Hyperinsulinemic-euglycemic clamp to assess insulin sensitivity in conscious mice
Mouse preparation and basal glucose metabolism
Following overnight fast, a mouse will be placed in a rat-size restrainer with its tail tape-tethered at one end, and blood samples will be obtained from the tail vessels during the experiments. D-[3-3H]glucose (0.05 µCi/min) will be infused using microdialysis pumps for 2 hours in awake mice to assess the basal rate of whole body glucose turnover. A blood sample will be collected at the end for the measurement of plasma glucose, insulin, and [3H]glucose concentrations (basal parameters).
Following the basal period, a 2-hr hyperinsulinemic-euglycemic clamp will be conducted with a primed (150 mU/kg body weight) and continuous infusion of human insulin at a rate of 15 pmol/kg/min to raise plasma insulin within a physiological range. Blood samples will be collected at 10~20 min intervals for the immediate measurement of plasma glucose, and 20% glucose will be infused at variable rates to maintain basal glucose levels. Insulin-stimulated whole body glucose metabolism will be estimated with a continuous infusion of [3H]glucose throughout the clamps (0.1 µCi/min). To estimate insulin-stimulated glucose uptake in individual organs, 2-[1-14C]deoxy-D-glucose (2-[14C]DG) will be administered as a bolus (10 µCi) at 75 min after the start of clamp. Blood samples will be taken at 80, 85, 90, 100, 110, and 120 min of clamp for the measurement of plasma [3H]glucose, 3H2O, and 2-[14C]DG concentrations. Additional blood sample will be taken at 120 min to measure plasma insulin concentrations (clamp parameters). At the end of clamp, mice will be anesthetized, and tissue samples will be taken for biochemical and molecular analyses. Samples may be transferred to the In Vitro Metabolism and Clinical Chemistry Core for clinical chemistry analysis (e.g., insulin, hormones) and in vitro measurement of glucose/lipid/protein metabolism in isolated organs.
Glucose concentrations during clamps will be analyzed using clinical glucose analyzer, and insulin levels will be measured by ELISA. Plasma [3H]glucose, 2-[14C]DG, and 3H2O concentrations will be determined following deproteinization of samples and using liquid scintillation counter on dual channels for separation of 3H and 14C. The radioactivity of 3H in tissue glycogen will be determined by precipitating glycogen with ethanol and counting for 3H-labeled glycogen in individual organs. Organ-specific 2-[14C]DG-6-phosphate concentrations will be determined using ion-exchange column. Intracellular triglyceride level will be measured by digesting tissue samples in chloroform-methanol and performing spectrophotometry using triglyceride assay kit.
Calculation of in vivo glucose metabolism
Basal whole body glucose turnover will be determined as the ratio of the [3H]glucose infusion rate to the specific activity of plasma glucose at the end of basal period. Insulin-stimulated whole body glucose uptake will be determined as the ratio of the [3H]glucose infusion rate to the specific activity of plasma glucose during the final 30 min of clamps. Hepatic glucose production during insulin-stimulated state (clamp) will be determined by subtracting the glucose infusion rate from the whole body glucose uptake. Whole body glycolysis will be calculated from the rate of increase in plasma 3H2O concentration from 90~120 min of clamps. Whole body glycogen plus lipid synthesis will be estimated by subtracting whole body glycolysis from whole body glucose uptake. Since 2-deoxyglucose is a non-metabolizable glucose analog, insulin-stimulated glucose uptake in individual organs will be estimated by determining the organ (i.e., skeletal muscle, adipose tissue, heart)-specific content of 2-[14C]DG-6-P. To this end, glucose uptake in individual organs will be calculated from plasma 2-[14C]DG decay profile and intracellular 2-[14C]DG-6-P content. Skeletal muscle glycogen synthesis will be calculated from 3H incorporation into muscle glycogen. Skeletal muscle glycolysis will then be estimated as the difference between muscle glucose uptake and glycogen synthesis rates.