Resveratrol 4 health, life

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Here's the paper from the abstract in message 24371, now available as a pdf.

Resveratrol improves health and survival of mice on a

high-calorie diet

Nature 444, 337-342 (16 November 2006)

ph A. Baur, J. Pearson, L. Price,

Hamish A. son, Carles Lerin, Avash Kalra,

Vinayakumar V. Prabhu, Joanne S. Allard, Guillermo

-Lluch, Kaitlyn , J. Pistell, Suresh

Poosala, G. Becker, Olivier Boss, Dana Gwinn,

Mingyi Wang, Sharan Ramaswamy, W. Fishbein,

G. Spencer, G. Lakatta, Le

Couteur, Reuben J. Shaw, Placido Navas, Pere

Puigserver, K. Ingram, de Cabo and

A. Sinclair

Resveratrol (3,5,4'-trihydroxystilbene) extends the

lifespan of diverse species including Saccharomyces

cerevisiae, Caenorhabditis elegans and Drosophila

melanogaster. In these organisms, lifespan extension

is dependent on Sir2, a conserved deacetylase proposed

to underlie the beneficial effects of caloric

restriction. Here we show that resveratrol shifts the

physiology of middle-aged mice on a high-calorie diet

towards that of mice on a standard diet and

significantly increases their survival. Resveratrol

produces changes associated with longer lifespan,

including increased insulin sensitivity, reduced

insulin-like growth factor-1 (IGF-I) levels, increased

AMP-activated protein kinase (AMPK) and peroxisome

proliferator-activated receptor- coactivator 1 (PGC-1)

activity, increased mitochondrial number, and improved

motor function. Parametric analysis of gene set

enrichment revealed that resveratrol opposed the

effects of the high-calorie diet in 144 out of 153

significantly altered pathways. These data show that

improving general health in mammals using small

molecules is an attainable goal, and point to new

approaches for treating obesity-related disorders and

diseases of ageing.

The number of overweight individuals worldwide has

reached 2.1 billion, leading to an explosion of

obesity-related health problems associated with

increased morbidity and mortality1, 2. Although the

association of obesity with increased risk of

cardiovascular disease and diabetes is well known, it

is often under-appreciated that the risks of other

age-related diseases, such as cancer and inflammatory

disorders, are also increased. At the other end of the

spectrum, reducing caloric intake by 40% below that of

ad libitum-fed animals (caloric restriction) is the

most robust and reproducible way to delay age-related

diseases and extend lifespan in mammals3, 4.

Experiments with Saccharomyces cerevisiae and

Drosophila melanogaster have implicated the

sirtuin/Sir2 family of NAD+-dependent deacetylases and

mono-ADP-ribosyltransferases as mediators of the

physiological effects of caloric restriction5. In

mammals, seven sirtuin genes have been identified

(SIRT1–7). SIRT1 regulates such processes as glucose

and insulin production, fat metabolism, and cell

survival, leading to speculation that sirtuins might

mediate effects of caloric restriction in mammals5. We

previously screened over 20,000 molecules to identify

25 that enhance SIRT1 activity in vitro6. Resveratrol,

a molecule produced by a variety of plants in response

to stress, emerged as the most potent.

Resveratrol has since been shown to extend the

lifespan of evolutionarily distant species including

S. cerevisiae, C. elegans and D. melanogaster in a

Sir2-dependent manner6, 7, 8, 9. A recent study found

that resveratrol improves health and extends maximum

lifespan by 59% in a vertebrate fish10. In mammalian

cells, resveratrol produces SIRT1-dependent effects

that are consistent with improved cellular function

and organismal health11, 12, 13, 14, 15. Whether

resveratrol acts directly or indirectly through Sir2

in vivo is currently a subject of debate16.

On the basis of the unprecedented ability of

resveratrol to improve health and extend lifespan in

simple organisms, we have asked whether it has similar

effects in mice. We hypothesized that resveratrol

might shift the physiology of mice on a high-calorie

diet towards that of mice on a standard diet and

provide the associated health benefits without the

mice having to reduce calorie intake. Cohorts of

middle-aged (one-year-old) male C57BL/6NIA mice were

provided with either a standard diet (SD) or an

otherwise equivalent high-calorie diet (60% of

calories from fat, HC) for the remainder of their

lives. To each of the diets, we added resveratrol at

two concentrations that provided an average of 5.2

0.1 and 22.4 0.4 mg kg-1 day-1, which are feasible

daily doses for humans. After 6 months of treatment,

there was a clear trend towards increased survival and

insulin sensitivity. Because the effects were more

prominent in the higher dose (22.4 0.4 mg kg-1 day-1,

HCR), we initially focused our resources on this group

and present the results of those analyses herein.

