非酯化脂肪酸对原代培养犊牛肝细胞胰岛素信号通路的影响

NEFAs impair the insulin signaling pathway of primary cultured bovine hepatocytes mediated by SIRT1

旨在探讨非酯化脂肪酸(nonestesterified fatty acids,NEFAs)对体外原代培养犊牛肝细胞胰岛素信号通路的影响及SIRT1在其中的调控作用。选择培养48h的原代犊牛肝细胞,添加不同浓度的NEFAs(0、0.6、1.2、1.8mmol/L),继续培养12h,胰岛素处理组在收样前用100nmol/L胰岛素处理30min,每个浓度3个重复。另外选取已培养48h的细胞,用SIRT1抑制剂Nicotinamide(10mmol/L)处理12h,胰岛素处理组在收样前用100 mmol/L胰岛素处理30min。运用免疫印迹Western blot方法,检测NEFAs和Nicotinamide对肝细胞胰岛素信号通路关键蛋白IR和Akt的磷酸化水平及SIRT1蛋白表达水平的影响。结果显示,与对照组相比,1.2、1.8 mmol/L NEFAs组胰岛素刺激的IR磷酸化水平显著降低(P<0.01),而Akt的磷酸化程度在NEFAs浓度为0.6 mmol/L时即显著降低(P<0.01),同时,SIRT1的蛋白表达水平在NEFAs为1.2、1.8mmol/L显著降低(P<0.01);添加SIRT1抑制剂显著降低SIRT1的蛋白表达水平(P<0.01),同时胰岛素刺激的Akt磷酸化水平也显著降低(P<0.01)。结果表明,高浓度的NEFAs可以损伤体外原代培养肝细胞的胰岛素信号通路,SIRT1在NEFAs损伤的胰岛素信号通路中发挥重要作用。

The objective of this study was to investigate the effect of NEFAs on the insulin signaling pathway of primary cultured bovine hepatocytes and the important role of SIRT1 in the NEFAs impaired insulin signaling pathway.After hepatocytes were cultured for 48 h,different concentrations of NEFAs（0,0.6,1.2,1.8mmol/L）were added with three replications in one group and cultured for another 12 h.Hepatocytes were treated with SIRT1 inhibitor nicotinamide（10mmol/L）for 12 h.The phosphorylation level of IR,AKT and protein expression level of SIRT1 were detected by Western blot.The results showed that phosphorylation level of IR,AKT in high concentration of NEFAs（1.2and 1.8mmol/L）were significantly decreased compared with the control group（P〈0.01）.SIRT1 protein level was gradually decreased in a concentration-dependent manner,with significant difference in the high concentration of NEFAs group（1.2and1.8mmol/L）（P〈0.01）.Compared with the normal group,SIRT1 protein expression level were significantly lower（P〈0.01）in the group treated with SIRT1 inhibitor,meanwhile,insulin-stimulated phosphorylation of AKT decreased significantly（P〈0.01）.In summary,high concentration of NEFAs can impair the insulin signaling pathway of bovine hepatocytes cultured in vitro,and SIRT1 plays an important role in the hepatocyte insulin signaling pathway impaired by NEFAs.