3T3-L1 cell culture and differentiation

To induce adipocyte differentiation, the 3T3-L1 cells were cultures in 6-well plates at 3×10⁵ cells/well to confluence. After 2 days, the cells were treated with a differentiation mixture containing 1 µM dexamethasone, 5 mM 3-isobutyl-1-methylxanthine and 1 µg/ml insulin (Sigma-Aldrich; Merck Millipore) in DMEM with 10% FBS (MDI) to induce the preadipocytes to differentiate. After 2 days, the medium was replaced with DMEM with 10% FBS and 1 µM insulin. Cultures were incubated for 2 days, after which the culture medium was replaced again with DMEM (10% FBS) and repeated at 2 day intervals until day 7. SB203580, a p38 mitogen-activated protein kinase (MAPK) inhibitor (Cell Signaling Technologies, Inc., Danvers, MA, USA) was used as the positive control. The triglyceride (TG; BioAssay Systems, Hayward, CA, USA; cat. no. ETGA-200) was detected by colorimetric method in the cell lysates at 570 nm using a microplate reader (Benchmark Plus; Bio-Rad Laboratories, Inc.). The leptin (R&D Systems, Inc.; cat. no. MOB00) concentrations were measured by ELISA in supernatant at 450 nm using a microplate reader.