LRPl6基因抑制IRS-1信号通路和过氧化物酶体增殖物激活受体γ转录活性导致C2-C12成肌细胞胰岛素抵抗

摘要

目的 探讨人白血病相关蛋白16(LRPl6)基因对C2-C12成肌细胞胰岛素抵抗的影响及机制.方法 构建过表达LRP16、抑制表达LRP16的细胞系和相应的对照细胞系.检测LRP16基因对C2-C12成肌细胞葡萄糖摄取率,胰岛素受体底物1(IRS-1)丝氨酸和酪氨酸磷酸化,磷脂酰肌醇3-激酶[P13K(p85)]表达,蛋白激酶B(Akt)磷酸化,过氧化物酶体增殖物激活受体γ(PPARγ)葡萄糖转运蛋白4(GLUT-4)表达以及PPARγ转录活性的影响.结果 过表达LRPl6基因的C2-C12成肌细胞胰岛素刺激的葡萄糖摄取率降低为对照组的46%(4700±97比10 200±347,P<0.01),抑制表达LRPl6基因后升高为对照组的1.73倍(17 600±466比10 200 ±91,P<0.05).过表达LRPl6促进IRS-1丝氨酸307位点磷酸化,抑制IRS-1的酪氨酸磷酸化、P13K(p85)表达、Akt的磷酸化和PPARγ、GLUT-4的表达.LRPl6剂量依赖性的降低PPARγ的转录活性,当pcDNA3.1-16为0.4和0.5 μg时,PPARγ的转录活性降低为对照组的43%(76±11比33±9,P<0.01)和27%(21 ±9比76 ±11,P<0.01).结论 LRPl6基因通过抑制IRS-1信号通路和PPARγ转录活性导致C2-C12成肌细胞胰岛素抵抗.%Objective To explore the effect of LRP(leukemia related protein)16 on insulin resistance in C2-C12 cells and explore its molecular mechanism.Methods Lipidosome transfection and lentivirus mediated siRNA(small interfering RNA)technology were used to establish LRP 16 overexpression and underexpression cell lines and their corresponding controol cell lines.And 2-deoxy-[3H]-glucose was used to measure the effect of LRP 16 on insulin-stimulated glucose uptake.The effects of LRPl6 on the phosphorylation of IRS(insulin receptor substrate)-1,Akt and the expressions of P13K(085),PPAR (peroxisome proliferator actived receptor)γ and GLUT-4 were detected by Western blot.Luciferase was used to study the effect of LRPl6 on the transcriptional activity of PPARγ Resuits Insulin.stimulated glucose uptake decreased to 46%of the control when LRPl6 was over-expressed ( 4700±97 vs 10 200±347.P<0.01).And the insulin.stimulated glucose uptake was 1.73 fold of control when the expression of LRPl6 was suppressed in C2-C12 cells(17 600±466 vs 10 200 ±91,P<0.05).The overexpression of LRPl6 attenuated the insulin.induced tyrnsine phosphorylation of IRS-1.the phosphorylation of Akt and the expressions of P13 K(085),PPARγ and GLUT-4.But it promoted the insulin-induced phosphorylation of IRS-1 at Ser307 in C2-C12 cells.LRPl6 decreased the transcriptional activity of PPART in a dosedependant manner.The transcriptional activity of PPARγdecreased to 43%and 27%of the control when the doses of pcDNA3.1-16 were 0.4 and 0.5μg(76±11 vs 33±9,P<0.01)and 27%(21±9 vs 76±11,P<0.01). Conclusion LRP16 gene causes insulin resistance in C2-C12 cells by inhibiting the IRS-1signaling and the transcriptional activity of PPARγ.