Obesity stems from a prolonged imbalance between the level of energy intake and energy expenditure, with the resultant surplus being stored as lipids predominantly in adipose tissue, but also in muscle and liver tissue, triggering features of the metabolic syndrome. Understanding the factors which regulate both energy intake and expenditure, such as environmental/lifestyle manipulations or pharmaceuticals, is an important step towards developing obesity treatments.

The physiological benefits of resveratrol (3,4',5-trihydroxystilbene or RSV), a natural polyphenol, are currently under intensive investigation. Resveratrol is produced by plants in response to infection by the pathogen Botrytis cinerea 1. It is also induced in response to a variety of stress conditions (climate, exposure to ozone, sunlight and heavy metals) 2. Presently, it has been detected in more than 70 plant species, including grapes, peanuts, berries and pines 2. Resveratrol is able to activate nicotinamide adenosine dinucleotide-dependent deacetylase SIRT1 (Silent mating type Information Regulation 2 homologue Type 1), one of the seven mammalian sirtuins 34 involved in glucose homeostasis and lipid metabolism. This activation of SIRT1 by RSV seems to prevent obesity by inducing oxidative mitochondrial metabolism 5 and reduced insulin resistance 6 in mice on a high-caloric diet. In the same way, RSV minimizes hyperglycaemia in diet-induced obese and diabetic mice 7, and dyslipidemia in the experimental model of obese Zucker rats. These rodent models are widely used as animal models of obesity and type 2 diabetes which present many features of the human metabolic syndrome 8. The validity of information obtained in primates, particularly in humans, is still under debate because the experimental protocols used in the preliminary studies were quite different from each other 9. Moreover, recent works have demonstrated that RSV supplementation mimics caloric restriction in mice 10, suggesting that RSV could be a good candidate for the development of obesity therapies.

However, most RSV studies focused on changes in energy expenditure and its related cellular mechanisms 1112 and were mainly obtained from genetically modified mouse models of obesity 13 or in the context of a high-fat diet 1411. The effects of RSV on spontaneous food intake and its regulation are still unclear and still unknown in primates.

The aim of the present study was thus to determine the effects of a four-week RSV supplementation on energy metabolism and spontaneous food intake in a non-human primate, the grey mouse lemur (Microcebus murinus), which demonstrates seasonal spontaneous obesity. Grey mouse lemurs present high photoperiod-dependent variations in body mass associated with energy saving mechanisms, in particular a phase of heterothermia during their daily resting period. When exposed to a short photoperiod, mouse lemurs display a fast and linear body-mass gain (about 6-8 g/week) associated with a high caloric intake. In just a few weeks, a near 100% increase in body mass is observed due to high levels of fat storage, while the duration and depth of daily hypothermia bouts increase 1516.

The effect of RSV supplementation on body mass, resting metabolic rate (RMR), daily body temperature (Tb) variations and spontaneous food intake in the grey mouse lemur was explored to investigate of the hypothesis that RSV supplementation will affect the time courses of pre-wintering fattening mechanisms in this primate. Because previous studies on mouse lemurs have suggested that several gut hormones may be related to the pre-wintering fattening phase and to the daily heterothermia expression 17, we also tested the effects of RSV supplementation on several gut-derived peptide hormones that are involved in numerous aspects of fuel homeostasis 18. We measured glucagon-like peptide 1 (GLP-1), pancreatic polypeptide (PP), peptide YY (PYY) and glucose-dependent insulinotropic polypeptide (GIP). Glucagon-like peptide 1 and PYY were shown to decrease energy expenditure and food intake 19. Conversely, PP and GIP induced an increase in energy expenditure, motor activity and mobilization of fat stores 20. Consequently, possible relationships between the key parameters involved in energy balance regulation and gut hormones in grey mouse lemurs were also investigated.

