摘要目的 探讨固醇调节元件结合蛋白-1c (SREBP-1c)对骨骼肌细胞胰岛素受体底物-1(IRS-1)的调控机制,以明确SREBP-1 c在高脂诱导骨骼肌胰岛素抵抗中的作用.方法 L6细胞经2％胎牛血清(FBS)诱导分化成肌管细胞,经500 μmol/L棕榈酸(PA)处理建立胰岛素抵抗模型.采用Western印迹检测L6肌管细胞经PA处理后0.5、1、3、6、12、24、48 h的SREBP-1c、p-IRS-1(Tyr608/612)、IRS-1、p-AKT(Ser473)、AKT的蛋白表达.检测L6肌管细胞过表达SREBP-1 c或用肝X受体(LXR)激动剂TO901317(5 μmol/L)处理后SREBP-1 c、脂肪酸合成酶(FAS)及胰岛素信号通路相关分子的蛋白表达.采用双荧光素酶报告基因实验分析SREBP-1 c转录因子对IRS-1启动子区域的调控作用.结果 L6肌管细胞经PA处理后,SREBP-1c蛋白表达在1h后即显著增高,胰岛素信号通路蛋白p-IRS-1(Tyr608/612)、IRS-1及p-AKT(Ser473)表达在6h后显著下降,AKT蛋白表达无变化.L6肌管细胞经LXR激动剂TO901317处理后SREBP-1c、FAS基因及蛋白表达增高,IRS-1基因表达下降,p-IRS-1(Tyr608/612)、IRS-1及p-AKT(Ser473)/AKT蛋白表达下降.L6肌管细胞过表达SREBP-1c后相关蛋白出现与LXR激动剂干预相似的变化.双荧光素酶报告基因实验结果显示SREBP-1c显著抑制IRS-1的启动子活性,SREBP-1c DNA结合域的酪氨酸突变成丙氨酸后则对IRS-1启动子无作用.结论 SREBP-1c通过抑制IRS-1基因转录表达而影响胰岛素信号通路,在高脂诱导骨骼肌胰岛素抵抗中起关键作用.

abstractsObjective Sterol regulatory element binding protein-1c (SREBP-1c) is a master regulator of fatty acid synthase and controls lipogenesis.And insulin receptor substrate-1 (IRS-1) is a key insulin signaling mediator in skeletal muscle.The present study was conducted to explore the mechanism of SREBP-1c in the regulation of IRS-1 in skeletal muscle cells and elucidate the role of SREBP-1c in high fatinduced skeletal muscle insulin resistance.Methods L6 cells differentiated into myotubes in differentiation medium with 2％ FBS.An in vitro insulin resistant model in L6 myotubes was established by 500 μmol/L of palmitate acid (PA).SREBP-1c,p-IRS-1 (Tyr608/612),IRS-1,p-AKT(Ser473) and AKT were detected by Western blot after incubating L6 myobutes with 500 μmol/L of PA for 0.5,1,3,6,12,18 or 24 h.SREBP-1c,FAS and molecules related to insulin signaling pathway were detected by Western blot when L6 myotubes over-expressed SREBP-1c or after a treatment of liver X receptor (LXR) agonist (TO901317,5 pmol/L).The regulatory effects of transcription factor SREBP-lc on promoter region of IRS-1 were assessed by dualluciferase reporter assay.Results SREBP-1 c protein expression increased significantly after 1-hour exposure to PA.The protein levels of p-IRS-1 (Tyr608/612),IRS-1 and p-AKT(Ser473) decreased significantly after a 6-hour incubation of PA.However AKT protein levels were unaffected.The protein expressions of SREBP-1 c and FAS were up-regulated by LXR agonist treatment versus controls.By contrast,LXR agonist treatment led to decreased expressions of IRS-1,p-IRS-1 (Tyr608/612) and p-AKT(Ser473)/AKT proteins versus controls.The expressions of related proteins were similar to the observations made with LXR agonist intervention.The results of dual-luciferase reporter assay indicated that IRS-1 promoter activity was repressed significantly by SREBP-lc over-expression or TO901317 treatment whereas the dominant negative form of SREBP-1 c (a mutant of Tyr320Ala lacking the ability of binding DNA) had no effect.Conclusion SREBP-1c may suppress IRS-1 expression and the subsequent insulin signaling pathway.And it plays a key role in PA-induced insulin resistance of skeletal muscle.