游离脂肪酸引起肝细胞胰岛素抵抗及缺陷位点研究

Study on Free Fatty Acid-induced Insulin Resistance and Defective Locus in a Human HepG2 Cell Line

目的研究游离脂肪酸诱导人肝细胞(HepG2)引起胰岛素抵抗及信号转导缺陷位点。方法用含0.25mmol/L的软脂酸(PA)或100nmol/L胰岛素的DMEM培养基培养HepG2细胞24h后,用100nmol/L胰岛素刺激不同时间后,分别测定培养液的葡萄糖浓度,细胞内的糖原含量,磷酸烯醇式丙酮酸羧激酶(PEPCK)活性以及磷酸化的蛋白激酶B(P鄄Ser473PKB)的蛋白水平。磷酯酰肌醇3激酶(PI鄄3K)的抑制剂Wortmannin加入培养基,终浓度10-6mol/L。结果与正常对照组相比,PA处理组和高胰岛素组培养液中葡萄糖含量增高(P<0.05);PA处理组胰岛素刺激的PEPCK活性高于正常对照组(P<0.01),P鄄Ser473PKB蛋白水平低于正常对照组(P<0.01)。用Wortmannin处理后,正常对照组中PEPCK活性差别有统计学意义(P<0.01),PA处理组中的PEPCK活性差别无统计学意义(P>0.05);正常对照组和PA处理组中P鄄Ser473PKB差别均有统计学意义(P<0.01)。结论0.25mmol/L的AP培养24h后,细胞产生了胰岛素抵抗并且胰岛素信号转导途径存在障碍,可能蛋白激酶B(PKB)及下游分子缺陷参与了肝胰岛素抵抗的形成。

Objective To explore free fatty acid-induced insulin resistance and defective locus in HepG2 cells. Methods HepG2 cells were incubated in DMEM medium with Palmitic Acid (PA) (0.25 mmol/L) or insulin (100 nmol/L) for 24 hours before the cells were stimulated with 100 nmol/L insulin for different hours. Glucose concentration in medium, glycogen contents, PEPCK activity and protein levels of P-Ser473 PKB in cell lysates were measured, respectively. 10-6 mol/L Wortmannin (WT) was added to medium in some groups. Results In PA treatment groups compared with control group, glucose concentration and insulin-stimulated PEPCK activity were significantly increased (P<0.05), glycogen contents and protein levels of P-Ser473 PKB were significantly decreased (P<0.01). With the presence or absence of WT, PEPCK activity were no significant difference in PA groups (P>0.05), but were marked difference in control groups (P<0.01). Protein levels of P-Ser473 PKB were significant difference between PA treatment group and the control (P<0.01). Conclusion After HepG2 cells were incubated with PA, insulin resistance was induced because of the impairment of insulin receptor signaling. Defects of PKB and its downstream molecules may be responsible for the occurrence of hepatic insulin resistance.