Adipocyte Differentiation and Inflammation

Murine 3T3-L1 pre-adipocytes, [32 (link)] were grown, maintained, and induced, in order to differentiate using a standard protocol [33 (link)]. Fully differentiated adipocytes were maintained in DMEM (Sigma-Aldrich, St Louis, MO, USA) supplemented with 10% FBS (Sigma-Aldrich) until two days before experimentation, when cells were fed with 10% calf serum (Sigma-Aldrich). Prior to treatments, media was changed to low-glucose (5.5 mM) DMEM (Sigma-Aldrich) and 1% calf serum overnight. Cells were treated with 5 nM TNF-α (R&D Systems) for 8 h (TNF), 30 min with 10 nM insulin either alone (Ins) or followed by TNF treatment (Ins + TNF).