Establishment Of Methods In Insulin Sensitivity Assessment And Their Application In Drug Research

【Objective】Assessing insulin sensitivity accurately is of great importance for studying the mechanism of glucose metabolism regulation, and development of new drugs that can improve insulin sensitivity. Fistly, this study aims at establishing effective, reliable methods of assessing insulin sensitivity in rats and mice by comparing the impact of different experimental conditions; Secondly, through makeing use of those technologies, this stydy is to assess the insulin sensitivity of AMPKα2 knockout animal models and evaluate the effect of new drug treatment of PNU-282987, a selective agonist ofα7 nAChR, on AMPKα2 knockout mice.【Methods】Part I: Establishment of methods in evaluating insulin sensitivity in mice and rat. First, we established the gold standard of assessing insulin sensitivity hyperinsulinemic-euglycemic clamp in anesthetized rats, conscious rats and anesthetized mice through exploring the best dose of sodium pentobarbital to anesthetize animals, finding the proper position for cannulation, catheter length and insertion depth, the liquid filled in catheters, catheter fixation after surgery in conscious animal methods, the effect of anesthesia and recovery time after surgery on glucose infusion rate. Finally, we further improved insulin tolerance test (ITT), oral glucose tolerance test (OGTT), homeostasis model assessment (HOMA) and other methods which had been initially established already in our laboratory and wrote standard operating procedures (SOP) of each method.PartⅡ: Applying those methods of assessing insulin sensitivity in drug research. First, we evaluated insulin sensitivity of AMP-activated kinase-α2 (AMPKα2) knockout mice and its wild type (WT) through insulin tolerance test, glucose tolerance test, HOMA, and hyperinsulinemic-euglycemic clamp. Next we further detected whether long term treatment of selectiveα7nAChR agonist could improve insulin sensitivity of AMPKα2 mice through those established methods. Tests were designed as follows: AMPKα2 mice were divided into two groups: One group of mice were treated with Saline (AMPK Sal), and the other were treated with PNU-282987 (AMPK PNU). After 6 weeks of treatment with PNU-282987 (0.53mg/kg) and saline, the insulin sensitivity was assessed with insulin tolerance test, glucose tolerance test, HOMA-IR value.【Results】PartⅠ: Establishment of methods of assessing insulin sensitivity.1. Hyperinsulinemic-euglycemic clamp was successfully established in anesthetized Sprague-Dawley rat (SD), conscious SD rats, anesthetized C57BL/6J mice (C57). Glucose infusion rate clamp from anesthetized SD rats was 26.9±2.5mg/(kg?min), conscious SD rats was 29.4±1.0mg/(kg?min), anesthetized AMPK knockout mice was 59±6.6 mg/(kg?min).2. Standard operating procedures (SOP) of those methods that assessing insulin sensitivity, including insulin tolerance test, glucose tolerance test and hyperinsulinemic-euglycemic clamp were written down.PartⅡ:1. The systemic insulin sensitivity characteristics of AMPKα2 knockout mice.(1) HOMA: The HOMA-IR value of Wild-type group was 3.37; AMPK knockout HOMA value was 1.50. There was no significant difference between them.(2) Insulin tolerance test: After intraperitoneal injection of insulin, compared with wild type group, the blood glucose at 30 minute and 45 minute of AMPK knockout group were significantly increased.(3) Intraperitoneal glucose tolerance test: Compared with wild-type group, blood glucose of AMPKα2 knockout group at 60 minute and 90 minute after glucose injection were significantly increased;(4) Hyperinsulinemic-euglycemic clamp test: The GIR of AMPKα2 mice was 55.8±5.4mg/(kg·min), and the GIR of wild type was 82.6±9.2 mg/(kg·min), with significant difference between them.2. The effect of PNU-282987 on insulin sensitivity of AMPKα2 knockout mice after 6 weeks intraperitoneal injection (0.53mg/kg).(1) HOMA: PNU-282987 could significantly improve HOMA-IR of AMPKα2 knockout mice.(2) Insulin tolerance test: Compared with AMPKα2 knockout saline group, blood glucose at 15, 30 and 45 minute after intraperitoneal injection of insulin were significantly higher than that in AMPKα2 knockout PNU group.(3) Glucose tolerance test: Compared with AMPKα2 knockout saline group, blood glucose level of AMPKα2 knockout PNU group were significantly lower at 0, 30, 60, 90min, while their insulin levels were not significantly different.【Conclusion】1. We have successfully established the gold standard of assessing insulin sensitivity hyperinsulinemic-euglycemic clamp in anesthetized SD rats, conscious rats as well as anesthetized mice.2. AMPKα2 mice are insulin resistant, glucose intorlerant, and have impaired glucose stimulated insulin secretion.3. Long term treatment with selectiveα7nAChR agonist, PNU-282987, can significantly enhance insulin sensitivity of AMPKα2, which suggests thatα7nAChR can improve insulin sensitivity independently, while AMPKα2 is not involved in.