In vivo and ex vivo lipolysis assay

To assess in vivo lipolysis, serum free fatty acids (FFAs) were measured in cardiac blood obtained from non-fasted mice before and 20 min after i.p. injection of CL316243 (1 mg/kg). Ex vivo and in vitro lipolysis was measured as described previously36 (link)50 (link) with some modifications. Briefly, for ex vivo analysis, adipose tissues were surgically removed from male and female mice, weighed, and washed with cold PBS; fat depots were incubated with CL316243 (1 μM) or vehicle in Krebs-Ringer Bicarbonate Buffer (KRBH, Sigma) containing 1% fatty acid-free BSA (GenDEPOT) and glucose (2.5 mM) for 2 h at 37 °C with mild shaking. After incubation, FFA and glycerol were measured using a glycerol-free reagent (Sigma) and the FFA quantification kit (BioVision, Mountain View, CA, USA) and normalized to the weight of adipose tissue samples.