Glucose assay
Primary hepatocytes were isolated from WT C57BL/6 J mice. Glucose assay was made in glucose free DMEM with lactate (100 mM), pyruvate (1 mM) and pen/strep (1%). The stimulation of glucose production was with glucagon.Primary mouse hepatocytes were treated with metformin (0.25 mM-2.5 mM) or vehicle. After 24 h of incubation period, 500 μl of media was collected and glucose concentration was determined by GAGO assay modified to 96 well plate. For amino acid supplementation, glucose production medium contained DMEM glucose free (D5030; Merck), 3.75 sodium bicarbonate, supplemented with 2 mM Glutamine, 10 mM lactate and 1 mM sodium pyruvate with or without Glucagon (100 ng/ml), Metformin (0.25 mM), leucine (0.8, 7, 15 mM), cells were incubated for 12 h at 37 °C and 5% CO2, 500 μl of medium was collected and glucose concentration was determined using glucose assay kit (GHK20, Merck) as described by the provider.