Cardiac and Muscle Glucose Metabolism

The LV of the heart, quadriceps skeletal muscle and epididymal adipose tissue (15-25 mg) were used to determine 2-²H-deoxyglucose clearance as we have previously described^{73 (link)}. To determine 1-¹⁴C-glucose incorporation into glycogen, ~10-15 mg of LV tissue was digested in 1 M KOH at 70 °C for 20 min and glycogen was precipitated with saturated Na₂SO₄, washed twice with 95% ethanol and resuspended in acetate buffer (0.84% sodium acetate, 0.46% acetic acid, pH 4.75) containing 0.3 mg/mL amyloglucosidase (Sigma). Glycogen was allowed to digest overnight at 37 °C before being assayed for glucose content using the glucose oxidase method^{76 (link)}. Digested glycogen was also assessed for ¹⁴C-glucose incorporation by scintillation counting. To determine 1-¹⁴C glucose incorporation into lipids, 5–10 mg of LV tissue was homogenised in chloroform/methanol (2:1) and mixed overnight at room temperature. Organic and inorganic phases were separated by addition of 0.6% NaCl and the lower organic phase was collected and evaporated under N₂ at 45 °C. The dried extract was resuspended in absolute ethanol and TG content was assayed using TG GPO-PAP reagent (Roche) and ¹⁴C-glucose incorporation by scintillation counting.