锌α_2糖蛋白通过增强脂肪细胞自噬活性改善胰岛素抵抗的作用及机制研究

Effect of zinc-α2-glycoprotein improved insulin resistance by enhancing autophagy activity in adipocytes and its mechanism

目的研究锌α_2糖蛋白(ZAG)对胰岛素抵抗3T3-L1细胞自噬相关基因7和哺乳动物雷帕霉素靶蛋白基因和蛋白表达的影响,探讨ZAG调控脂肪细胞自噬活性改变的作用及分子机制。方法将3T3-L1脂肪细胞随机分为正常对照、胰岛素抵抗、ZAG、氯喹、ZAG+氯喹5组,应用地塞米松诱导细胞胰岛素抵抗,ZAG组加入ZAG,氯喹组加入氯喹,ZAG+氯喹组同时加入ZAG和氯喹。以葡萄糖氧化酶法测定5组细胞上清液中葡萄糖消耗量,观察ZAG对脂肪细胞葡萄糖摄取的影响;应用免疫印迹技术测定脂肪细胞Atg7和哺乳动物雷帕霉素靶蛋白(mTOR)的蛋白表达变化;应用实时荧光定量聚合酶链反应(PCR)技术检测脂肪细胞Atg7和m TOR的mRNA表达变化。结果与正常对照组相比,胰岛素抵抗组的Atg7蛋白和m RNA表达明显下降(P均<0.05),而mTOR的蛋白和mRNA表达显著升高(P均<0.05),经ZAG干预后,Atg7蛋白和m RNA表达水平有一定升高,mTOR的蛋白和mRNA表达降低,与正常对照组比较差异有统计学意义(P均<0.05)。结论 ZAG通过调控脂肪细胞Atg7及mTOR的表达,增强脂肪细胞自噬活性,进而改善胰岛素抵抗。

Objective To determine the effect of zinc-α2-glycoprotein(ZAG)on the mRNA and protein expressions of autophagy-related gene 7(Atg7)and mammalian target of Rapamycin(mTOR)in insulin-resistant 3T3-L1 cells,and to investigate the effect of ZAG in regulating autophagy activity in adipocytes and its molecular mechanism.Methods 3T3-L1 adipocytes were randomly divided into the normal control group,insulin resistance group,ZAG group,Chloroquine group and ZAG+Chloroquine group.Dexamethasone was used to induce cellular insulin resistance.The ZAG group,Chloroquine group and ZAG+Chloroquine group were added with ZAG,Chloroquine,and ZAG+Chloroquine,respectively.The glucose consumption in the supernatant of the five groups was measured by glucose oxidase method,and the effect of ZAG on glucose uptake by adipocytes was determined.The protein and mRNA ex-pression levels of Atg7 and mTOR in adipocytes were determined by Western blotting and real-time fluorescence quantitative PCR,respectively.Results Compared with the normal control group,the protein and mRNA expression levels of Atg7 significantly decreased(both P<0.05),whereas the protein and mRNA expression levels of mTOR significantly increased(both P<0.05),in the insulin resistance group.After ZAG intervention,the protein and mRNA expression levels of Atg7 increased,whereas the protein and mRNA expression levels of mTOR decreased,with significant differences compared with those in the normal control group(all P<0.05).Conclusion ZAG may enhance the autophagy activity by regulating the expression of Atg7 and mTOR in adipocytes,thereby improving insulin resistance.