Analyses of the other groups will be presented at a

later date.

Increased survival

Mice on the HC diet steadily gained weight until 75

weeks of age, after which average weight slowly

declined (Fig. 1a). Although mice on the HCR diet were

slightly lighter than the HC mice during the initial

months, there was no significant weight difference

between the groups from 18–24 months, when most of our

analyses were performed. There was also no difference

in body temperature (Table 1), food consumption

(Supplementary Fig. 1a, , total faecal output or

lipid content (Supplementary Fig. 1c, d), or

post-mortem body fat distribution (Supplementary Fig.

2).

Table 1. Effects of a high-fat diet and resveratrol on

various biomarkers in plasma.

============================================================

Parameter Standard diet High calorie High calorie +

resveratrol Fed or Fasted

============================================================

Free fatty acids (mequiv.)

0.27 (0.04) 0.59 (0.06)^# 0.53 (0.03)^# Fed

0.83 (0.10) 0.45 (0.20) 0.54 (0.05) Fasted

Triglycerides (mg/dl) 76.6 (6.8) 81.4 (6.6) 88.2

(10.8) Fasted

Cholesterol (mg/dl) 135 (7) 183 (20)^# 204 (16)^#

Fasted

Insulin (ng/ml)

1.77 (0.64) 9.21 (1.95)^# 2.46 (0.47)^## Fed

0.73 (0.14) 2.70 (0.36)^# 1.06 (0.30)^## Fasted

Glucose (mg/dl)

129.0 (5.4) 118.3 (4.7) 114.8 (6.3) Fed

94.5 (3.3) 125.3 (11.6)^# 85.6 (10.3)^## Fasted

IGF-I (ng/ml)

346 (40) 534 (12)^# 482 (21)^#^### Fed

625 (33) 999 (102)^# 929 (81)^# Fasted

IGFBP-1 (AU)

1.0 (0.3) 1.7 (0.3) 1.7 (1.0) Fed

1.0 (0.2) 0.5 (0.3) 0.3 (0.1)^# Fasted

IGFBP-2 (AU) 1.0 (0.2) 0.7 (0.04) 0.9 (0.1) Fasted

Leptin (ng/ml) 2.0 (1.1) 21.6 (7.2) 11.6 (6.5) Fasted

Adiponectin (microg/ml) 12.1 (1.6) 9.5 (0.5) 9.0 (0.8)

Fed

Amylase (U/l) 2,060 (150) 2,960 (320)^# 2,190 (230)^##

Fasted

Ala aminotransferase (U/l) 347 (119) 390 (61) 446 (88)

Fasted

Asp aminotransferase (U/l) 448 (85) 425 (90) 512 (46)

Fasted

Creatine phosphokinase (U/l) 4,260 (1820) 2,010 (810)

2,520 (680) Fasted

Lactate dehydrogenase (U/l) 1,530 (240) 1,610 (170)

2,020 (180) Fasted

Alkaline phosphatase (U l-1) 43.8 (3.4) 44.6 (6.0)

34.2 (1.4) Fasted

Bilirubin (mg/dl) 0.16 (0.02) 0.10 (0.03) 0.16 (0.02)

Fasted

Albumin (g dl-1) 2.78 (0.16) 2.88 (0.19) 2.66 (0.14)

Fasted

Creatinine (mg/dl) 0.54 (0.02) 0.48 (0.04) 0.46 (0.04)

Fasted

Cyclo-oxygenase (liver, AU/mg) 1.00 (0.14) 0.80 (0.11)

0.83 (0.11) Fed

Citrate synthase (liver, AU/mg) 141 (14) 128 (21) 138

(11) Fed

Body temperature (°C) 34.71 (0.14) 35.52 (0.17)^#

35.57 (0.15)^# Fed

============================================================

Values shown are mean (s.e.m.). AU, arbitrary units; U

l-1, units per litre.

^## P < 0.05 versus high calorie.

^# P < 0.05 versus standard diet.

^### P < 0.05 versus high calorie by one-tailed

Student's t-test.

At 60 weeks of age, the survival curves of the HC and

HCR groups began to diverge and have remained

separated by a 3–4-month interval (Fig. 1b). A similar

effect on survival was observed in a previous study of

one-year-old C57BL/6 mice on caloric restriction,

ultimately resulting in a 20% extension of mean

lifespan17. With the present age of the colony at 114

weeks, 58% of the HC control animals have died (median

lifespan 108 weeks), as compared to 42% of the HCR

group and 42% of the SD controls. Although we cannot

yet confidently predict the ultimate mean lifespan

extension, proportional hazards regression shows

that resveratrol reduced the risk of death from the HC

diet by 31% (hazard ratio = 0.69, P = 0.020), to a

point where it was not significantly different from

the SD group (hazard ratio = 1.03, P = 0.88).

