Pyruvate

Blood samples and physiological test samples were collected after an overnight fast (16 hours). For glucose tolerance tests (GTT) and insulin tolerance tests (ITT), mice were fasted then glucose (2g/kg body weight, Sigma-Aldrich, St. Louis, USA) or insulin (0.25U/kg Actrapid, Novo Nordisk, Sydney Australia) was given by intraperitoneal injection (IP). A tail nick was made and glucose levels were measured at the time-points shown using an Optium glucometer (Abbot Diabetes Care, Doncaster, Australia). For the pyruvate challenge test (PCT), mice were fasted and 2g/kg of pyruvate (Sigma-Aldrich, St. Louis, USA) dissolved in phosphate-buffered saline was given IP. Mice were sacrificed after an overnight fast unless otherwise specified, at least 1 week after the last physiological test. Livers were divided for formalin fixation or snap-freezing in liquid nitrogen for gene expression and lipid studies with the same lobe for each use, every time.

For the insulin and glucose tolerance tests, mice were fasted for 6 hours and insulin Humalog (i.p. 0.75 or 0.5 U/kg body weight, Lilly, Indianapolis, Indiana) or glucose (2 g/kg body weight, Sigma-Aldrich, St. Louis, Missouri) was given, respectively. For the pyruvate tolerance test, mice were fasted for 15 hours and pyruvate (2 g/kg body weight, Sigma-Aldrich, St. Louis, Missouri) was administered. Blood glucose was measured with a blood glucose meter (Accu-Check, Roche Diabetes Care, Indiana polis, Indiana).

After 6‐hr fast, WT and G609G mice (n = 8) were injected intraperitoneally with glucose (2 g/kg of fat free mass), insulin (0.75 U/kg of fat free mass), or pyruvate (2 g/kg of fat free mass; Sigma‐Aldrich, St Louis MO, USA) to perform glucose tolerance test (GTT), insulin tolerance test (ITT) or pyruvate tolerance test (PTT) respectively. Blood was collected by tail snip and glucose was measure before the injection (t = 0 min) and 15, 30, 60, and 120 min after injection using OneTouch Ultra®2 glucometer.

The glucose tolerance tests were performed on 11-week-old males as previously described^{83 (link)}. Briefly, after 6 h of fasting, mice (N ≥ 8) were weighed and given 2 g/kg of glucose (Sigma-Aldrich, St. Louis, MO) through a gavage needle (18 G, 5.08 cm; Cadence Science, Cranston, RI). A drop of blood was sampled from each mouse at 0, 15, 30, 60, 90, and 120 min after oral gavage and the glucose concentration was determined immediately using the ACCU-CHEK Nano SmartView glucometer (Roche Diagnostics, Indianapolis, IN). For pyruvate tolerance testing, mice were fasted overnight, followed by intraperitoneal injection of pyruvate (1.5 g/kg, Sigma-Aldrich, St. Louis, MO). The blood glucose levels were measured as above.

For glucose profile measurement, the blood glucose and insulin level were measured by collecting the blood samples from the tip of the tail using Accu-Chek glucometer (Roche Diagnostics, IN) as described (Wong et al., 2014 (link)). For the glucose tolerance test (GTT), insulin tolerance test (ITT), and pyruvate tolerance test (PTT), mice were fasted overnight prior to intraperitoneal injection of glucose (1 g/kg body weight [BW]; Sigma, St. Louis, MO), 0.75 U/kg BW insulin (Novolin R, Novo Nordisk, Bagsvaerd, Denmark), or 1 g/kg BW pyruvate (Sigma). Blood glucose levels were measured from the tip of tail vein at 15, 30, 60, 90, and 120 min after injection. For plasma and hepatic lipid level, serum levels of triglyceride (TG) and total cholesterol (CHO) were measured using commercial kit (BioSino, Biotechnology and Science Inc, China) according to the manufacturer’s instructions. Hepatic lipids were extracted using Folch methodology, and liver extract was dissolved in ethanol for TG and CHO measurement. Both serum and hepatic levels of free fatty acid (FFA) were measured using commercial kit (Solarbio, China) according to the manufacturer’s instructions. For liver function assay, the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured in serum using commercial kits (Stanbio) according to the manufacturer’s instructions.