Resveratrol suppresses body mass gain in a seasonal non-human primate model of obesity

We used six male grey mouse lemurs (Microcebus murinus, Cheirogaleidae, primates) born in a laboratory breeding colony in Brunoy, France (Agreement # 962773) from a population originally caught 40 years ago on the southwest coast of Madagascar. The animals were 3-year-old adults and presented a mean ± standard error of the mean (SEM) body mass of 133 ± 4 g. Conditions were constant in respect to ambient temperature (25°C), relative humidity (55%), and ad libitum food availability. Behavioural and physiological seasonal changes in mouse lemurs are dependent on the photoperiod and are reproduced in captivity using an artificial photoperiodic regimen. In the breeding colony, animals were exposed to an artificial photoperiodic regimen consisting of alternating six-month periods of Malagasy winter-like short-day lengths (light:dark 10:14) and Malagasy summer-like long-day lengths (light:dark 14:10). Animals were studied during the five weeks following the shift from long to short day lengths corresponding to the pre-wintering fattening period. To minimize social influences, the animals were housed individually in cages (0.4 × 0.4 × 0.6 m) provided with nesting materials, and were separated from each other by wooden partitions. Each week and just before the daily food allotment, animals were weighed inside their nest box, to avoid any stress, using a balance accurate to 1 g.

All animals were fed ad libitum during one week, with fresh fruit (bananas and apples) and a mixture of cereals, milk and eggs, providing them with a total of 126 kJ per day. The cereals were composed of 60% carbohydrates, 10% proteins and 30% lipids. The cereals were primarily wheat flour (96%). The mixture was introduced 1 h before the beginning of the animal's nocturnal active phase (Figure 3A). Water was always given ad libitum. During the following four weeks (RSV1, RSV2, RSV3 and RSV4), the animals were fed the same as above but 200 mg/kg of RSV (Sequoia Research Products, United Kingdom) per day was added to the mixture. Prior to the study, we monitored the food intake of the six animals presented with a choice between a bowl of control food and a bowl of food supplemented with 200 mg/kg/day of RSV. Food was provided ad libitum in both bowls. Food intake was assessed by weighing the daily leftovers and correcting for water evaporation (calculated previously with the control mixture under the same environmental conditions). The average daily amounts of food eaten were similar between control and RSV supplemented groups (t = 1.738, df = 5, p = 0.143), evidencing no significant difference in the palatability of the mixtures. To determine the exact quantity of food ingested by the animals, daily leftovers were weighed and corrected for water evaporation as with the palatability control. All the procedures were carried out in accordance with the European Communities Council Directive (86/609/EEC). All the experiments were done under personal licence to experiment on mouse lemurs, delivered by the Ministry of Education and Science after approval of a local Ethics Committee (n° 91-305, issued 20 July, 2006).

Oxygen consumption was measured in a closed indirect calorimetry system. Measurements on post-absorptive animals were made daily during their resting period, 4-6 h after the beginning of the light period to minimize the influence of circadian variations (Figure 3A). The animals were trained to nest in 2.5 l opaque respiratory chambers with a woven floor to absorb any urine. The respiratory chamber was placed in a cabinet at a controlled ambient temperature of 25.0 ± 0.5°C, a value within the thermoneutral zone defined for the mouse lemur [38]. After a 20 min habituation phase with a constant air flow (2 l/min) drawn through the respirometry chamber from bottom to top, the chamber was closed for a 40-min period. The air was dried prior to analysis using a silica gel. The VO₂ consumed by the animal was calculated from initial and final concentrations of O₂ in the chamber which were measured from dried gas using a SERVOMEX 570A paramagnetic gas analyser (Servomex Ltd., England) (accuracy 0.01% O₂). Calculations were made in Standard Temperature and Pressure, Dry and, the daily atmospheric humidity was accounted for. The oxygen analyser was routinely calibrated with N₂ and atmospheric air assuming 21.00% O₂ as recommended by the manufacturer. Oxygen consumption was expressed as ml O₂/h. The animals were weighed before each measurement. Oxygen consumption was corrected for the body weight of the animal [39]. Body temperature was not measured before the experiment to avoid disturbing sleeping animals but males never enter deep torpor at an ambient temperature of 25°C [40]. However, O₂ consumption at 25°C cannot be defined as basal because the body temperature varies in the grey mouse lemur, thus values were referred to as resting metabolic rate (RMR) [15]. The values are given as mean ± SEM.

