To prepare adipocyte and SVF fractions, 10–15 g of fresh SAT or VAT was washed thoroughly with Hanks balanced salt solution (HBSS, Sigma), minced, and after clots and connective tissue was removed, subdivided into two 50 mL polypropylene conical tubes. Each tube contained 25 mL of HBSS supplemented with 40 mM HEPES (pH 7.2), 1.0% BSA (Fraction V, Bioworld), 2 mM CaCl2 and 0.1% collagenase (Type 1, Worthington, OH, USA). Tubes were gently agitated on an orbital shaker for 3 h at room temperature, centrifuged at 12 × g for 5 min, and the floating adipocyte layer and undigested fat was removed. This fraction was sent through a 500 µm steel mesh filter to remove undigested fat and washed once with HBSS. Approximately 0.1–0.2 mL of this fraction was treated with 0.9 mL of TRIzol. The SVF was centrifuged at 300 × g and then treated with 1.0 mM EDTA-HBSS in at 37 C for 15 min and centrifuged at 300 × g. Pelleted cells were resuspended in HBSS in 1.0 mM EDTA, sent through a 70 µm filter to remove large adipocytes, centrifuged, and solubilized with 1.0 mL of TRIzol. For both adipocytes and SVF, RNA was purified and cDNA prepared as described above.