游离脂肪酸引起肝细胞胰岛素抵抗及其机制的研究

目的研究游离脂肪酸（FFA）诱导人肝癌细胞（HepG2）引起胰岛素抵抗及其分子机制.方法应用含0.25 mmol/L的软脂酸（PA组）或100 nmol/L胰岛素（Ins组）与不含软脂酸和胰岛素（正常组）的DMEM培养基培养HepG2细胞24 h,正常组和PA组中又分加与不加磷酯酰肌醇3激酶（PI3K）抑制剂Wort-mannin两个亚组,100 nmol/L胰岛素刺激后分别测定培养液中的葡萄糖浓度、细胞内的糖原含量、磷酸烯醇式丙酮酸羧激酶（PEPCK）活性及胰岛素受体底物2（IRS-2）的蛋白水平.结果PA组和Ins组培养液中葡萄糖含量显著高于正常组（P＜0.01）,而细胞内糖原含量显著减少（P＜0.01）;PA组胰岛素刺激的PEPCK活性显著高于正常组（P＜0.01）,IRS-2蛋白水平显著低于正常组（P＜0.01）.无论应用Wortmannin处理与否,PA组中的PEPCK活性及IRS-2蛋白水平差异无显著性（P＞0.05）,而正常组中PEPCK活性及IRS-2蛋白水平的差异有显著性（P＜0.01）.结论加0.25 mmol/L的软脂酸培养24 h后,肝细胞可能由于胰岛素信号转导障碍产生胰岛素抵抗;胰岛素抵抗的形成可能与IRS-2及PI3K相关分子缺陷有关.

Study on free fatty acid-induced insulin resistance and molecular mechanism in a human hepatoma cell line,HepG2cell

Objective To explore the free fatty acid(FFA)-induced insulin resistance and molecular mechanism in HepG2 cell.Methods HepG2 cells were incubated in DMEM medium with or without 0.25 mmol/L palmitate (PA) or 100 nmol/L insulin before the cells stimulated with 100 nmol/L insulin; the normal ones were taken as controls. Glucose concentration in the medium, glycogen contents, phosphoenolpyruvate carboxykinase(PEPCK) activity and protein level of IRS-2 in the cell lysates were measured, respectively. Wortmannin was added to the medium in some groups.Results In PA and insulin treatment groups, the glucose concentrations were significantly increased (P<0.01),but glycogen contents were significantly decreased in comparing with those of the control group(P<0.01) .Insulin-stimulated PEPCK activity was significantly higher than that of the control group; In the presence or absence of Wortmannin,PEPCK activity and protein levels of insulin receptor substrate-2(IRS-2) were of no significant difference in PA groups(P>0.05); however, there was marked difference in the normal groups(P<0.01). Conclusion After the HepG2 cells incubated with PA for 24h, insulin resistance induced is due to impairment of insulin receptor signalling pathway. Defects of PI3K-associated molecules and IRS-2 might be responsible for the occurrence of free fatty acid-induced insulin resistance.