Hyperinsulinemic-Euglycemic Clamps in Mice

Hyperinsulinemic–euglyclemic clamps were carried in age-matched conscious unrestrained catheterized mice as described^{84 (link),85 (link)}. To this end, catheters were surgically implanted 7 days prior to the experiment in the right jugular vein and exteriorized above the neck using a vascular access button (Instech Laboratories Inc., Plymouth Meeting, PA). Mice were fasted for 3 h, followed by an infusion for 2 h of [3-³H] glucose (0.05 μCi min⁻¹; Cat. #NET331, PerkinElmer). Continuous insulin infusion (1.5 mIU kg⁻¹ body weight min⁻¹, Umuline, Lilly France) was used for the induction of hyperinsulinemia. Upon reaching steady state, the insulin-stimulated glucose uptake in tissues was determined in vivo using a 10 μCi bolus injection of 2-[¹⁴C] deoxyglucose (Cat. #NEC720A250UC, PerkinElmer). After 30 min, mice were rapidly killed by cervical dislocation and tissues were removed and stored at –80 °C until use.
Glucose concentration was measured using the glucose oxidase method (GLU, Roche Diagnostics) and insulin using an ELISA commercial kit (Cat. #90080, CrystalChem Inc.). Measurements of 2-[¹⁴C] deoxyglucose-6-phosphate concentration in individual tissues allowed calculation of the glucose utilization index in tissues. After 30 min, tissues were removed and stored at −80 °C as above.