42 kcal?g?1, three subgroups) for 6 weeks. After 3 weeks of the different diets (i.e. at the age of 10 weeks), rats fed the standard diet received a daily i.p. injection of vehicle, whereas the three subgroups etc of animals fed the HF diet were treated as follows: i.p. injection of vehicle for the control and the pair-fed groups, or Sirolimus at a dose of 2 mg?kg?1?day?1. Respiratory exchange ratio and locomotor activity Analyses were performed at the end of the 3 week i.p. injection in rats fed a HF diet. We used the 12-cage LabMaster system (TSE Systems GmbH, Berlin, Germany) of the Small Animal Phenotyping Core Facility (CMU, University of Geneva, Geneva), under controlled temperature (22 �� 1��C) and lighting (12 h light�Cdark cycle). Before the recording, animals were allowed a 4 day acclimatization period in training cages.

Glucose tolerance test (GTT) Rats were food-deprived for 4 h (08.30�C12.30 h), and a glucose load of 1.5 g?kg?1 was administered i.p. Blood samples were collected for glycaemia measurements using Glucotrend? Active (Roche, Basel, Switzerland) and for further analyses of insulin concentrations. The GTT was performed in both standard and HF-diet-fed animals after 10 days of treatment. The last injection of vehicle or Sirolimus was administered 4 h before the GTT. Euglycaemic hyperinsulinaemic clamps Overnight-fasted rats were anaesthetized with i.p. sodium pentobarbital (75 mg?kg?1). Euglycaemic hyperinsulinaemic clamps were performed using an insulin infusion of 18 mU?kg?1?min?1 known to completely suppress hepatic glucose production (Terrettaz et al.

, 1986), and the glucose infusion rate (GIR) was measured (Vettor et al., 1994). Once in steady state, a bolus Entinostat of 2-deoxy-d-[1-3H]-glucose (30 ��Ci) was injected to determine the in vivo glucose utilization index of insulin-sensitive tissues, such as skeletal muscles and adipose tissue (Vettor et al., 1994). Rats were then killed by rapid decapitation, and tissues were rapidly removed, frozen and stored at ?80��C. Tissue concentrations of 2-deoxy-d-[1-3H]-glucose-6-phosphate were used to calculate the in vivo glucose utilization index, expressed in ng?mg?1?min?1. These experiments were performed in animals fed the standard diet after 20 days of treatment. In that case, the last injection of vehicle or Sirolimus was administered 24 h before the beginning of the clamps. Body composition In both standard and HF-fed animals, an EchoMRI-700 quantitative NMR analyser (Echo Medical Systems, Houston, TX, USA) was used to measure total fat mass and lean body mass at the beginning and at the end of treatments (days 0 and 20).