Molecular and developmental biological analysis of impact of genetic and environmental factors on the development of insulin resistance

1.为了探讨自然IRS-1基因变异是否参与2型糖尿病的发生发展，我们研究了四个IRS-1基因突变(P170R、M209T、S809F和G971R)对胰岛素信号转导的影响。将野生型(WT)或4个突变型IRS-1基因分别导入稳定高表达人IR而缺乏内源性IRS-1的32D-IR细胞，并进行分析。与WT相比，表达P170R、M209T和G971R的细胞的胰岛素作用显著降低，而表达S809F的细胞则无此作用。这些数据表明IRS-1变异体对2型糖尿病的发生有贡献。为探讨缓激肽(BK)增强胰岛素信号的分子机制，将缓激肽B2受体(BK2R)和/或胰岛素受体和/或IRS-1基因导入32D细胞，并对其进行分析。在这些细胞中，BK增强了胰岛素刺激的胰岛素受体和IRS-1的酪氨酸磷酸化，并增加了IRS-1相关的PI 3-激酶活性。此外，BK显著降低细胞颗粒因子中针对胰岛素受体的蛋白酪氨酸磷酸酶(PTPase)活性，提示BK的作用部分是通过抑制颗粒组分中的PTPase(S)实现的。我们的结果表明BK通过BK2R介导的信号通路增强胰岛素信号通路。为了阐明肥胖对IRS-1异常个体的影响，我们创建了肥胖的IRS-1(+/-)小鼠，并对其进行了特征分析。肥胖的IRS-1(+/-)小鼠的血浆胰岛素水平是肥胖的WT小鼠的两倍。腹膜糖耐量试验显示肥胖IRS-1(+/-)小鼠60min血糖高于肥胖WT小鼠，而腹膜胰岛素抵抗试验显示肥胖IRS-1(+/-)小鼠胰岛素抵抗高于肥胖WT小鼠。这些结果表明，基因异常，如IRS-1基因变异，在正常体重下不会影响胰岛素抵抗，但当它们合并肥胖时，可能会加剧体内的胰岛素抵抗。

1. To examine if the natural IRS-1 gene variants contribute to the development of type 2 diabetes, we have studied the impact of four mutant IRS-1s (P170R, M209T, S809F and G971R) on insulin signaling. 32D-IR cells, stably overexpresing human IR and lack endo-genous IRS-1, were transfected with wild-type (WT) or four mutant IRS-1 cDNAs, and analyzed. Cells expressing P170R, M209T and G971R exhibited significant decrease in insulin actions as compared with WT, while S809F did not. These data suggested the contribution of IRS-1 variants to the development of type 2 diabetes.2. To determine the molecular mechanism of bradykinin (BK) enhancement of the insulin signal, 32D cells were transfected with BK B2 receptor (BK2R) and/or insulin receptor and/or IRS-1 cDNA, and analyzed. In these cells, BK enhanced insulin-stimulated tyrosine phosphorylation of the insulin receptor and IRS-1 and increased IRS-1 associated PI 3-kinase activity. Furthermore, protein tyrosine phosphatase (PTPase) activity against insulin receptor in particulate factor of the cells was significantly reduced by BK, suggesting that effect of BK was in part mediated by inhibition of PTPase(s) localized in particulate fraction. Our results demonstrated that BK enhanced insulin signal cascade through the BK2R mediated signaling pathway.3. To clarify the contribution of obesity in individuals with IRS-1 abnormality, we created obese IRS-1 (+/-) mice and characterized. Obese IRS-1 (+/-) mice showed twice higher plasma insulin than obese WT mice. Intraperitoneal glucose tolerance test showed higher blood glucose at 60 min in obese IRS-1(+/-) mice compared to obese WT mice, and intraperitoneal insulin toletance test showed higher insulin resistance in obese IRS-1 (+/-) mice compared to obese WT mice. These results suggest that gene abnormality, such as IRS-1 gene variants, which does not affect insulin resistance at normal body weight, could worsen insulin resistance in vivo when they complicated obesity.