During the control week prior to RSV supplementation, the studied animals presented an average body-mass gain of 6.2 ± 1.3% (1.2 g/d) from their initial mass (133 ± 4 g). At the end of the four weeks of RSV supplementation, the body-mass gain was gradually suppressed to 1.0 ± 0.3% (0.5 g/d, F = 14.0, df = 4, p = 0.007, Figure 1A). A 13% decrease in spontaneous food intake was noted from the third week of RSV treatment (from 121.4 kJ/d to 104.7 kJ/d, F = 10.4, df = 4, p = 0.035, Figure 1B). The RMR increased by 29% after four weeks of RSV treatment compared to the control period (F = 12.7, df = 4, p = 0.013, Figure 1C). Total daily locomotor activity (LA) was not modified by RSV treatment (164 ± 23 a.u. in the control week vs. 168 ± 12 a.u. in the fourth week of treatment, F = 0.4, df = 4, p = 0.980, Figure 1D). A representative profile of the weekly average of variations in Tb of one mouse lemur is represented in Figure 2A. The mouse lemurs exhibited a significant increase in their mean Tb during the diurnal phase (Tb day) with a gain of 1.0 ± 0.3°C between the control and the fourth week of treatment (34.7 ± 0.5°C vs. 35.6 ± 0.1°C, F = 14.3, df = 4, p = 0.006). The minimal Tb value (Tb min) of the animals increased by 1.5 ± 0.6°C between the control and the fourth week of treatment (32.4 ± 0.8°C vs. 33.9 ± 0.4°C, F = 11.1, df = 4, p = 0.026). The amplitude of the phase of daily hypothermia was greatly reduced in response to RSV supplementation. On the other hand, no significant change in the mean Tb during the active nocturnal phase (Tb night) was found in response to RSV supplementation (36.4 ± 0.1°C vs. 36.6 ± 0.1°C, F = 3.3, df = 4, p = 0.504) (Figure 2B).

No significant time-course change was reported for GLP-1, PP and PYY levels. Only the GIP level was significantly increased by RSV after four weeks of supplementation (104 ± 14 pg/ml in the control week vs. 175 ± 39 pg/ml in the fourth week, F = 11.6, df = 4, p = 0.021) (Table 1). Although trends are visible, the increased inter-individual variability observed during the treatment (from 21% to 61%, F = 14.3, df = 4, p = 0.002) did not allow any significant conclusion to be reached. Lastly, no significant correlation could be found between the variations in gut hormones and the variations in body-mass gain, food intake or RMR (Table 2).

The main findings of this study were: 1) a four-week resveratrol supplementation had significant effects on energy metabolism in the mouse lemur, characterized by a reduction in seasonal body-mass gain associated with an increase in resting metabolic rate and a decrease in food intake; 2) the response to resveratrol supplementation mainly involved a strong reduction of daily heterothermia expression with no change in the daily amount of locomotor activity; 3) despite the fact that glucose-dependent insulinotropic polypeptide (GIP) values increased during resveratrol supplementation, no correlation was evidenced between the other tested gut hormones and changes in energy balance parameters.

Resveratrol supplementation was given to mouse lemurs at the very time of seasonal fattening induced by exposure to short day lengths. This period corresponds to an increase in both spontaneous food intake and frequency of deep torpor, leading to fast body mass gain through fat accumulation. Moreover, the resting metabolic rate and locomotor activity decrease, reinforcing the energy saving processes. Resveratrol supplementation appeared to limit all these energy saving mechanisms by acting more specifically on food intake and daily hypothermia bouts without change in locomotor activity.

The observed decrease in food intake in mouse lemurs cannot be related to the palatability of resveratrol as the mouse lemurs were not found to be averse to resveratrol prior to the experiment. It is more likely that an aversive palatability of resveratrol would have led to an immediate reduction in food intake which was not observed. However, the effects on food intake might have been related to the dose of resveratrol given. The dosage level administered in this study (200 mg/kg) was selected from studies in the literature on rodents, and was intermediate between the 40 mg/kg of Baur et al. 14 and the 400 mg/kg of Lagouge et al. 12. This dose was carefully mixed in the food and, since the animals tended to eat less than provided, the actual ingested dose of resveratrol was below 200 mg/kg. The amount of resveratrol ingested by the animals was 96.1% in the first week and 84% in the fourth week (corresponding to an equivalent of 192 mg/kg/day and 168 mg/kg/day, respectively). These amounts, based on the existing literature, appeared sufficiently high to be effective in mammals 212223. However, the observed satiety in resveratrol-supplemented mouse lemurs may be a sensibility response specific to this species or to primates.

Although the change in food intake could explain the reduction of seasonal body-mass gain, resveratrol supplementation significantly affected the body temperature patterns in the mouse lemur. Average values of energy savings by daily torpor in this species have been estimated to be about 40-70% as compared to the maintenance of normothermia 2425. Under resveratrol supplementation, the depth and the duration of daily torpor decreased and were associated with an increase in both body temperature and resting metabolic rate during the daily resting period. In contrast, body temperature values during the night active phase were not modified, and nor was locomotor activity. The decrease in energy savings during the daily diurnal rest would thus contribute to the reduction in body-mass gain under resveratrol supplementation.

