To isolate primary hepatocytes, mice were infused through the inferior vena cava with a calcium-free Hepes-phosphate buffer (pH 7.4) for 10 min followed by a collagenase solution (Liberase TM, Roche) for 10 min. The digested livers were excised, and hepatocytes were collected and washed five times in buffer by centrifuging at 70g for 5 min. Cells were further purified by centrifugation (2400g for 10 min) over a Percoll density gradient (1.06 g/ml). Primary mouse hepatocytes were allowed to attach for 6 hours on collagen-coated plates in Williams’ Medium E (Life Technologies, catalog no. 12551-032) fortified with nonessential amino acids, GlutaMAX (Life Technologies, catalog no. 35050-061), antibiotics, 10% fetal bovine serum, and dexamethasone (10 nM) and cultured overnight in the same medium without serum. Cultures were then washed in Hepes phosphate-salt-bicarbonate (HPSB) buffer (10 mM Hepes, 4 mM KCl, 125 mM NaCl, 0.85 mM KH2PO4, 1.25 mM Na2HPO4, 1 mM MgCl2, 1 mM CaCl2, and 15 mM NaHCO3) containing 0.2% FFA-free bovine serum albumin (BSA) and incubated in the same buffer containing GLP-1 (100 nM), Exendin-4 (30 nM), insulin (10 nM), and/or glucagon (10 ng/ml) and substrates for 3 hours in a 5% CO2 incubator. 14C-pyruvate (2 mM, 0.5 μCi pyruvate per incubation) was used as substrate. Incubations were carried out in 0.5-ml buffer in 24-well plates containing 0.25 million cells per well. At the end of incubation, the buffer solutions were transferred to 1.7-ml microcentrifuge tubes and added with 0.25 ml of 5% ZnSO4 and 0.25 ml of 0.3 N Ba(OH)2 suspensions to each tube, followed by addition of 0.5 ml of water. After centrifugation, supernatants were transferred to a fresh set of tubes and assayed for radiolabeled glucose released into the medium by separation of radiolabeled glucose by mixed-bed ion exchange resins, AG 501-X8 resins (Bio-Rad). Two hundred milligrams of resins was added to each tube, vortexed intermittently for 15 min, and centrifuged, and the supernatants were transferred to scintillation vials for counting radioactivity. Cells on the plates were dissolved in 1 N NaOH for protein estimation.

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