【摘要】 目的从蛋白激酶C(PKC)信号通路角度,探讨游离脂肪酸(FFA)引起肝脏胰岛素抵抗(IR)的可能机制。方法培养HepG2细胞,同时设立对照组、软脂酸(PA)组、高胰岛素组。软脂酸组、高胰岛素组分别用250μmol/L PA、5×10-7mol/L胰岛素处理24h。然后对照组、软脂酸组再根据胰岛素刺激前加(+)与不加(-)PKC抑制剂白屈菜红碱盐酸盐(chelerythrine chloride,CC)5μmol/L预处理1h,随机分为两亚组:对照组(-)、对照组(+)、PA组(-)、PA组(+)。葡萄糖氧化酶法测定胰岛素刺激后12h葡萄糖消耗量,蒽酮法测定胰岛素刺激后3h点细胞内糖原含量,Western blotting技术检测15min点细胞内P-Ser473PKB、P-Ser21/9GSK-3α/β水平。结果PA组(-)与高胰岛素组葡萄糖消耗量无统计学差异(P=0.523)。葡萄糖消耗量、细胞内糖原含量、P-Ser473PKB、P-Ser21GSK-3α、P-Ser9GSK-3β水平均显示,PA组(-)与对照组(-)比较显著降低(P值依次为0.000,0.000,0.004,0.004,0.028),对照组(+)与对照组(-)比较略有升高但无显著性差异;PA组(+)与PA组(-)比较显著升高(P值依次为0.000,0.014,0.043,0.041,0.035)。结论PA(250μmol/L)体外成功诱导了HepG2细胞产生IR,PKC信号通路在FFA引起肝脏IR中起着重要作用。更多还原

【Abstract】 Objective To explore the possible mechanism of insulin resistance(IR)for HepG2 cells induced by high concentrations of palmitate(PA)through protein kinase C(PKC)signaling pathway analysis.Methods The model of hepatic IR was established induced by PA.HepG2 cells were randomly divided into control group,PA group(250 μmol/L PA)and high insulin group(50 nmol/L insulin)and treated for 24 hours.Then both of control group and PA group were redivided into subgroups:control(-)、control(+)、PA(-)、PA(+).Only control(+)and PA(+)groups were pretreated by 5 μmol/L chelerythrine chloride(CC)of PKC inhibitor for l h before insulin stimulation.Insulin-stimulated glucose consumption was measured using the glucose oxidase method at 12-hour time point.Cell glycogen was measured with anthrone method at 3-hour time point and protein levels of phosphate-protein kinase B(P-Ser473 PKB)and phosphate-glycogen synthase kinase(P-Ser21/9 GSK-3α/β)at 15-min time point were determined in total cell lysates by Western-blotting.Results There was no significant difference in glucose consumption between PA(-)group and high insulin group(P=0.523).All levels of glucose consumption,glycogen content,P-Ser473 PKB,P-Ser21 GSK-3α and P-Ser9 GSK-3β in PA(-)group were reduced significantly(P=0.000,0.000,0.004,0.004,0.028 respectively)compared with those in control(-)group,and were increased significantly(P=0.000,0.014,0.043,0.041,0.035 respectively)in PA(+)group compared with those in PA(-)group.Conclusions The model of hepatic insulin resistance is established successfully.Overaction of PKC may play an important role in inducing IR in HepG2 cells.更多还原