Triglyceride determination kit

Cells were harvested in 200 μl lysis buffer (25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1% Triton X-100). An aliquot of the lysate was mixed with the same amount of Folch solution (2:1 v/v chloroform/methanol). The lower phase was collected, and TG content determined, using a Triglyceride Determination Kit (Wako). Another aliquot of the lysate was used for protein determination by BCA assay (Nacalai Tesque).

Liver samples (50 mg) were homogenised in 1 ml Folch solution (2:1 v/v chloroform/methanol) using a Precellys Evolution tissue homogeniser (Bertin). Homogenates were added with 200 μl 0.9% NaCl solution. The lower phase was collected, and TG content determined using the Triglyceride Determination Kit (Wako).

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).