No such effect of resveratrol has been shown in mice, for which no change in food intake or body temperature was observed 1113. However, these data originate from experimentally-induced obesity and it is likely that the responses observed in mouse lemurs rely on specific life-history traits of this primate, which has developed high seasonal energy saving mechanisms and exhibits pre-wintering spontaneous obesity.

Resveratrol activates mammalian sirtuins, more specifically SIRT1, which is involved in glucose homeostasis and lipid metabolism. In mice exposed to a high-fat diet or in ob/ob mice, resveratrol was shown to protect mice from diet-induced metabolic disorders 121326 by mimicking some effects of caloric restriction. Likewise, resveratrol and assimilated SIRT1 activators have been proposed as potential treatments for type 2 diabetes in mice models 14122728. The effects of resveratrol have been suggested to depend on an increase in oxidative mitochondrial metabolism 14 and on enhanced fat oxidation 11, leading to an increase in energy expenditure. Interestingly, SRT1720, which activates SIRT1 by four fold in comparison to resveratrol, induces a significant decrease in fat mass without a change in food intake in mice 11. In the mouse lemur, concomitantly to an increase in energy expenditure through decreasing hypothermia bouts and increasing metabolism, resveratrol might have a direct negative effect on fat storage.

Due to their involvement in satiety and satiation processes 182930, several gut hormones have been measured in resveratrol-supplemented mouse lemurs. No significant change was recorded after four weeks for the glucagon-like peptide 1 and the peptide YY, two gut hormones involved in the reduction of food intake and energy expenditure 31323334. Conversely, the two other gut hormones are known to induce an increase in energy expenditure. The pancreatic polypeptide increases satiety, thus leading to a loss in body mass 20 and the glucose-dependent insulinotropic polypeptide (GIP) was shown to induce an increase in exploratory behaviour and performance in some motor function tests in a transgenic mouse that over-expressed GIP 35. Only GIP significantly increased with resveratrol supplementation. Data emerging from studies in animal models and cultured human fat cells support a physiological role for GIP in fat cell metabolism, leading to the mobilization of fat stores 1736. Such effects might have contributed to the reduction in body-mass gain by limiting fat storage in supplemented mouse lemurs. The observed effect of resveratrol on food intake in the mouse lemur appears to be independent of changes in anorexic gut hormones, but the high observed inter-individual variability in our study does not allow definite conclusions to be reached. Moreover, because sampling was performed at a single point in fasted animals during the diurnal rest, changes may have been missed.

Resveratrol has been studied as a potential mimetic of caloric restriction 4. However, caloric restriction decreases the metabolic rate 37, whereas resveratrol had the opposite effect 12. From previous studies using moderate caloric restriction in wintering mouse lemurs 17, the effects of resveratrol significantly differed from changes observed in caloric-restricted animals. In the mouse lemurs, caloric restriction led to an increase in the duration and depth of daily torpor bouts. These body temperature adjustments were efficient at preventing body-mass loss and were not associated with changes in gut hormones 17.

In conclusion, we demonstrated for the first time the short-term effects of resveratrol on the metabolism of an heterothermic primate. Resveratrol activates energy expenditure by inducing an increase in resting metabolic rate and a decrease in torpor patterns that play key roles in energy saving in this primate. Moreover, resveratrol had a satiety effect in this primate that reduced their spontaneous food intake. All of these changes participated in the limitation of pre-wintering fattening processes in which the increase in the glucose-dependent insulinotropic polypeptide levels would play an additive role. These results provide novel information on the potential effects of resveratrol on energy metabolism and control of body mass in a primate.

a.u.: arbitrary unit; GIP: glucose-dependent insulinotropic polypeptide; GLP-1: glucagon-like peptide 1; LA: locomotor activity; PP: pancreatic polypeptide; PYY: peptide YY; RMR: resting metabolic rate; RSV: resveratrol; RSV1: first week of RSV supplementation; RSV2: second week of RSV supplementation; RSV3: third week of RSV supplementation; RSV4: fourth week of RSV supplementation; SEM: standard error of the mean; SIRT1: Silent mating type Information Regulation 2 homologue Type 1; Tb: body temperature; Tb night: mean Tb during the active nocturnal phase; Tb day: mean Tb during the resting diurnal phase; Tb min: minimal Tb value.

AD performed the experimental protocol, analysed the data and drafted the manuscript. SB participated in the design of the study and drafted the manuscript. FA conceived the study, participated in its design and coordination, performed the statistical analyses and drafted the manuscript. All authors read and approved the final manuscript.