蛋白激酶C在软脂酸诱导HepG_2细胞胰岛素抵抗中的作用

Role of protein kinase C in insulin resistance induced by palmitate in HepG_2 cells

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。

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 beth 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 1 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-Set473 PKB） and phosphate-glycogen synthase kinase （P-Ser21/9 GSK-3α/13） at 15-min time point were determined in total cell lysates by Westem-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-Set473 PKB, P-Ser21 GSK-3et and P-Set9 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.