二甲双胍在慢性高胰岛素处理的Hep G2细胞对胰岛素受体后信号转导的调节作用

摘要

目的:研究慢性高胰岛素对胰岛素受体后信号转导的影响及其与胰岛素抵抗的关系,并探讨二甲双胍治疗胰岛素抵抗是否通过胰岛素信号转导途径的介导.方法:人类肝癌细胞系(Hep G2)在无血清条件下首先与100 nmol/L高浓度胰岛素预温育16 h以产生胰岛素抵抗细胞模型,并观察不同浓度(0.01-10 mmol/L)的二甲双胍对胰岛素受体后磷酯酰肌醇3激酶(PI3K)途径信号转导的影响.结果:高浓度胰岛素100nmol/L慢性处理引起了IRβ、IRS1和IRS2的酪氨酸磷酸化和蛋白表达水平的下调,p85与IRS的相互作用也有显著降低.生理治疗浓度的(0.01-0.1 mmol/L)二甲双胍逆转了这种慢性高胰岛素引起的信号下调,细胞用0.1 mmol/L二甲双胍预温育后,IRβ、IRS1和IRS2的磷酸化反应水平分别增加了2.7倍(P＜0.01),6.8倍(P＜0.01)和2.3倍(P＜0.01),p85与IRS1的相互作用从34%增加至86%(P＜0.01),与IRS2的相互作用从30%增加至92%(P＜0.01).相反,药物浓度(1-10 mmol/L)的二甲双胍进一步抑制了IRβ、IRS的磷酸化及IRS与p85的相互作用.结论:高浓度胰岛素慢性处理可引起胰岛素受体后PI 3K途径信号转导的下调,二甲双胍对胰岛素信号转导的作用可能是其治疗胰岛素抵抗的主要分子机制.%AIM: To study the effect of chronic insulin treatment on insulin post-receptor signaling transduction and whether the effects of metformin are transmitted throughout the cascade of insulin signaling intermediates in a human hepatoma cell line (Hep G2). METHODS: Hep G2 cells were incubated in serum free media containing either insulin 100 nmol/L or insulin 100 nmol/L plus different concentrations (0.01-10 mmol/L) of metformin for 16 h and then were stimulated with insulin 100 nmol/L for 1 min. RESULTS: Chronic treatment of insulin 100 nmol/L induced a significant reduction in the phosphorylation and protein expression of IRB, IRS 1 and IRS2, which therefore resulted in a downregulation of association of PI3K with IRS. Therapeutic concentrations (0.01-0.1 mmol/L)of metformin prevented the changes induced by chronic insulin treatment in these post-receptor components of insulin signaling pathway. Tyrosine phosphorylation of IRβ, IRS1, and IRS2 was increased by 2.7 fold (P＜0.01),6.8 fold (P＜0.01), and 2.3 fold (P＜0.01) of chronically insulin-treated cells alone, respectively, after metformin 0.1 mmol/L was added. The association of p85 with IRS 1 and IRS2 was also increased from 34 % to 86 % (P＜0.01) and from 30 % to 92 % (P＜0.01), respectively. In contrast, metformin in pharmacological concentration (1-10 mmol/L) further inhibited tyrosine phosphorylation of IRβ, IRS 1, IRS2 and the interaction of PI3K with IRS.The association of IRS1 with p85 was further decreased by 58 % (P＞0.05) and of IRS2 by 30 % (P＜0.05).CONCLUSION: Chronic insulin exposure of Hep G2 cells induces the downregulation of insulin signal transduction via PI3K pathway. The effect of metformin on insulin signaling transduction represent a primary mechanism of metformin action in insulin resistant state