Free glycerol reagent

FFA2-deficient (FFA2^−/−) mice were obtained from Deltagen (San Mateo, CA). Genotypes of wild-type and FFA2^−/− mice were identified by PCR analysis of genomic DNA from tail biopsies. Epididymal adipose tissues from male mice were harvested, cut into ∼15-mg sections and maintained in 96-well cell culture plates containing Krebs–Ringer buffer, 25 mmol/L glucose and 1% BSA for up to 1 h. For lipolysis assay, explants were transferred to fresh 96-well cell culture plates containing compounds, isoproterenol or insulin in Krebs–Ringer buffer supplemented with 25 mmol/L glucose and 1% BSA for 1 h at 37°C. Glycerol released into the supernatant during treatment was measured by free glycerol reagent (Randox, Crumlin, UK). All animal procedures were ethically reviewed and carried out in accordance with European Directive 86/609/EEC and the GSK Policy on the Care, Welfare and Treatment of Animals.

Primary human subcutaneous preadipocytes (Zenbio, RTP, NC; sex unknown) were cultured and differentiated into adipocytes as per vendor instructions. Differentiated adipocytes were preincubated in Krebs–Ringer buffer (123 mmol/L NaCl, 5.4 mmol/L KCl, 20 mmol/L NaHCO₃, 1 mmol/L NaH₂PO₄, 1 mmol/L CaCl₂, 0.8 mmol/L MgCl₂) supplemented with 25 mmol/L glucose and 0.5% (v/v) fatty-acid free BSA (Sigma Chemical Co., St. Louis, MO) for 2 h. Cells were then treated with compounds, pertussis toxin (PTX, 100 ng/mL), isoproterenol or insulin (both 200 nmol/L) in Krebs–Ringer buffer for 4 h. Glycerol released into the supernatant during treatment was measured by free glycerol reagent (Randox). Preadipocytes were sourced ethically and their research use was in accord with the terms of the informed consents.