慢性胰岛素刺激对胰岛素受体后不同信号转导途径的下降调节  

Down-regulation of different post-receptor signal transduction pathways by chronic insulin stimulation

作　　者：袁莉[1] Reinhard ZIEGLER Andreas HAMANN 

机构地区：[1]华中科技大学同济医学院附属协和医院内分泌科,武汉430022 [2]Department of Endocrinology and Metabolism

出　　处：《中华内分泌代谢杂志》2003年第4期308-312,共5页Chinese Journal of Endocrinology and Metabolism

摘　　要：目的　研究胰岛素慢性刺激对人肝癌细胞株 (HepG2 )胰岛素受体后不同信号转导途径的影响。方法　HepG2 细胞在无血清条件下与不同浓度的胰岛素 ( 0～ 10 0nmol/L)温育 16h ,然后用 10 0nmol/L胰岛素急性刺激 1min。这些细胞的溶解物中的胰岛素受体 β亚单位 (IRβ) ,胰岛素受体底物 (IRS) 1,IRS 2 ,磷酯酰肌醇 3激酶 (PI3K)的调节亚单位P85 ,有丝分裂原激活蛋白激酶 (MAPK)的蛋白表达水平和MAPK的磷酸化水平通过Western免疫印迹法测定 ,IRβ、IRS 1/ 2的蛋白磷酸化水平以及IRS 1/ 2与P85的结合反应用特异性抗体的免疫沉淀法。结果　胰岛素 1min急性刺激能迅速导致IRβ、IRS 1、IRS 2的酪氨酸磷酸化和MAPK的磷酸化 ,以及IRS 1( 2 )与P85的相互作用而激活PI3K。高浓度胰岛素慢性刺激显著降低了IRβ、IRS 1和IRS 2的酪氨酸磷酸化。细胞用 10 0nmol/L胰岛素预温育 16h后 ,IRβ、IRS 1和IRS 2的磷酸化水平降至最低值 ,分别为对照水平的 2 2 .2 % (P <0 .0 1)、10 .9% (P <0 .0 1)和 2 2 .0 % (P<0 .0 1) ,与IRS的磷酸化变化相平行 ,IRS 1和IRS 2与PI3K的相互作用分别降低至对照水平的 3 4.3 %(P <0 .0 1)和 3 0 .0 % (P <0 .0 1) ,MAPK的磷酸化水平降低至对照水平的 16.4% (P <0 .0 1)。Objective To study the effect of chronic insulin stimulation on different signal transduction pathways of insulin post-receptor in a human hepatoma cells line (HepG2). Methods HepG2 cells were incubated in serum-free media in either presence or absence of various concentrations of insulin (0-100 nmol/L) for 16 h before the cells were stimulated with 100 nmol/L insulin for 1 min. Protein levels of insulin receptor β-subunit (IRβ), insulin receptor substrate (IRS)-1, IRS-2, P85 subunit of phosphatidylinositol 3-kinase (PI3K), mitogen activated protein kinase (MAPK) and phosphorylated proteins of MAPK were determined in total cell lysates by Western-immunoblot, and determination of phosphorylated proteins IRβ, IRS-1, IRS-2 and interaction of PI3K with IRS-1/-2 were performed by immunoprecipitation. Results Insulin stimulation for 1 min rapidly induced tyrosine phosphorylation of IRβ, IRS-1 and IRS-2 and phosphorylation of MAPK, which in turn, resulted in binding of P85 with IRS-1/-2 and activation of MAPK. After 100 nmol/L insulin treatment for 16 h, there was a more marked reduction in the phosphorylations of IRβ,IRS1andIRS2inHepG2 cells, reaching 22.2% (P<0.01) and 10.9% (P<0.01) and 22.0% (P<0.01) of control levels, respectively, and the binding between IRS-1, IRS-2 and PI3K was decreased to 34.3% (P<0.01) and 30.0% (P<0.01) of control level, respectively, and the phosphorylation of MAPK was decreased to 16.4% of control level (P<0.01). Chronic insulin (1-100 nmol/L) treatment induced a dose-dependent decrease in the protein level of IRβ and a slight decrease in the protein level of IRS-1 and IRS-2. There were no significant changes in protein levels PI3K and MAPK. Conclusion Chronic high insulin stimulation to HepG2 cells results in down-regulation of insulin signal transduction linked to PI3K and MAPK pathways, in which the impaired tyrosine phosphorylation of IRS may play a critical role, these changes seem to be important to the insulin resistance.

关 键 词：胰岛素受体 信号转导 调节 胰岛素抵抗 高胰岛素血症 

分 类 号：R587[医药卫生—内分泌]