13C-glucose isotope metabolic tracing analysis

After a 6-h fast, mice underwent a glucose tolerance test in which 10% of [U-¹³C₆]-glucose (99% CLM-1396 D-Glc-U-¹³C₆ from Cambridge Isotope) was administered via intraperitoneal injection. Tissues were collected at 60 min following glucose administration and rapidly frozen in liquid nitrogen. Glucose (Sunny and Bequette 2010), organic, and amino acid (Sobolevsky et al. 2003) ¹³C-mass isotopomer enrichments were determined as described previously. M+3 enrichments of tissue lactate and pyruvate and total mass enrichment of TCA cycle organic acids were used as semiquantitative indicators of glucose oxidation.