Cell Metabolism 2011 (Nov 2); 14 (5): 612–622 ~ FULL TEXT
Silvie Timmers, Ellen Konings, Lena Bilet, Riekelt H. Houtkooper,
Tineke van de Weijer, Gijs H. Goossens, Joris Hoeks, Sophie van der Krieken,
Dongryeol Ryu, Sander Kersten,
Top Institute Food and Nutrition (TIFN),
Comments from Dr. Stephen G. Chaney, Ph.D.
Full Professor of Biochemistry, Biophysics and Nutrition
University of North Carolina, Chapel Hill
Although this was a relatively small study, but it was very well designed. 11 obese men (average age 52, average weight = 220 pounds, average BMI = 31) were enrolled in the study.
The study was what scientists call a randomized, double-blind crossover study. In plain English that means that half of the men received 150 mg of resveratrol during the first 30 days and the other half received a placebo. This was followed by a four week washout period to remove resveratrol from the bloodstream. Then in the final 30 days of the trial the groups were switched. Those that received the placebo during the first 30 days were given resveratrol and vice versa.
The strength of this kind of study is that each subject serves as their own control – which eliminates a lot of individual variability.
The result of just 30 days on resveratrol were impressive:
1) The same gene regulators (AMPK, SIRT1 and PGC-1a) were activated in this study as are activated by caloric restriction and resveratrol in mice and endurance training in humans.
2) Blood glucose levels and blood insulin levels were decreased and insulin sensitivity was improved.
3) Triglyceride levels and levels of inflammation markers (eg IL-6 and TNFa) were decreased.
4) Systolic and average blood pressure was decreased.
5) Both gene expression and metabolic studies showed that mitochondrial efficiency was increased – especially the ability of mitochondria to generate energy from fat stores. In addition, fat stores in the muscle fibers responsible for endurance exercise were increased.
6) Fat stores in the liver (a pathological condition associated with obesity that can lead to liver damage) were decreased and blood markers of liver health were improved.
7) No adverse effects of resveratrol supplementation were observed.
The authors concluded “[This study] shows that resveratrol supplementation exerts favorable metabolic adaptations that in many aspects mimic the effects of caloric restriction and/or endurance training.”
Resveratrol is a natural compound that affects energy metabolism and mitochondrial function and serves as a calorie restriction mimetic, at least in animal models of obesity. Here, we treated 11 healthy, obese men with placebo and 150 mg/day resveratrol (resVida) in a randomized double-blind crossover study for 30 days. Resveratrol significantly reduced sleeping and resting metabolic rate. In muscle, resveratrol activated AMPK, increased SIRT1 and PGC-1a protein levels, increased citrate synthase activity without change in mitochondrial content, and improved muscle mitochondrial respiration on a fatty acid-derived substrate. Furthermore, resveratrol elevated intramyocellular lipid levels and decreased intrahepatic lipid content, circulating glucose, triglycerides, alanine-aminotransferase, and inflammation markers. Systolic blood pressure dropped and HOMA index improved after resveratrol. In the postprandial state, adipose tissue lipolysis and plasma fatty acid and glycerol decreased. In conclusion, we demonstrate that 30 days of resveratrol supplementation induces metabolic changes in obese humans, mimicking the effects of calorie restriction.
From the FULL TEXT Article:
In our western society, the number of age-related chronic diseases such as obesity, diabetes and cancer increases progressively (Crews, 2005).
