Collagen 1 coated

Male Sprague-Dawley rats were obtained from Charles River Laboratories at 200–230 g (~6–7 wks old*). Hepatocytes were isolated by the Yale University Liver Center from overnight fasted rats (maintained on regular chow unless HFD is specified, in which case rats were treated with HFD for 3 days [see above]). Isolated hepatocytes were suspended and washed two times in recovery medium containing DMEM high glucose (20 mM) (Sigma, D5648) with 10% FBS, 1 nM insulin, 1 nM dexamethasone, and antibiotics (10,000 units/ml penicillin and 10 mg/ml streptomycin, Invitrogen). Cell count and viability were estimated by trypan blue exclusion. Primary hepatocytes were cultured under 5% CO₂ and 95% O₂ in air at 37°C. For glucose production assays, the cells were plated at 5 × 10⁵ cells/cm² in collagen-I-coated (BD Biosciences) 6-well plates in recovery medium. For all other experiments, cells were plated at 2.5 × 10⁶ cells/cm² in collagen-I-coated (BD Biosciences) 10cm dishes. After 4 h, cells were washed with PBS, and the medium was changed to DMEM low glucose (5 mM) (Sigma, D5648) with 10% FBS, 1 nM insulin, 1 nM dexamethasone, and antibiotics (10,000 units/ml penicillin and 10 mg/ml streptomycin, Invitrogen).

Primary hepatocytes from regular chow rats were isolated at the Yale Liver Center. Cells were washed three times with recovery media (DMEM with high glucose plus 10% FBS), and an equal number of cells were seeded in each well of a 6-well plate. Isolated hepatocytes were suspended and washed two times in recovery medium containing DMEM high glucose (20 mM) (Sigma, D5648) with 10% FBS, 1 nM insulin, 1 nM dexamethasone, and antibiotics (10,000 units/mL penicillin and 10 mg/mL streptomycin, Invitrogen). Cell count and viability were estimated by trypan blue exclusion. The cells were plated at 5 × 10⁵ cells/cm² in collagen-I-coated (BD Biosciences) 6-well plates in recovery medium and were cultured under 5% CO₂ and 95% O₂ in air at 37°C. After 4 h, cells were washed with PBS, and the medium was changed to DMEM low glucose (5 mm) (Sigma, D5921) with supplemented antibiotics but no hormones prior to gluconeogenesis assays. Hepatocytes were incubated in the presence and absence of substrate (Pyruvate 1mM, lactate 9 mM) and drug as indicated in DMEM (D5030, Sigma) with 2 mM L-glutamine and 24 mM bicarbonate without glucose. After 3 h the medium was removed for enzymatic analysis of glucose, background subtracted and normalized to the protein content. PK activity in the lysate was measured as described.