Test of Counter-regulatory Mechanisms Against Hypoglycemia
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 clamp starts at t = 0 with a continuous infusion of human insulin (Novo Nordisk) at 16 or 20 mU/kg/min. Hypoglycemia (50~60 mg/dL) is achieved and 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 are collected at t = 0, 30, and 120 min for determination of changes in counter-regulatory hormones and/or metabolites.
A continuous infusion of erythrocytes obtained from donor mice via cardiac puncture is given at 4 μl/min throughout the experiment period to prevent anemia from the repeated blood sampling.