The only non-pharmacological intervention known to date to alleviate these deleterious conditions is calorie restriction. Reduction of calorie intake to 30-50% below ad libitum levels, or every-other-day feeding, can delay the onset of age-related diseases, improve stress resistance, and decelerate functional decline (Barger et al., 2003; Goodrick et al., 1990; McCay et al., 1935). Although short-term dietary restriction has metabolic effects in humans such as lowering metabolic rate (Heilbronn et al., 2006), improving insulin sensitivity (Larson-Meyer et al., 2006; Lim et al., 2011) and reducing cardiovascular risk factors (Lefevre et al., 2009), eating less for the sake of creating a desirable metabolic profile is unlikely to gain widespread compliance. As such, the focus has been on the development of calorie restriction mimetics that evoke some of the benefits of calorie restriction without an actual reduction in calorie intake. In that respect, sirtuins are considered an important molecular target (Canto and Auwerx, 2009). Indeed, it was suggested that the yeast Sir2 gene (Lin et al., 2000) or its worm (Tissenbaum and Guarente, 2001) and fly (Rogina and Helfand, 2004) orthologues are required for the effects of calorie restriction, although the relevance of the role of Sir2/SIRT1 as a strictu sensu longevity regulator is debated (Burnett et al., 2011). What is clear, however, is that mammalian SIRT1 plays a context-dependent role in health span regulation, for instance by mediating effects in metabolic stress situations, such as high-fat diet-induced obesity (Baur et al., 2006; Lagouge et al., 2006; Pearson et al., 2008). As such, SIRT1 confers protection against ageing-associated metabolic diseases such as glucose intolerance and cancer (Herranz et al., 2010; Pearson et al., 2008; Rutanen et al., 2010). In light of the growing number of patients suffering from metabolic diseases, compounds that activate SIRT1 directly or indirectly might offer protection against the onset of metabolic damage and promote healthy ageing.
To this end, Howitz and colleagues performed an in vitro screen to identify small molecule activators of SIRT1 (Howitz et al., 2003). Resveratrol, a natural polyphenolic compound present in various dietary components such as mulberries, peanuts, grapes and red wine, was identified as the most potent activator of SIRT1 (Howitz et al., 2003). Recently, it was shown, however, that resveratrol may not activate SIRT1 directly (Beher et al., 2009; Pacholec et al., 2010), but rather exerts its effects on SIRT1 through activation of AMPK (Baur et al., 2006; Canto et al., 2009; Canto et al., 2010; Feige et al., 2008; Hawley et al., 2010; Um et al., 2010), although additional direct SIRT1 activation is not completely excluded (Dai et al., 2010). Regardless of the mode of activation, resveratrol treatment in mice fed a high calorie diet consistently improved various health parameters including glucose homeostasis, endurance and survival (Baur et al., 2006; Lagouge et al., 2006; Pearson et al., 2008; Sun et al., 2007), and has therefore been suggested to act as a calorie restriction mimetic. However, so far no human studies have been reported that have systematically examined the metabolic effects of resveratrol in vivo.
Here, we gave obese but otherwise healthy subjects a dietary supplement containing 99% pure trans-resveratrol (resVida™), during 30 days and examined whole-body energy expenditure, substrate utilization, ectopic lipid storage, mitochondrial function, and lipolysis in adipose tissue and skeletal muscle using a combination of in vivo and ex vivo measurements. Our data show that like calorie restriction, resveratrol supplementation lowers energy expenditure, improves metabolic profile, as well as global health parameters.
Resveratrol, which was discovered in a small-molecule screen as a potent SIRT1 activator (Howitz et al., 2003), has been extensively studied in animal and cellular studies with promising results (Baur et al., 2006; Lagouge et al., 2006). Here, we show that resveratrol supplementation in humans exerted favorable metabolic adaptations that in many aspects mimic the effects of calorie restriction and/or endurance training (Civitarese et al., 2007; Heilbronn et al., 2006; Larson-Meyer et al., 2006; Larson-Meyer et al., 2008; Lefevre et al., 2009). These metabolic adaptations include a reduction in sleeping metabolic rate, blood pressure and hepatic lipid content, an improvement in skeletal muscle intrinsic mitochondrial function and several plasma markers of general health, an increase in intramyocellular lipid content, as well as an increase in skeletal muscle PGC-1α protein content. These data extend findings, which so far have only been observed in cell and rodent models, to the human situation, showing that resveratrol has promising beneficial metabolic effects, and suggests that resveratrol has the potential to improve metabolic health in subjects at risk for developing the metabolic syndrome.
Resveratrol exerts significant effects on energy metabolism. Sleeping metabolic rate (SMR) was significantly lower following 30 days of resveratrol supplementation, without changing 24-hour energy expenditure. It should be noted that SMR is the component of human energy metabolism that is most sensitive to metabolic changes—as it is not affected by physical activity—and small differences in sleeping energy expenditure can be detected with high accuracy. In line, we observed that basal and postprandial energy expenditure was also lower after 30 days of resveratrol supplementation. The 2-4% reduction in energy expenditure upon resveratrol treatment is consistent with the effects observed after calorie restriction (6% reduction) (Heilbronn et al., 2006; Martin et al., 2007).
