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

1.为了研究IRS-1基因的天然变异是否与2型糖尿病的发生有关，我们研究了4种突变IRS-1（P170 R、M209 T、S809 F和G971 R）对胰岛素信号传导的影响。用野生型（WT）或四种突变型IRS-1 cDNA转染稳定过表达人IR且缺乏内源性IRS-1的32 D-IR细胞，并进行分析。与WT相比，表达P170 R、M209 T和G971 R的细胞表现出显著的胰岛素作用降低，而S809 F则没有。这些数据表明IRS-1变异体对2型糖尿病的发展有贡献。为探讨缓激肽（BK）增强胰岛素信号的分子机制，用BK B2受体（BK 2 R）和/或胰岛素受体和/或IRS-1 cDNA转染32 D细胞，并进行分析。在这些细胞中，BK增强胰岛素刺激的胰岛素受体和IRS-1的酪氨酸磷酸化，并增加IRS-1相关的PI 3-激酶活性。BK还能显著降低细胞颗粒因子中胰岛素受体的蛋白酪氨酸磷酸酶（PTR）活性，提示BK的作用部分是通过抑制颗粒因子中的PTR介导的。我们的结果表明BK通过BK 2 R介导的信号通路增强胰岛素信号级联反应.为了阐明肥胖在IRS-1异常个体中的作用，我们创建了肥胖IRS-1（+/-）小鼠并进行表征。肥胖IRS-1（+/-）小鼠的血浆胰岛素水平是肥胖WT小鼠的两倍。腹膜内葡萄糖耐量试验显示肥胖IRS-1（+/-）小鼠60 min时血糖高于肥胖WT小鼠，腹膜内胰岛素耐受性试验显示肥胖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，在正常体重时不影响胰岛素抵抗，但当它们合并肥胖时，可能会加重体内胰岛素抵抗。

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.