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Following overnight fast or fed state (5-hr after food removal), [3H]glucose (0.05 µCi/min) will be intravenously infused for 2 hrs in awake mice to assess basal whole body glucose metabolism. Basal glucose uptake in individual organs will be measured using an intravenous injection of 2-[14C]deoxyglucose and taking blood samples at 5-min intervals for 30 min to determine plasma 2-[14C]deoxyglucose concentrations. At the end of experiment, mice will be anesthetized, and tissue samples will be taken for organ-specific levels of 2-[14C]deoxyglucose-6-phosphate. Basal whole body and organ-specific glucose metabolism will be calculated as described in the Hyperinsulinemic-euglycemic clamp study.
[14C(U)]palmitate (10 µCi) will be intravenously injected to assess basal lipid uptake into individual organs. Blood samples will be collected at 0, 0.5, 1, 2, 3, 4, and 5 min following the injection for the measurement of plasma [14C]palmitate concentrations. Additional blood samples will be taken at the end to measure plasma fatty acids levels. At the end of experiment, tissue samples will be taken to measure [14C]palmitate incorporation into organ-specific triglyceride. Briefly, tissues will be homogenized with chloroform/methanol, and lipid layer will be extracted using H2SO4. Organ-specific triglyceride levels will be measured using spectrophometry, and [14C]-labeled triglyceride will be measured using liquid scintillation
In vivo rates of protein synthesis in individual organs (heart, skeletal muscle, liver, adipose tissue, lung, and kidney) will be measured in awake mice by determining the amount of L-[3H]Phenylalanine incorporation into proteins. [3H]-L-phenylalanine (0.2 µCi/ml/µmol, 30 µCi/100 g/body wt; 1 ml/100 g/body wt) will be intravenously bolus injected into awake mice. At 15-min following the injection, the mouse will be anesthetized, and samples will be collected. Collected blood will be used for determination of phenylalanine concentrations and radioactivity. The phenylalanine concentrations will be measured by HPLC analysis of supernatants from trichloroacetic acid extracts of plasma.