It is important to note that basal and postprandial energy expenditure were reduced by resveratrol in our human study rather than increased, as was true for mice (Lagouge et al., 2006). Although our energy expenditure data are opposite to the effects seen in mice, a lowering of resting and sleeping metabolic rate is likely a reflection of improved metabolic efficiency, and completely in line with the endurance training or calorie restriction-like effects of resveratrol (Heilbronn et al., 2006; Martin et al., 2007). Although the dose used in our human study was ~200 fold lower than doses used in the mouse studies, we reached similar plasma resveratrol concentrations. We cannot exclude, however, that metabolism of resveratrol is different between mouse and man, and possibly more importantly that timing of treatment (30 days in our study vs 4-6 months in mice (Baur et al., 2006; Lagouge et al., 2006)) significantly impacts physiological outcome.
Prolonged calorie restriction (during six months) has also been suggested to increase the expression of genes encoding proteins involved in mitochondrial biogenesis and function (Civitarese et al., 2007). Similarly, our microarray data indicate increased mitochondrial gene expression in muscle following resveratrol supplementation. In fact, we showed that this induction is likely to be mediated by AMPK—which is activated by resveratrol—and resulted in increased SIRT1 and PGC-1α protein content, accompanied by increased citrate synthase activity, suggesting that mitochondrial activity was effectively improved. Moreover, detailed mitochondrial characterization revealed that resveratrol had beneficial effects on mitochondrial respiration when octanoyl-carnitine was used a substrate, but not when only glutamate was used. These data suggest that resveratrol specifically improves muscle fat oxidative mitochondrial capacity. Interestingly, we have recently found comparable effects in PGC-1α overexpressing mice, showing an improved intrinsic mitochondrial function (i.e. respiration per mitochondrion) but only when fatty acids are used as a substrate (Hoeks et al., 2011). The fact that mtDNA copy number, OXPHOS protein content, and PCr recovery were not changed in resveratrol-treated subjects suggests that 30 days resveratrol treatment mostly affects mitochondrial efficiency, not abundance. It is well possible that more long-term treatment would cause these parameters to change as well.
Interestingly, IMCL content was markedly increased after 30 days of resveratrol supplementation. Together with the improvement in muscle fat oxidative capacity and the other beneficial metabolic adaptations like lowering of circulating triglyceride and glucose levels, the present data hint towards endurance training-like effects of resveratrol (Dube et al., 2008; Meex et al., 2010). Consistent with data in rats, where resveratrol has been shown to reduce hepatic lipid synthesis (Ahn et al., 2008; Arichi et al., 1982), we observed reduced IHL content. Recent data suggest that endurance training is also able to lower IHL content (Kantartzis et al., 2009), as was calorie restriction, although this effect was mainly attributed to a reduction in body weight (Larson-Meyer et al., 2006; Lim et al., 2011). Taken together with the reduced plasma triglycerides and increased muscle fatty acid oxidation, we hypothesize that fat is liberated from peripheral depots to be metabolized by the muscle. Again, here our data suggest that resveratrol mimics the effect of calorie restriction and endurance training. Also, we have investigated postprandial metabolism, but only found a reduction in total energy expenditure that was reflected by a lower fat oxidation during the late postprandial phase—a change that is reminiscent of the role of SIRT1 in regulating the efficient switch in energy substrate utilization (Canto et al., 2009; Canto et al., 2010). Although postprandial lipolysis tended down, this does not refute our hypothesis of increased fat mobilization, but rather confirms the idea of improved metabolic flexibility.
A striking finding in our study was the resveratrol-induced reduction of systolic blood pressure by 5 mmHg. In rodents, resveratrol is also vasoactive and has been shown to cause vasodilatation and to improve aortic endothelial function in diabetic mice (Wang et al., 2011), to reduce heart rate (Lagouge et al., 2006) and even to lower blood pressure (Rivera et al., 2009). Also in human obese subjects endothelial function and cardiovascular health, dose-dependently improved after one week of resveratrol supplementation (Wong et al., 2010). Similarly, healthy non-obese individuals also improved their cardiovascular risk after six months of calorie restriction as evidenced by a reduction in blood pressure (Lefevre et al., 2009). Another indication that global health was improved upon resveratrol treatment was the decreased expression levels of genes of inflammatory pathways, as were plasma levels of several inflammatory markers, and leukocyte numbers.
