2.7. Lipolysis assay

For the in vitro lipolysis assay, 15 mg of adipose tissue was collected and cultured in 100 μl of lipolysis buffer (Krebs buffer plus 0.1% glucose and 3.5% fatty-acid-free BSA) in a 96-well plate for 2 h at 37 °C, and then glycerol or FFAs were measured. 1-h glycerol release was examined for the indicated time in freshly changed medium. Plasma FFAs and glycerol were measured in mice after 24 h of fasting. FFAs were quantified by a NEFA assay (Wako Diagnostics, Japan), and glycerol was quantified by an enzyme-coupled colorimetric assay (GPO Trinder reaction) with a colorimetric assay kit (Applygen Technologies, Beijing, China).