug metabolism, use of the 
following drugs will be exclusionary for this study: anti-epileptics, mexilitene, quinidine, cyclorpsorine, 
tacrolimus, HIV protease inhibitors. Dosages of up to 1000 mg/day have been widely used without 
reports of adverse effects.  
 
3. POTENTIAL BENEFITS OF THE PROPOSED RESEARCH TO THE SUBJECTS AND OTHERS 
The study will be of no direct benefit to the subject, although subjects may gain a better 
understanding of their own metabolic processes. The results of the study will contribute to 
generalizable knowledge about the effects of resveratrol on insulin action, mitochondrial function, 
muscle strength and neurocognitive function. 
4.  IMPORTANCE OF THE KNOWLEDGE TO BE GAINED (Risk: Benefit Ratio): 
The risks involved are minimal as all procedures are well established. The study may result in a better 
understanding of the role of resveratrol in determining insulin action in humans. As described above, 
a number of mechanisms exist to ensure safety of subjects. All studies will be performed by a 
physician and nurse in attendance in the CRC procedure unit with emergency equipment readily 
accessible. The PI, coinvestigators, and research team have a combined experience of over 10 years 
in human investigation, and sufficient care in choice of subjects should minimize the risks. The 
scientific value of these studies is to develop an understanding of the mechanism of an important 
clinical and potentially-exciting device for the treatment of diabetes. 
 
 
INCLUSION OF WOMEN  
As mentioned in the human subject characteristics section above, the study will exclude pregnant 
women because of the use of radioactive tracer. Otherwise, an anticipated 50% of subjects will be 
female based on preliminary data and the population distribution in the Bronx.  
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INCLUSION OF MINORITIES 
We anticipate that 50% of our study subjects will be from minority communities, based on our 
preliminary data and the population distribution in the Bronx. Recruitment strategies to encourage 
minority enrollment include advertisements in ethnic newspapers and magazines.  
 
INCLUSION OF CHILDREN 
The purpose of this study is to determine effects of resveratrol on age-related parameters in middle-
aged subjects and in those with Type 2 diabetes and hence no children will be studied. 
 
 
DATA SAFETY AND MONITORING PLAN (DSMP): 
 
All human subject research being conducted at the Einstein GCRC must have a DSMP. The 
guidelines for Data and Safety Monitoring have been established by the GCRC Research 
Subject Advocate (RSA), and approved by the Assistant Dean for Compliance who oversees 
human 
research 
subject 
safety 
at 
the 
Albert 
Einstein 
College 
of 
Medicine. 
http://gcrcweb.aecom.yu.edu/gcrc/dsm.htm 
Adverse Event Monitoring: 
Process: Data and safety monitoring for this study will be performed by the principal investigator on 
an ongoing basis.  All volunteers will be seen by the P.I. or her research staff. Laboratory data will be 
reviewed by the P.I. within 24 hours of receipt. 
Reporting: All adverse events will be compiled, and reported in summary form, on an annual basis to 
the IRB, and at the conclusion of the study.  Unanticipated (non-serious) adverse events will be 
reported to the IRB within 30 days and serious adverse events will be reported to the IRB within 48 
hours by phone, email or fax 
 
Recruitment Monitoring: 
 
Process: The P.I. will assess the recruitment and retention of study subjects on an ongoing basis.  
The recruitment goal for this protocol is 40 subjects to complete all of the proposed studies. 
Reporting: Summary statistics regarding recruitment and retention of study subjects will be reported 
to the IRB on an annual basis, and at the conclusion of the study. 
 
Early Study Termination: 
 
Process: The P.I. will determine if the study is to be terminated prior the scheduled study conclusion.  
Early study termination will be considered in the event of an unanticipated serious adverse event 
determined to be possibly, probably or definitely related to the study. The P.I. may also terminate the 
study if a positive drug screening test at any time during the study is found. Payment will be reduce to 
$50 and the subject will be withdrawn from the study 
Reporting: The P.I. will report the decision to terminate the study to the IRB within 48 hours of this 
determination.  The P.I. will submit a narrative description of the reasons for early termination of the 
study within 10 days. 
 
DATA SHARING 
 
We respect that the rights and privacy of people who participate in NIH-sponsored research must be 
protected at all times.  Thus, data intended for broader use will be free of identifiers that would permit 
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linkages to individual research participants. We and our collaborators will make the data and associated 
documentation available to users only if they provide: (1) a commitment to using the data only for research 
purposes and not to identify any individual participant; (2) a commitment to securing the data using 
appropriate computer technology; and (3) a commitment to destroying or returning the data after analyses 
are completed. We expect that the results of this study will be presented at national scientific meetings and 
published in peer reviewed journals.  
 
 
 
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20 
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