Sodium oxamate

EGCG, sodium oxamate and unlabelled glucose were purchased from Sigma-Aldrich (St. Louis, MO). Stable [1, 2- ¹³C₂]-d-glucose isotopes were purchased from Isotec, Inc. (Miamisburg, OH) with 99% purity and 99% isotope enrichment for each position. Recovery standards [U-¹³C₆]-glucose and [U-¹³C₃]-lactate were purchased from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA). Isotope incubation and treatment were performed as described previously (Harris et al., 2012 ).

U-¹³C-glutamine (CLM-1822), U-¹³C-Glucose (CLM-1396-5), U-¹³C-Pyruvate (CLM-2440) and α-¹⁵N-glutamine (NLM-1016) were from Cambridge Isotopes Laboratories; ¹³C-bicarbonate (372382), L-aspartate (A8949) and Sodium Oxamate (O2751) and all remaining reagents were obtained from Sigma-Aldrich.

80–200 μL of blood was collected via tail vein of C57BL/6 WT and Mrs2 KO mice and placed into K₂ EDTA-coated tubes. Following collection, blood was spun at 10,000×g for 5 min at 4°C and plasma was removed from the top layer. Using a 1:3 dilution with DNAse/RNAse-free distilled water, glucose and lactate measurements were taken using a YSI 2900 Series Biochemistry Analyzer per the manufacturer’s instructions and reported as mg/dL.
To measure extracellular lactate, hepatocytes from WT and Mrs2 KO mice were plated in collagen-coated 6-well plates (BioCoat, Corning) at a density of 1.2 × 10⁶ cells per dish in hepatocyte culture media. Cells were then treated with 100 μg/mL LPS and 20 mM sodium oxamate (Sigma-Aldrich), alone or in combination. 150 μL of media from the cells was collected at the following time points: 0 hours (2 hours post-plating), 8 hours, and 24 hours after the addition of compounds. Extracellular lactate production was then measured by subjecting collected media samples to analysis using a YSI 2900 Series Biochemistry Analyzer per the manufacturer’s instructions and results reported as mg/dL.