Our data also point towards favorable effects on glucose homeostasis after 30 days of resveratrol supplementation in obese subjects. Indeed, HOMA-index was improved after resveratrol, suggesting favorable effects on insulin sensitivity. These beneficial effects of resveratrol on metabolism are in concordance with several findings of resveratrol supplementation in animals (Baur et al., 2006; Canto et al., 2009; Lagouge et al., 2006; Pearson et al., 2008; Sun et al., 2007). Unfortunately, we could not determine if these effects resulted in improved whole body insulin sensitivity.
In conclusion, we demonstrate beneficial effects of resveratrol supplementation for 30 days on the metabolic profile in healthy obese males, which seems to reflect effects observed during calorie restriction (table S4). Although most of the effects that we observed were modest, they were very consistently pointing towards beneficial metabolic adaptations. Furthermore, there were no effects on safety parameters and no adverse events were reported. Therefore, resVida was safe and well tolerated at the tested concentration. Future studies should investigate the long-term and dose-dependent metabolic effects of resveratrol supplementation in order to further establish whether resveratrol supplementation has the potential to overcome the metabolic aberrations that are associated with obesity in humans.
The study protocol was reviewed and approved by the Medical Ethical Committee of Maastricht University Medical Centre (MUMC+). All study participants gave written informed consent before initiation of the study.
Eleven healthy, obese, male volunteers without family history of diabetes or any other endocrine disorder participated in this study (Table 1). None of the subjects were on medication or were engaged in sports activities for more than two hours per week. Body composition was determined by a dual-energy X-ray absorptiometry scan (DXA, Discovery A; Hologic Corp, Bedford, MA) and maximal aerobic capacity was measured as described (Kuipers et al., 1985).
Clinical study design
Subjects participated in two experimental trials: a placebo and a resVida™ (150mg/day trans-resveratrol (99.9%) (provided by DSM Nutritional Products, Ltd) condition, in a randomized double-blind crossover design with a four-week wash-out period. Subjects were instructed to take the first supplement on the day after baseline measurements (d1) and the last supplement in the evening on day 29. The subjects were instructed to abstain from alcoholic beverages and foods containing substantial amounts of resveratrol (e.g. wine, red grapes, peanuts and berries) and were advised not to take any other food supplements during the study period. Compliance with these instructions was confirmed by verbal declaration of the subjects. Subjects were advised to maintain their normal living-, activity-, and sleeping-pattern during the intervention period. At the start (day 0) and end (day 30) of both intervention periods (resveratrol and placebo), blood samples were analyzed for general safety parameters including clinical chemistry, hematology and coagulation values. A twelve lead electrocardiogram (ECG) (Laméris, Veenendaal, The Netherlands) was performed at the beginning and end of both the resveratrol and placebo intervention. Each experimental trial lasted four weeks during which the subjects came on a weekly basis (day 0, 7, 14, 21, 30) to the university. The weekly check-up took place in the morning after an overnight fast, and included a measurement of body mass and withdrawal of a small blood sample for the analysis of resveratrol (original and metabolites) to confirm compliance to the protocol. On day 28 in the evening, subjects came to the university for 1H-MRS measurements of the liver, to quantify intrahepatic lipid content, and post-exercise PCr recovery rate was examined by 31P-MRS to estimate in vivo mitochondrial function (Schrauwen-Hinderling et al., 2007). To standardize food intake, subjects had lunch with the same food items in the two conditions and after lunch stayed fasted until the start of the measurement at 17 h.
After the MRS measurements on day 28, subjects stayed in the respiration chamber during 36 h to allow measurement of 24 h substrate oxidation and energy expenditure (Schoffelen et al., 1997). Before their stay in the respiration chamber, a standardized evening meal was provided. In the respiration chamber subjects were fed in energy balance (2332.5 kcal/day, 13.1 g of protein, 54.0 g of carbohydrate, 32.9 g of fat) and followed an activity protocol as described (Schrauwen et al., 1997). In the morning of day 30, after subjects left the respiration chamber, a muscle biopsy was taken to investigate ex vivo mitochondrial respiration, which was followed by the withdrawal of a fasting blood sample for the analysis of the effect of resveratrol on circulating substrates. Hereafter, adipose tissue and skeletal muscle lipolysis was examined by means of microdialysis.
