Hyperinsulinemic-Euglycemic Clamp in Mice

Clamps were performed according to recent recommendations of the Mouse Metabolic Phenotyping Center Consortium (15 (link)). After surgical implantation of an indwelling catheter in the right jugular vein, the mice were allowed to recover for 1 week prior to clamp experiments. Following an overnight 14-h fast, the mice were infused with 3-[³H]glucose at a rate of 0.05 μCi/min for 120 min to determine basal glucose turnover. Next, a primed infusion of insulin and 3-[³H]glucose was administered at 7.14 milliunits·kg⁻¹·min⁻¹ and 0.24 μCi/min, respectively, for 4 min, after which the rates were reduced to 3 milliunits·kg⁻¹·min⁻¹ insulin and 0.1 μCi/min 3-[³H]glucose for the remainder of the experiment. Blood was collected via tail massage for plasma glucose, insulin, and tracer levels at set time points during the 140-min infusion, and a variable infusion of 20% dextrose was given to maintain euglycemia. Glucose turnover was calculated as the ratio of the 3-[³H]glucose infusion rate to the specific activity of plasma glucose at the end of the basal infusion and during the last 40 min of the hyperinsulinemic-euglycemic clamp study. Hepatic glucose production represents the difference between the glucose infusion rate and the rate of glucose appearance. A 10-μCi bolus injection of [¹⁴C]2-deoxyglucose was given at 90 min to determine tissue-specific glucose uptake, which was calculated from the area under the curve of [¹⁴C]2-deoxyglucose detected in plasma and the tissue content of [¹⁴C]2-deoxyglucose-6-phosphate, as previously described (16 (link)). Following collection of the final blood sample, the mice were anesthetized with an intravenous injection of 150 mg/kg pentobarbital, and tissues were harvested and froze with aluminum forceps in liquid nitrogen. All of the tissues were stored at −80 °C until later use.