Primary hepatocytes isolation and glucose output assay

Primary hepatocytes were isolated from adult F1 female and male mice using the modified two-step perfusion method [26]. Perfusion buffer (HBSS containing 500 μm ethylene glycol tetraacetic acid and 50 µg/mL heparin) and digestion buffer (HBSS containing 0.5 mg/mL collagenase and 0.04 mg/mL trypsin inhibitor) was successively perfused into the liver under anesthesia through a catheter in the inferior vena cava. Cells were separated from liver tissue using cell scrapers and passed through a 70 μm cell strainer. Isolated cells were washed in Dulbecco′s Modified Eagle′s Medium (DMEM) and purified in the mixture of DMEM and 45% Percoll (Amersham Biosciences AB, Piscataway, NJ) using the density-gradient centrifugation method. Primary hepatocytes were seeded in collagen-coated plates with DMEM containing 10% FBS and 1% penicillin-streptomycin for about 4 h to allow attachment. For glucose output assay, primary hepatocytes were starved in glucose output media (4.7 mM KCl, 118 mM NaCl, 1.2 mM CaCl₂, 1.2 mM MgSO₄, 20 mM NaCO₃,1.2 mM KH₂PO₄, 20 mM HEPES, and pH 7.4) for 1 h immediately after attachment, followed by treatment with substrates containing 1 mM pyruvate and 10 mM lactate for 2 h. Glucose release into the output media was measured using the Hexokinase Liquid Reagents Kit (Fisher Scientific G7517120) and was normalized to the total protein content of the cell lysates.