To check compliance, resveratrol metabolites were measured by mass spectrometry in plasma on day 0, 7, 14, 21, 29 and 30 as described in the supplemental methods.
In the morning of day 30 - after a standardized overnight fast for 12 hours - and during the postprandial microdialysis test, blood samples were withdrawn for the determination of plasma metabolites according to standard procedures. Full experimental detail is described in the supplemental methods.
On day 0 and day 30, blood pressure was measured after an overnight fast. By placing an automatic inflatable cuff (Omron Healthcare, Hamburg, Germany) on the non-dominant arm in the resting condition, systolic and diastolic blood pressure was measured in triplicate. Mean systolic and diastolic blood pressure values were used to calculate the mean arterial pressure.
Lipid accumulation in liver
On day 28, before subjects underwent the respiration chamber measurement, proton magnetic resonance spectroscopy (1H-MRS) was used to quantify hepatic lipid content (IHL) on a 3 T whole body scanner (Achieva; Philips Healthcare, Best, The Netherlands) using a five-element coil as described (Hamilton et al., 2011), however with a repetition time = 4000 msec, echo time = 37 msec, and number of averages = 64). To minimize motion artifacts, subjects were asked to breathe in the rhythm of the measurement and to be at end-expiration during acquisition of spectra. To determine the intensity of the lipid peak, the water signal was suppressed using frequency-selective prepulses. The unsuppressed water resonance was used as internal reference (number of averages=32) and spectra were fitted with AMARES (Vanhamme et al., 1997) in the jMURI software (Naressi et al., 2001). Values are given as T2-corrected ratios (according to (Hamilton et al., 2011)) of the CH2 peak, relative to the unsuppressed water resonance (as percentage).
31P-MRS-based measurement of mitochondrial function
On day 28, 31P-MRS measurements were performed in vastus lateralis muscle on a 1.5 T whole-body scanner (Intera; Philips Health Care, Best, The Netherlands) essentially according to an established methodology (Schrauwen-Hinderling et al., 2007).
On day 30, when subjects left the respiration chamber, a muscle biopsy was taken from the vastus lateralis muscle under local anesthesia (2% lidocaïne), as previously described (Phielix et al., 2008). A portion of the muscle tissue was directly frozen in melting isopentane and stored at –80°C until assayed. Another portion (~30 mg) was immediately placed in ice-cold preservation medium for determination of ex vivo mitochondrial respiration (Phielix et al., 2008).
Molecular and protein expression
Mitochondrial DNA copy number, gene expression by microarray, and protein expression by Western blot, were performed according to standard procedures as described in the supplemental methods.
High resolution respirometry
Permeabilized muscle fibers were immediately prepared from the muscle tissue collected in the preservation medium, as described elsewhere (Boushel et al., 2007; Phielix et al., 2008). Subsequently, the permeabilized muscle fibers (~2.5 mg wet weight) were analyzed for mitochondrial function using an oxygraph (OROBOROS Instruments, Innsbruck, Austria) (Hoeks et al., 2010).
Fresh cryosections (5 µm) were stained for intramyocellular lipids (IMCL) by Oil Red O staining combined with fibertyping and immunolabeling of the basal membrane marker laminin to allow quantification of IMCL, as described (Koopman et al., 2001).
Postprandial substrate utilization and tissue lipolysis
In 10 subjects, the lipolytic effects of resveratrol in adipose tissue and skeletal muscle were successfully determined by microdialysis, essentially according to (Goossens et al., 2004). A full description of the microdialysis method is provided as supplemental information.
Kolmogorov-Smirnov normality test was performed to evaluate normality distribution. Student’s paired t-test was used to compare placebo and resVida supplementation in normally distributed data, otherwise Wilcoxon signed-rank test was used. For the microdialysis test day, postprandial area under the curve (AUC) of plasma and interstitial metabolites and indirect calorimetry data were calculated using the trapezium rule. In addition to the total AUC (0h-6h after meal ingestion), also the early (0h-2h), mid (2h-4h) and late (4h-6h) AUCs were calculated to obtain more detailed information about the time course of postprandial responses. A P-Value < 0.05 was considered statistically significant. Data are reported as mean ± SEM. Statistical analyses were performed using the statistical program SPSS 16.0 for Mac OS X.
Raw microarray datasets have been submitted to NBCI Gene Expression Omnibus (GSE32357).