Abstract: Objective To establish a ¹³C isotope-labeled hyperinsulinemic-euglycemic clamp model for the assessment of insulin sensitivity in mice. Methods The mouse model of insulin resistance was established by high-fat diet feeding. The phosphorylation level of downstream insulin signaling protein, Protein Kinase, also known as Akt was assessed. Glucose metabolism was evaluated using glucose tolerance test, insulin tolerance test, and pyruvate tolerance tests and the area under the curve of the respective testes over 2 hours was calculated to quantify overall blood glucose level. Mice underwent jugular vein catheterization surgery, and a ¹³C isotope-labeled hyperinsulinemic-euglycemic clamp experiment was conducted to monitor blood glucose levels and to calculate the glucose infusion rate(GIR).Tail vein serum was collected for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to determine glucose disposal rate(GDR) and hepatic gluconeogenesis rate(HGP). Following the inhibition of glycogenolysis in mice, Akt phosphorylation level was measured to evaluate insulin signaling. The clamp test was repeated to calculate GIR, and tail vein blood serum was analyzed by LC-MS/MS to determine GDR and HGP. Results After one week of high-fat diet feeding, mice exhibited significantly elevated blood glucose level (P＜0.001) accompanied by reduced p-Akt level in liver and muscle. Glucose tolerance tests, insulin tolerance tests and pyruvate tolerance test demonstrated a significant increase in blood glucose level (P＜0.05) and a higher area under curves (AUC) (P＜0.001) in high-fat-fed mice. During the ¹³C-labeled hyperinsulinemic-euglycemic clamp experiment, after the blood glucose levels were stable, the GIR of high-fat-fed mice was significantly reduced (P＜0.001), GDR was decreased (P＜0.0001)and hepatic gluconeogenesis rate was increased(P＜0.01). After pharmacological inhibition of glycogenolysis, mice showed elevated blood glucose level(P＜0.001) and further reductions in p-Akt level in liver and muscle. The ¹³C-labeled clamp experiment revealed that in the treated group, the GIR decreased(P＜0.01) while GDR was reduced (P＜0.000 1)and HGP increased (P＜0.01). Conclusions An improved hyperinsulinemic-euglycemic clamp model was developed to assess insulin sensitivity in mice.

Key words: insulin resistance, glucose clamp experiment, jugular vein catheterization surgery, D-glucose-¹³C6