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),
6700 Wageningen,
The Netherlands
The Abstract:
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:
Introduction
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.
Discussion
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-1? 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-1? 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-1? 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.
