摘要
目的分析在α-亚麻酸(ALA,C18:3)干预后,胰岛素抵抗Hep G2(insulin resistance Hep G2,IR-Hep G2)细胞模型脂类合成关键基因表达水平的变化。方法 Hep G2细胞造模期分为两组,由无血清培养基培养的对照组(normal control group,NC)以及含有0.25 mmol/L软脂酸的无血清培养基培养的软脂酸组(palmitic acid,PA),培养24 h后鉴定细胞胰岛素敏感性并测定细胞内胆固醇(total cholesterol,TCH)、甘油三酯(Tryglyceride,TG)水平,然后用ALA替代20%的PA培养12 h,再次鉴定细胞胰岛素敏感性并检测细胞内TCH、TG水平,real time q PCR检测TG、TCH合成关键基因mRNA水平,Western blot检测TG、TCH合成关键基因蛋白表达量。结果 IR-Hep G2细胞模型建立成功,并且PA组TCH水平显著高于NC组(P=0.0016),出现了脂代谢紊乱;ALA干预IR-Hep G2细胞12 h后,细胞活性有所恢复,与PA组相比,ALA组胰岛素诱导基因(insulin induced genes,INSIGs)及脂类合成关键蛋白的mRNA表达无明显变化;与基因表达结果不一致的是,ALA干预后可以特异性提升细胞内INSIG-2蛋白相对表达量,但对INSIG-1蛋白相对表达量没有明显影响。同时,ALA可以显著抑制55k Da固醇调节元件结合蛋白-2(sterol regulatory element-binding protein-2,SREBP-2)、HMG-Co A还原酶(3-hydroxy-3-methyl glutaryl coen-zyme A reductase,HMGCR)及脂肪酸合酶(fatty acid synthase,FASN)蛋白的表达,且ALA组细胞内TG水平显著降低(P=0.0119)。结论 0.05 mmol/L ALA干预IR-Hep G2细胞后,通过提升INSIG-2蛋白的表达,抑制SREBP-2从内质网到高尔基体的剪切成熟活化,降低细胞内55k Da SREBP-2水平及HMGCR的表达,并且抑制FASN表达,从而抑制了TG/TCH的合成。
Objective We investigate the effect of ALA on lipid biosynthesis-related genes in animal and cell IR models. Method HepG2 cells were divided into two groups, normal control group （NC） and palmitic acid group （PA） in which cells were firstly cultured for 24 h in the medium contained 0.25 mmol/L palmitic acid. The glucose uptake, cellular TG and TCH level were determined and once there was significant difference in glucose uptake, ALA took 20 percent the place of PA for another 12 h culture. Celluar TCH and TG level were detected and mRNA and protein expression of genes related to lipid synthesis were examinated by real-tlme qPCR and Western blot. Result After incubation with 0.25 mmol/L PA for 24 h, the glucose uptake of PA group was significantly lowered than NC group indicating the IR-HepG2 cell model was established characterized by extremely high TCH level. ALA selectively upregulated the protein expression of INSIG-2 and decreased the TG level in IR-HepG2 cells, and there were no statistically significant differences in TCH level and INSIG-1 protein expression. ALA had no significant effect on the abundance of the transcriptionally active （65 kDa） form of Srebp-1c while the Fasn protein reduced by 21% after ALA intervention. When compared with PA, ALA reduced 55 -kDa cleaved form of SREBP-2 and Hmgcr protein. Conclusion ALA selectively upregulated the protein expression of INSIG-2 in IR-HepG2 cells, rather than Insig-1. ALA reduced the abundance of post-translational form of SREBP-2 and its target Hrngcr and Fasn by increasing the expression of INSIG-2.
出处
《实验动物科学》
2016年第6期5-10,共6页
Laboratory Animal Science
基金
国家自然科学基金面上项目(No.31272386)
关键词
Α-亚麻酸
脂类合成
胰岛素抵抗
胰岛素诱导基因
Alpha-Linolenic acid
lipids synthesis
insulin resistance
insulin induced genes
