Rapamycin

Rapamycin (LC laboratories) was dissolved in 10% Tween-20/90% ethanol, and 20 h before assays, Rapamycin was added to conditioning plates or test plates at a final concentration of 100 μM. Control plates were prepared to contain the solvents at the same concentration as that in the Rapamycin-containing plates.

Mice were placed on NCD (10% kcal fat; Research Diets D12450Bi) or HFD (45% kcal fat; Research Diets D12451i) starting at 4–6 weeks of age for 12 to 18 weeks. Mice were weighed weekly, and food consumption was tracked. Body composition, including body fat, lean mass, and fluid, was measured via TD-NMR using the Bruker Minispec Body Composition Analyzer. Energy expenditure, movement, VO₂ max, VCO₂ max, respiratory exchange ratio (RER), and food and drink consumption were measured using CLAMS metabolic cages, which was performed by the Metabolic Phenotyping Core. Serum was collected from mice treated with HFD for 0, 2 or 17 weeks and Leptin levels were measured via MAGPIX, using the mouse metabolic hormone panel (Millipore). For the rapamycin experiments, mice were placed on HFD for four weeks prior to administration of rapamycin, and then kept on HFD and intraperitoneally (i.p.) injected with rapamycin (LC Laboratories) three times per week for 10 weeks. rapamycin was prepared in a solution of 100% ethanol, 10% PEG400, and 10% Tween80 and was injected at 4 mg/kg based on body weight.

For rapamycin treatment, rapamycin (LC Laboratories, R-5000) was dissolved in DMSO and mixed into the media when preparing food vials. The rapamycin dose was 50 µM.