Test of Insulin Resistance (i.e. whole body glucose utilization, hepatic glucose production and glucose uptake in peripheral tissues, etc.)
Mice are surgically implanted with dual carotid and jugular catheters 5-7 days before the clamp and subjected to the clamp procedures under conscious and unrestrained conditions after fasted for 5-6 hours .
The protocol consists of a 90min tracer equilibration period (t = -90 to 0 min), followed by a 120min experimental period (t = 0 to 120 min). At t = -90, a bolus infusion of 1.0 μCi (20 μCi for rat) of [3-3H]glucose (HPLC purified; PerkinElmer) is given, followed by a 0.05 μCi/min (0.2 μCi/min for rat) infusion for 90 minutes. At t = -10min, a blood sample (~100 μl) is taken for assessment of basal levels of insulin and glucose turnover rate. The insulin clamp begins at t = 0 with a prime-continuous infusion (16 mU/kg bolus, followed by 4.0 mU/kg/min or 24 pmol/kg/min) of human insulin (Novo Nordisk). The infusion of [3-3H]glucose is increased to 0.10 μCi/min (0.4 μCi/min) for the rest of the experiment to minimize changes of specific activity from the equilibration period.
Euglycemia (120~130 mg/dL) is maintained during the clamp by measuring blood glucose every 10 min starting at t = 0 and infusing 50% glucose at variable rates accordingly. Blood samples (40~50 μl) are collected during a steady-state of glucose infusion at t = 80, 85, 90, 100, 110 and 120 min for determination of glucose specific activity. Blood insulin concentrations are determined from samples taken at t = -10 and 120 min. For mouse study, a continuous infusion of erythrocytes obtained from donor mice via cardiac puncture is given at 4 ul/min throughout the experiment period to prevent anemia from the repeated blood sampling.
To estimate insulin-stimulated glucose uptake in individual tissues, a bolus injection of [1-14C]-2-deoxyglucose ([14C]2DG; PerkinElmer) (10 μCi for mouse and 30 μCi for rat) is given at t = 78 min while continuously maintaining the hyperinsulinemic-euglycemic steady-state. [14C]2DG is a nonmetabolizable glucose analogue and after transported into cells it is phosphorylated to [14C]2DG -6-phospate ([14C]2DGP), which is trapped in tissues. Blood samples are taken at 2, 7, 12, 22, 32, and 42 min after the injection (or at t = 80, 85, 90, 100, 110 and 120 min as stated above) for determination of plasma [14C]2DG radioactivity . At the end of the experiment, animals are anesthetized with an intravenous infusion of sodium pentobarbital and tissues are collected and immediately frozen in liquid nitrogen for later analysis of tissue [14C]2DGP radioactivity.