Hepatocyte Isolation and Glucose Output Assay

Mouse hepatocytes were isolated from chow-fed wild-type mice 4 days after tail vein injection of Rec2-Ad36E4ORF1 (2 × 10¹⁰ vg/mouse) or AAV buffer as a control according to the published method with modifications (17). Briefly, mice were anesthetized and perfused at a rate of 3 mL/min by cannulation through the inferior vena cava with perfusion buffer (115 mmol/L NaCl, 5 mmol/L KCl, 25 mmol/L HEPES, 0.5 mmol/L EGTA, and 25 mmol/L d-glucose, pH 7.4) followed by Liver Digestion Medium (Thermo Fisher Scientific). The portal vein was cut to allow the flow of the solution through the liver. The perfusion temperature was kept at 37°C. The liver was dissected, and the capsule peeled off. Hepatocytes were dispersed by mechanical dissociation and filtered through a 100-μm strainer. The hepatocytes were rinsed twice in ice-cold DMEM supplemented with 10% FBS, penicillin/streptomycin, 10 nmol/L insulin, and 100 nmol/L dexamethasone; resuspended in the same culture medium; and then seeded in a collagen-coated six-well plate. The cell culture was continued for 24 h before starved overnight in serum-free DMEM without insulin and dexamethasone. The glucose output was measured in glucose- and phenol red–free DMEM supplemented with 20 mmol/L sodium lactate and 2 mmol/L sodium pyruvate. After a 5-h incubation, the supernatant medium was collected and briefly spun. The glucose concentration in the medium was determined by colorimetric glucose assay (Eton Bioscience, San Diego, CA). The glucose readout was normalized to the total protein content from each well cell lysate.