The grey mouse lemur is a nocturnal species exhibiting high levels of locomotor activity (LA) during the dark period and complete rest during the light period. Under exposure to short days, the daily rhythm of body temperature (Tb) in the mouse lemur is characterized by high Tb values during the active period and, before the onset of the light phase, a rapid and linear drop in Tb reaching minimal values after 3-4 hours. This hypothermia bout is followed by a spontaneous re-warming to normothermic Tb levels until the following dark phase (Figure 3A).

The recording of LA and Tb was obtained by telemetry at a constant ambient temperature of 25°C. A small telemetric transmitter weighing 2.5 g (model TA10TA-F20, DataScience Co. Ltd, Minnesota, USA) was implanted into the visceral cavity under ketamine anaesthesia (Imalgene, 100 mg/kg ip, Merial, France). Telemetry surgeries were performed 15 days before the beginning of the short day period. After surgery, the animals were returned to their home cage and allowed to recover for 15 days before starting the experiment and the continuous recording of Tb from the beginning of the short day period. Total recovery was checked by visual inspection to ensure complete healing of the surgical incision and by verification of a stable daily pattern of Tb variations. Recording began in the first week following the shift to the short photoperiod (Figure 3B). A receiver board was positioned in the cage and LA (expressed in arbitrary units, a.u.) was continuously recorded during this interval by two antennas located in the receiver board which detected vertical and horizontal movements (X-Y coordinate system, Dataquest Lab Pro v. 3.0, Data Science Co. Ltd, Minnesota, USA). The Tb (in°C) of the animals was recorded every 10 min. The following parameters were analysed: daily amount of LA, mean Tb during the active nocturnal phase (Tb night), mean Tb during the resting diurnal phase (Tb day) and the minimal Tb value (Tb min).

Blood sampling was carried out once a week during the control period and in each week of RSV treatment 4-6 h after the beginning of the light period to minimize the influence of circadian variations (Figure 3A). The blood samples were taken via the saphenous vein, without anaesthesia, before food allotment. The volume of blood sampled was 100 μl and that represented less than 2% of the total blood volume of the animals. The blood samples were centrifuged at 7000 rpm at 4°C for 30 min. The plasma was stored at -20°C according to the assay procedure. Levels of glucagon-like peptide 1 (GPL-1), pancreatic polypeptide (PP), peptide YY (PYY) and glucose-dependent insulinotropic polypeptide (GIP) were measured in duplicate using the human gut hormones multiplex panel (LincoplexTM Multiplex Assays, Bioscience) and Luminex technology at the Saint Antoine Hospital in Paris dedicated to micro-assays in small animals (IRSSA, INSERM, IFR65, France), as previously described [17]. The average recoveries of known quantities of standard peptides (taken at the low, medium and high concentrations given in the assay) added to mouse lemur plasma were 89% for GIP, 83% for GLP1, 88% for PP and 107% for PYY. Inter-assay and intra-assay coefficients of variation were < 19% and < 11%, respectively.

All values are expressed as mean ± SEM. We used the Friedman test to assess significant variations in all of the studied parameters. Comparisons were considered to differ significantly when p < 0.05. Possible correlations between the energy balance parameters and gut hormone levels were assessed by Spearman's correlation test. All statistical computations were performed using SYSTAT for Windows (V9, SPSS Inc., Illinois, USA).