Although resveratrol increased survival, it was

important to ascertain whether quality of life was

maintained. One way to assess this was to measure

balance and motor coordination, which we did by

examining the ability to perform on a rotarod.

Surprisingly, the resveratrol-fed HC mice steadily

improved their motor skills as they aged, to the point

where they were indistinguishable from the SD group

(Fig. 1c). It is possible that the improved rotarod

performance might have been due to minor differences

in body weight but we view this as unlikely because we

found no correlation between body weight and

performance within groups and rotarod performance was

also improved for resveratrol-treated SD mice (R.deC.

and K.P., unpublished data). These data are

reminiscent of the resveratrol-mediated increase in

motor activity in older individuals of the vertebrate

fish species Nothobranchius furzeri10.

Increased insulin sensitivity

In humans, high-calorie diets cause numerous

pathological conditions including increased glucose

and insulin levels leading to diabetes, cardiovascular

disease and non-alcoholic fatty liver disease, a

condition for which there is no effective treatment18.

The HC-fed mice had alterations in plasma levels of

markers that predict the onset of diabetes and a

shorter lifespan, including increased levels of

insulin, glucose and IGF-1 (Table 1). The HCR group

had significantly lower levels of these markers,

paralleling the SD group. An oral glucose tolerance

test indicated that the insulin sensitivity of the

resveratrol-treated mice was considerably higher than

controls (Fig. 2a–d). Homeostatic model assessment,

which is used to quantify insulin resistance, gave

scores of 2.5 for SD, 8.8 for HC and 3.5 for HCR,

confirming improved sensitivity (HCR versus HC, P =

0.01). Although the persistence of high glucose levels

for more than 60 min following an oral dose is unusual

for young mice, it is typical for older animals19.

Compared to the HC controls, the areas under the

curves for both glucose and insulin levels were

significantly decreased in the resveratrol-fed HC

group and were not significantly different from mice

in the SD group (Fig. 2b, d).

Next, we investigated possible mechanisms behind these

metabolic effects. AMPK is a metabolic regulator that

promotes insulin sensitivity and fatty acid oxidation.

Its activity correlates tightly with phosphorylation

at Thr 172 (p-AMPK). Chronic activation of AMPK occurs

on a calorically restriction diet and has been

proposed as a longevity strategy for mammals20.

Consistent with this idea, additional copies of the

AMPK gene are sufficient to extend lifespan in C.

elegans21. Because we and others22 have observed that

resveratrol can activate AMPK in cultured cells

through an indirect mechanism (Fig. 2e; see also

Supplementary 3a–d), we examined whether AMPK

activation occurred in the livers of the

resveratrol-fed group. Resveratrol showed a strong

tendency towards inducing phosphorylation of AMPK

(Fig. 2f), as well as two downstream indicators of

activity, namely phosphorylation of acetyl-coA

carboxylase at Ser 79 and decreased expression of

fatty acid synthase (Supplementary Fig. 3e, f).

Decreased organ pathology

At 18 months of age it was apparent that the

high-calorie diet greatly increased the size and

weight of livers and that resveratrol prevented these

changes (Fig. 3a–c; see also Supplementary Fig. 4a,

without altering plasma lipid levels (Table 1).

Histological examination of liver sections by staining

with haematoxylin and eosin or oil red O revealed a

loss of cellular integrity and the accumulation of

large lipid droplets in the livers of the HC but not

the HCR group. Blinded scoring of the liver sections

for overall pathology on a scale of 0–4 (with 4 being

the most severe) gave mean values of 1.3 for the SD

group, 2.8 for the HC group and 0.8 for the HCR group

(Fig. 3b). Plasma amylase, which can indicate

pancreatic damage, was elevated in the HC group and

was significantly reduced by resveratrol (Table 1).

The reasons for the elevation of plasma amylase levels

in the HC group are unclear given that pancreatic

sections of all animals revealed no damage to the

pancreas or decrease in islet area (data not shown).

Differences in the weights of other organs did not

reach statistical significance.

The ability of resveratrol to improve motor function

and increase insulin sensitivity indicated that its

effects were not confined to the liver. To test

directly whether other organs also benefited, we

examined heart tissue of the SD, HC and HCR mice.

