丝氨酸蛋白酶抑制剂对棕榈酸诱导的3T3-L1脂肪细胞胰岛素抵抗的影响及机制

摘要

目的 观察脂肪细胞因子丝氨酸蛋白酶抑制剂(Vaspin)是否通过IRS/PI3K/Akt/Glut信号通路改善3T3-L1脂肪细胞胰岛素抵抗.方法 以棕榈酸(PA)培养3T3-L1脂肪细胞构建胰岛素抵抗模型,分为PA组、PA+ 100 ng/ml Vaspin组、PA+200 ng/ml Vaspin组、PA+400 ng/ml Vaspin组及PA+400 ng/ml Vaspin+Wortmannin(PI3K抑制剂)组.运用2-脱氧-H3-D-葡萄糖掺入法及葡萄糖氧化酶-过氧化物酶法分别检测细胞葡萄糖摄取和消耗量.以RT-PCR和Westem blot检测胰岛素信号通路中、酪氨酸磷酸化胰岛素受体底物1(p-IRS-1)、Akt、磷酸化蛋白激酶B(p-Akt)和Glut-4的mRNA和蛋白表达水平.结果 与PA组相比,随着Vaspin浓度增加,葡萄糖摄取和消耗量逐渐增加(P＜0.05),IRS-1和Akt的mRNA和蛋白磷酸化水平逐渐上升(P＜0.05),p-IRS-1/IRS-l、p-Akt/Akt比值逐渐增加,同时Glut-4的mRNA和蛋白表达也相应上调(P＜0.05).与400 ng/ml Vaspin干预组相比,PA+400 ng/ml Vaspin+Wortmannin组葡萄糖摄取和消耗量降低,IRS-1和Akt的mRNA和蛋白磷酸化水平下降(P＜0.05),p-IRS-1/IRS-1、p-Akt/Akt比值下降,且Glut-4的mRNA和蛋白表达下调(P＜0.05).结论 Vaspin通过激活IRS/PI3K/Akt/Glut胰岛素信号通路,改善3T3-L1脂肪细胞胰岛素抵抗.%Objective To investigate the effects of vaspin on insulin resistants of 3T3-L1 adipocyte through the insulin receptor substrates (IRS) /phosphatidylinositol 3-kinase (PI3K) /protein kinase B (Akt) /glucose transporter (Glut) signaling pathway.Methods 3T3-L1 cells cultured by palmitic acid (PA) were used to establish insulin resistance models,which were divided into PA group,PA + 100 ng/ml vaspin group,PA+200 ng/ml vaspin group,PA+400 ng/ml vaspin group and PA+400 ng/ml vaspin+wortmannin (PI3K inhibitor) group.Glucose uptake and consumption were assessed by 2-deoxy H3-D-glucose incorporation and glucose oxidase-peroxidase respectively.IRS/PI3K/Akt/Glut signaling pathway was evaluated using reverse transcription polymerase chain reaction and Western blot analysis.Results Compared with PA group,glucose uptake and consumption increased gradually with the increasing of vaspin concentration in other groups (P ＜ 0.05).mRNA levels of IRS-1,Akt and Glut 4 increased gradually as vaspin concentration increasing (P＜0.05),and the ratios of p-IRS-1 to IRS-1,p-Akt to Akt and Glut 4 protein level also showed the same trends (P＜0.05).However,glucose uptake and consumption in PA+400 ng/ml vaspin+wortmannin group were less than that of PA +400 ng/ml vaspin group (P＜0.05).PA+400 ng/ml vaspin+wortmannin group showed lower mRNA and protein phosphorylation levels of IRS-1,Akt and Glut 4 (P＜0.05),and that the ratios of p-IRS-1 to IRS-1,p-Akt to Akt and Glut 4 protein levels decreased (P＜0.05).Conclusions Vaspin can improve the insulin sensitivity of 3T3-L1 adipocyte by activating IRS/PI3K/Akt/Glut signaling pathway.