Hyperinsulinemic-euglycemic clamp + 2 tracers (3H-glucose + 14C-2DG)

Test Number

MI2017

Test Description

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.

Price

$447.20/per mouse

Service Provided By