Blinded scoring of overall pathology—taking into

account subtle changes in the abundance of fatty

lesions, cardiac muscle vacuolization, degeneration

and inflammation—on a relative scale of 0–4 (with 4

being the most severe) gave mean values of 1.6 for the

SD group, 3.2 for the HC group and 1.2 for HCR group

(Fig. 3d; see also Supplementary Fig. 4c).

Improvements in the morphology of the aortic elastic

lamina were also apparent (Supplementary Fig. 4d).

Increased mitochondria

Exercise and reduced caloric intake increase hepatic

mitochondrial number23, 24 and we wondered whether

resveratrol might produce the same effect. The livers

of the resveratrol-treated mice had considerably more

mitochondria than those of HC controls and were not

significantly different compared to those of the SD

group (Fig. 3e, f). There was also a trend towards

higher citrate synthase activity in the

resveratrol-fed mice (an indicator of increased

mitochondrial content) although the effect was not

significant (Table 1). Culturing FaO hepatoma or HeLa

cells in the presence of resveratrol increased

mitochondrial number (Fig. 3g, h), similar to the

previously reported effect of culturing cells in serum

from calorically restricted rats24.

Mitochondrial biogenesis in liver and muscle is

controlled, in large part, by the transcriptional

coactivator PGC-125, 26, the activity of which, in

turn, is positively regulated by SIRT1-mediated

deacetylation27, 28. Hence, the acetylation status of

PGC-1 is considered a marker of SIRT1 activity in

vivo27. Because this assay required more tissue than

was available, we examined a separate cohort of

one-year-old mice on the HC diet that had been treated

with resveratrol for 6 weeks at 186 mg kg-1 d-1. The

acetylation status of PGC-1 in the resveratrol-fed

mice was threefold lower than the diet-matched

controls (Fig. 3i, j). There was no detectable

increase in SIRT1 protein levels in

resveratrol-treated mice (data not shown), suggesting

that SIRT1 enzymatic activity was enhanced by

resveratrol.

Microarrays and pathway analysis

These data demonstrate that resveratrol can alleviate

the negative impact of a high-calorie diet on overall

health and lifespan. To determine to what extent

resveratrol had shifted the physiology of the

high-calorie group towards the lower calorie group, we

performed whole-genome microarrays and pathway

analysis on liver samples. Z ratios were calculated as

described previously29 and a subset of expression

changes was verified by polymerase chain reaction with

reverse transcription (RT–PCR) (Supplementary Fig. 5).

In the HCR group, expression patterns for 782 out of

41,534 (<2%) individual genes changed significantly

relative to the diet-matched controls (Fig. 4a, .

Notably, within the top 12 most highly elevated

transcripts were serum amyloid proteins (Saa1–3),

major urinary proteins (Mup1 and Mup3), and both forms

of hydroxysteroid dehydrogenase that degrade

testosterone (Hsd3b4, Hsd3b5). The list of 12 most

highly downregulated transcripts included three

cytochrome p450 enzymes (Cyp2a4, Cyp2a5 and Cyp2b9)

that are known to activate pro-carcinogens30. The

complete data set is available at

http://www.grc.nia.nih.gov/branches/rrb/dna/index/dnapubs.htm#2.

We next performed parametric analysis of gene set

enrichment (PAGE), a computational method that

determines differences between pathways using a priori

defined gene sets31, 32. PAGE analysis indicated that

resveratrol caused a significant alteration in 127

pathways, including the TCA cycle, glycolysis, the

classic and alternative complement pathways, butanoate

and propanoate metabolism, sterol biosynthesis and

Stat3 signalling (Supplementary Fig. 6; for a complete

list see Supplementary Fig. 7). Some of the most

highly downregulated pathways in the resveratrol-fed

group are known to extend lifespan in model organisms

when attenuated, including insulin signalling, IGF-1

and mTOR signalling, oxidative phosphorylation and

electron transport33, 34, 35, 36. Downregulation of

glycolysis is a well known marker of caloric

restriction37 and has been proposed as a mechanism by

which caloric restriction works38. The increase in

Stat3, a transcription factor involved in cell

survival and liver regeneration39, is of note because

its activity is known to be suppressed in the liver by

high caloric diets and shows an age-related decline in

activity that is attenuated by caloric restriction40,

41.

A few of the pathway changes were unanticipated.

Although we had observed an increase in mitochondrial

number in the HCR group, there was a decrease in the

transcription of numerous mitochondrial genes,

suggesting that the turnover of mitochondrial proteins

was reduced. This result was unexpected, but is

consistent with a previous report showing that

SIRT1-dependent activation of PGC-1 does not enhance

transcription of mitochondrial genes27. Upregulation

of complement, which occurs in obese and aged mice,

was also observed in the HCR group for reasons that

are currently unclear.

