4.4. Lipolysis Assay

Freshly isolated adipocytes were incubated in plastic vials in a total volume of 300 μL of buffer A containing 0.05 mg/mL adenosine deaminase (Sigma, St. Louis, MO, USA). After 2 min of pre-incubation, adipocytes were incubated for 30 min with or without 50 μM isoproterenol to investigate their lipolytic responses. Then, the cell-free incubation medium was removed and assayed for glycerol release as an index of lipolysis. Glycerol release was determined using an Adipolysis Assay Kit (Cayman, Ann Arbor, MI, USA) according to the manufacturer’s instruction. Lipolysis was expressed as nmol of glycerol/mg of total protein/hour, because we previously confirmed that the same protocol of exercise training as that used in this study did not affect the protein contents per unit of cells (g/10⁵ cells) [10]. The protein in adipocytes was extracted as follows: Adipocytes were washed twice with phosphate-buffered saline and homogenized in 20 mM HEPES pH 7.5, 1% NP-40, 0.1% sodium dodecyl sulphate (SDS), 0.5% deoxycholic acid, and 150 mM sodium chloride, supplemented with protease and phosphatase inhibitors (ATTO, Tokyo, Japan). Protein was measured using a commercially available kit (BCA protein assay kit, Funakoshi, Tokyo, Japan).