摘要
目的:研究叉头框蛋白O1(FOXO1)表达和磷酸化过程对高糖刺激下人肾小管上皮细胞脂质沉积的影响。方法:体外培养人肾小管上皮细胞株HKC,给予高糖刺激后免疫荧光及Western blotting检测总FOXO1、磷酸化FOXO1和固醇调节元件结合蛋白1(SREBP1)的表达,油红O染色测定细胞内脂质沉积;构建野生型和磷酸化位点突变型FOXO1质粒,转染入HKC细胞后给予高糖刺激,采用免疫荧光、Western blotting和油红O染色确定FOXO1磷酸化位点突变对肾小管上皮细胞脂质代谢的影响。结果:高糖刺激HKC细胞48 h后,总FOXO1在正常糖组和高糖组的表达未见差异,而磷酸化FOXO1及SREBP-1表达明显上调,细胞内脂滴显著增加。将构建的野生型FOXO1质粒转染人细胞后,上调了总FOXO1和磷酸化FOXO1的表达,增加了SREBP-1表达和细胞内脂滴含量;而磷酸化位点突变的FOXO1质粒避免了高糖诱导引起的SREBP-1上调和细胞内脂质沉积。结论:磷酸化FOXO1参与了高糖诱导的肾小管上皮细胞SREBP-1上调和细胞内脂质聚集;磷酸化位点的突变可避免高糖引起的肾小管上皮细胞SREBP-1上调和脂质沉积。
AIM: To study the effect of forkhead box O1 (FOXO1) expression and phosphorylation on lipid accumulation in high glucose-stimulated human renal tubular cell line HKC. METHODS: HKC cells were stimulated with high glucose. The total FOXO1, phospho-FOXO1 and sterol-regulatory element-binding protein 1 (SREBP-1) were detected by the methods of immunofluorescence and Western blotting. The cellular lipid deposit was measured by oil red O staining. Moreover, the wild-type FOXO1 vector or mutant FOXO1 vector was transfectcd into HKC cells followed by the treatment with high glucose. Immunofluorescence, Western blotting and oil red O staining were used to determine the effect of the phosphorylation site mutation on the lipid metabolism in renal tubular cells. RESULTS: No difference in total FOXO1 expression between normal glucose group and high glucose group was observed 48 h after HKC cells were stimulated with high glucose. However, phospho-FOXO1 was significantly increased in high glucose-treated HKC cells. Subsequently, SREBP-1 and cellular lipid deposit were up-regulated. The wild-type FOXO1 vector increased total FOXO1, phosphoFOXO1, SREBP-1 and cellular triglyceride in high glucose-treated HKC cells. However, mutant FOXO1 vector at the phosphorylation site attenuated the effect of high glucose on SREBP-1 and cellular lipid deposit. CONCLUSION: The phosphorylation of FOXO1 is involved in high glucose-induced up-regulation of SREBP-1 and cellular lipid accumulation in renal tubular cells. In addition, the mutation at the phosphorylation site prevents high glucose-induced enhancement of SREBP-1 and lipid deposit.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2013年第11期2001-2005,共5页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.81100517)
河北省自然科学基金资助项目(No.H2012206008)