It is notable that resveratrol opposed the effects of

high caloric intake in 144 out of 153 significantly

altered pathways (Fig. 4c). In fact, the PAGE

signature of the HCR group was considerably more

similar to that of the SD group than the HC controls.

Principal component analysis yielded values of -1.82

(SD), -1.41 (HCR) and 3.22 (HC), with 88.4% of the

variability assigned to the first principal component,

making the HC group the clear outlier (Fig. 4d).

We next compared our PAGE results to a pre-existing

caloric restriction data set for C57BL/6 mice known as

AGEMAP, hypothesizing that the comparison of changes

induced by these two paradigms might reveal pathways

common to the enhancement of health and longevity. Of

the 36 different pathways identified by AGEMAP as

being significantly altered by caloric restriction,

there was sufficient overlap to compare 19 of them to

our data (Fig. 4e). Pathways altered in the same

direction by caloric restriction and resveratrol

included the downregulation of IGF-1 and mTOR

signalling, downregulation of glycolysis, and

upregulation of Stat3 signalling. One interesting

difference was that cell cycle checkpoint and

apoptotic pathways were elevated in the caloric

restriction group but downregulated by resveratrol. We

do not favour the interpretation that the

resveratrol-treated livers were undergoing less

apoptosis because levels of AST and ALT, two

indicators of hepatic apoptosis, were unchanged (see

Table 1). Perhaps the downregulation of cell cycle

checkpoints is linked to the recent discovery that

inhibition of checkpoint function in C. elegans

increases stress resistance and lifespan42. Although

the statistical power of this analysis is limited by

the overlap in data sets, the results suggest that

more comprehensive comparisons of the effects of

resveratrol and caloric restriction are warranted.

Discussion

The ability of resveratrol to prevent the deleterious

effects of excess caloric intake and modulate known

longevity pathways suggests that resveratrol and

molecules with similar properties might be valuable

tools in the search for key regulators of energy

balance, health and longevity. As a case in point, the

most highly upregulated gene in the HC group and

second most highly downregulated gene in the HCR group

was Cidea, which regulates energy balance in brown fat

and provides resistance to obesity and diabetes when

knocked out43.

Taken together, the findings in this study show that

resveratrol shifts the physiology of mice consuming

excess calories towards that of mice on a standard

diet, modulates known longevity pathways, and improves

health, as indicated by a variety of measures

including survival, motor function, insulin

sensitivity, organ pathology, PGC-1 activity, and

mitochondrial number. Notably, all these changes

occurred without a significant reduction in body

weight. Whether these effects are due to resveratrol

working primarily through SIRT1, which is the case for

simpler metazoans, or through a combination of

interactions, as predicted by the xenohormesis

hypothesis6, 44, remains to be determined. In either

case, this study shows that an orally available small

molecule at doses achievable in humans can safely

reduce many of the negative consequences of excess

caloric intake, with an overall improvement in health

and survival.

.... one-year-old male C57BL/6NIA mice were maintained

on AIN-93G standard diet (SD), AIN-93G modified to

provide 60% of calories from fat (HC), or HC diet with

the addition of 0.04% resveratrol (HCR). ... Male

C57BL/6NIA mice at 11 months of age ... were

maintained on a standard purified mouse diet (AIN-93G)

for one month prior to the start of the experiment.

The groups presented in this study were fed the

standard AIN-93G diet or AIN-93G plus 0.04%

resveratrol ad libitum for an additional six weeks

while high-calorie diets were prepared. Animals on

AIN-93G either continued on the standard diet (SD), or

were switched to high-calorie AIN-93G (modified by the

addition of hydrogenated coconut oil to provide 60% of

calories from fat, HC), and the group receiving

AIN-93G plus 0.04% resveratrol, was switched to the

high-calorie diet plus 0.04% resveratrol (HCR).

Coconut oil was chosen to avoid high levels of dietary

cholesterol associated with animal fats such as lard

and because it is solid at room temperature so that a

large quantity can be added to the food pellets

without much change in the consistency. This helps

prevent overgrowth of the teeth, which can happen with

high fat paste-like diets over an extended period.

For the PGC-1a experiments, mice at a similar age were

fed the HC diet or HC plus resveratrol at a

concentration that delivered ~ 186 mg/kg/day for six

weeks. Resveratrol (> 98%) was purchased from Orchid

Pharmaceuticals (Aurangabad, India) ...

-- Al Pater, alpater@...

-- Al Pater, PhD; email: Alpater@...

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