Free fatty acid determination kit

eWAT, iWAT and iBAT fat pads were isolated, weighed (about 50 mg for WAT, about 20 mg for BAT), cut into small pieces, and incubated in 600 μl of the lipolysis medium (DMEM (GIBCO 21063029), 2% BSA) with or without 10 μM isoproterenol (Sigma-Aldrich) for 3 h at 37 °C. After that, the medium was collected, and glycerol or NEFA concentration was measured using a free glycerol reagent (Sigma-Aldrich) or a Free Fatty Acid Determination Kit (Wako), according to the manufacturer’s instructions, respectively. The content of Glycerol or NEFA was normalised to the initial tissue weight.

Body weight and food intake were measured every week. Whole-body O₂ consumption and CO₂ production were monitored using an O₂/CO₂ metabolism measuring system for small animals (MK-5000RQ, Muromachi Kikai). In GTT experiments, 17-week-old mice were injected intraperitoneally with glucose (1 g per kg body weight) after a 4-h fast. In ITT experiments, 19-week-old mice were injected intraperitoneally with insulin (0.75 U per kg body weight) after a 4-h fast. Blood glucose levels were measured at the indicated time points using Glutest Neo alpha (Sanwa Kagaku). Unless otherwise specified, blood samples were collected under 4-h-fasted conditions. The following kits were used to determine metabolic parameters: Triglyceride Determination Kit (Wako), Total Cholesterol Kit (Wako), Free Fatty Acid Determination Kit (Wako), Insulin ELISA kit (Morinaga), Leptin ELISA kit (Morinaga) and Adiponectin ELISA kit (R&D Systems).