Isolation of adipocytes and lipolysis assay in isolated adipocytes

Adipocytes were isolated as described previously [26], with few modifications. The fragments of mesenteric fat for adipocyte isolation were collected and washed in warmed (37 °C) Krebs–Ringer–Henseleit buffer containing 5.5 mM of glucose, 10 mg/ml of bovine serum albumin (fatty acid free), and 20 mM of Hepes/Na (pH 7.4) (all chemicals provided by Sigma Aldrich). The fragments of fat were cut into small pieces and incubated in Krebs–Ringer–Henseleit buffer enriched with 1 mg/ml collagenase (Clostridium histolyticum type II collagenase from Sigma) for 1 h at 37 °C, with gentle shaking. Then, the material was filtered through 180-µm nylon filters (Millipore), and the adipocytes that have been collected on the filters were rinsed three times with Krebs–Ringer–Henseleit buffer. Mesenteric adipocytes from each rat were divided into three 0.2-ml cell suspension aliquots in 1.8 mL of Krebs–Ringer–Henseleit buffer and subjected to lipolysis as described recently [25]. Lipolysis was stimulated with forskolin (final concentration of 10 µM) and dibutyryl-cAMP (final concentration of 0.2 mM).