不同浓度胰岛素对人肾小管上皮细胞SREBP-1、FAS表达及脂质形成的影响

目的:探讨不同浓度胰岛素对人肾小管上皮细胞(HKC)固醇调节元件结合蛋白-1(SREBP-1)和脂肪酸合成酶(FAS)表达以及细胞内脂滴形成的影响。方法:分别给予0nmol/L、1nmol/L、10nmol/L、100nmol/L和200nmol/L胰岛素刺激HKC细胞6h。RT-PCR技术检测SREBP-1和FASmRNA表达,免疫细胞化学和Westernblotting检测SREBP-1蛋白的表达,油红O染色检测细胞内脂滴。结果:与0nmol/L胰岛素组相比,10nmol/L、100nmol/L和200nmol/L组HKC细胞SREBP-1和FASmRNA表达均升高,其中100nmol/L组升高最明显。免疫细胞化学技术显示SREBP-1蛋白定位于HKC细胞的胞浆,在10nmol/L、100nmol/L和200nmol/L组HKC细胞其表达明显增强。Westernblotting检测显示10nmol/L、100nmol/L和200nmol/L组HKC细胞SREBP-1蛋白的前体和成熟片段表达增强,其中100nmol/L组表达最强,差异显著。油红O染色显示胰岛素刺激6h后只有100nmol/L胰岛素组HKC细胞内可见明显红染脂滴颗粒。结论:高浓度胰岛素引起HKC细胞SREBP-1和FAS表达上调并最终导致细胞内脂滴沉积,这可能是代谢综合征引起肾脏脂质沉积的重要机制之一。

Concentration- dependent effect of insulin on expression of SREBP-1,FAS and lipid droplet formation in HKC cells

AIM: To investigate the effect of insulin at different concentrations on the expression of sterol regulatory element binding protein-1 (SREBP-1), fat acid synthase (FAS) and lipid droplet formation in human renal proximal tubular epithelial cell line (HKC). METHODS: HKC cells were treated with insulin at concentrations of 0 nmol/L, 1 nmol/L, 10 nmol/L, 100 nmol/L and 200 nmol/L respectively for 6 h. The analysis of SREBP-1 and FAS mRNA was performed by RT-PCR and the protein level of SREBP-1 was detected by Western blotting and immunocytochemistry. Oil red O staining was used to determine the cellular lipid droplet formation. RESULTS: Compared to HKC cells under the condition without insulin treatment (0 nmol/L group), the expression of SREBP-1 and FAS mRNA was significantly increased in HKC cells treated with insulin at concentrations of 10 nmol/L, 100 nmol/L or 200 nmol/L. Furthermore, the highest expression of SREBP-1 and FAS mRNA was observed in 100 nmol/L group. The SREBP-1 protein was located in the plasma of the HKC cells and was significantly upregulated in the cells treated with insulin at concentrations of 10 nmol/L, 100 nmol/L or 200 nmol/L. The results of Western blotting showed that the precursor and mature segments of SREBP-1 protein were increased in the cells of 10 nmol/L group, 100 nmol/L group and 200 nmol/L group, and those in 100 nmol/L group were the highest. The result of oil red O staining showed that the markedly deposited lipid droplet was only observed in 100 nmol/L group. CONCLUSION: The results suggest that insulin at high concentration up-regulates SREBP-1 and FAS, resulting in the formation and deposit of cellular lipid droplet in HKC cells, which may play an important role in the pathogenesis of renal lipid accumulation in metabolism syndrome.