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Establishment of Methods in Insulin Sensitivity Assessment and Their Application in Drug Research

Author: GuoLingLing
Tutor: ZuoChaoYu
School: Second Military Medical University
Course: Pharmacology
Keywords: Insulin sensitivity Hyperinsulinemic - euglycemic clamp AMPKα2 α7 nAChR PNU-282987
CLC: R587.1
Type: Master's thesis
Year: 2011
Downloads: 108
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Abstract

[Objective] To a true and accurate assessment of insulin sensitivity, is of great significance for the study of the sugar metabolic regulation mechanisms as well as the research and development of insulin-sensitizing drugs. This project intends to establish the overall level evaluation of insulin sensitivity in rats and mice, the gold standard \operating procedures. On this basis, the application has established various methods of system study adenosine monophosphate-activated protein kinase α2 catalytic subunit (Adenosine Monophosphate-Activated Protein Kinase Catalytic Subunitα2, AMPKα2) knockout mouse insulin sensitivity. Finally, evaluation of new targets for drug nicotine acetylcholine α7 receptor, (α7 nicotinic acetylcholine receptor, α7 nAChR) agonist PNU-282987 treatment of long-term improvement of insulin sensitivity. 【Methods】: insulin sensitivity in the establishment of evaluation methods. First anesthetic dose, the catheter indwelling position, the length of the cannula, the depth and other conditions were investigated to determine the hyperinsulinemic - the best experimental program glucose clamp method in anesthetized rats and mice. Then by comparing the intubation catheter after fixation methods and postoperative recovery time clamp experimental results in order to determine the the conscious rats hyperinsulinemic - euglycemic clamp method best experimental program. Finally, to improve our group pre-established insulin tolerance test glucose tolerance test, blood glucose and insulin determination experimental method and writing standard operating procedures. Part II: insulin sensitivity evaluation methods in drug research. First, with established insulin tolerance test, glucose tolerance test, HOMA-IR, insulin - is glucose clamp method AMPKα2 gene knock out mice and wild-type mice, insulin sensitivity comprehensive evaluation. Investigated α7 nAChR receptor specific agonist PNU-282987 long-term treatment to improve the role of insulin resistance. The AMPKα2 knockout mice were divided into two groups, respectively, by intraperitoneal injection of saline and PNU-282987 (0.53mg/kg), once a day for six weeks of treatment, the indicators investigated by detecting changes in insulin sensitivity. [Results] The first part: insulin sensitivity in the establishment of evaluation methods. 1. Determine the best solution anesthetized rats, mice and conscious rats hyperinsulinemic - euglycemic clamp experiments. The experiments showed that the average of anesthesia Sprague Dawley (SD) rats glucose input rate (GIR) was 26.9 ± 2.5mg / (kg · min), conscious SD rats was 29.4 ± 0.8mg / (kg · min), anesthesia C57BL / 6J (C57) in mice is 82.6 ± 9.2 mg / (kg · min). Completion of anesthetized rats, mice and conscious rats hyperinsulinemic - euglycemic clamp standard operating procedure to write; completion of rats and mice, the insulin tolerance test glucose tolerance test and other relevant methods writing of standard operating procedures. Part II: insulin sensitivity evaluation methods in drug research. No significant difference in 1. AMPKα2 knockout mice compared with wild-type HOMA-IR values. Insulin tolerance test in 30 minutes and 45 minutes after the administration of insulin, AMPKα2 knockout mice blood glucose decreased significantly smaller than wild-type. The oral glucose tolerance test, at 60 minutes and 90 minutes after giving glucose, AMPKα2 knockout mice blood glucose was significantly higher than wild-type. Meanwhile, in the glucose load after 30 minutes and 60 minutes time point, AMPKα2 top of the insulin concentration in mice was significantly lower than the wild-type gene knock. Hyperinsulinemic - euglycemic clamp experiments AMPKα2 knock-out mice GIR to 55.8 ± 5.4mg / (kg · min), wild-type GIR = 82.6 ± 9.2 mg / (kg · min), the difference between them significant. After six weeks after treatment, PNU-282987 significantly improve AMPKα2 gene knockout mice HOMA-IR index. Insulin tolerance test showed that the treatment group compared with the saline group, PNU-282987 in 15, 30, 45-minute blood glucose reduction. The glucose tolerance test shows that at 0, 30, 60, 90 minutes after the glucose load, PNU-282987 treatment group blood glucose was significantly lower than the saline treated group, while the two sets of insulin levels is always no significant difference. [Conclusion] on the topics in anesthetized rats, mice and conscious rats successfully established a standard method of evaluation of insulin sensitivity gold high insulin - glucose clamp method. \This chamber introduced AMPKα2 knockout mice have significant insulin resistance, impaired glucose tolerance, and glucose-stimulated insulin secretion diminished capacity characteristics. 3.α7 nAChR receptor agonist PNU-282987 long-term treatment can significantly improve AMPKα2 gene knock inter insulin sensitivity in mice, suggesting that α7 nAChR receptor-mediated insulin sensitizing effect, and the role of AMPKα2 independently.

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