CAJ | 학술논문

目的观察Kruppel样因子14 (KLF14)基因过表达对肝细胞胰岛素信号通路以及AMP活化的蛋白激酶(AMPK)关键信号分子的影响,探讨KLF14影响糖代谢的分子机制.方法建立KLF14过表达的Hepa1-6肝癌细胞模型,分为空载组、胰岛素组、KLF14转染组和胰岛素+KLF14处理组,采用[3H]-2-脱氧-D-葡萄糖摄取法检测肝癌细胞对葡萄糖的摄取率.以Western印迹检测KLF14过表达对胰岛素信号通路中各信号分子的磷酸化水平及磷酸烯醇式丙酮酸激酶(PEPCK)蛋白表达的影响.结果胰岛素处理Hepa1-6细胞后,KLF14转染组与未转染组相比,葡萄糖摄取率明显升高(P＜0.01);胰岛素受体(InsR)、胰岛素受体底物(IRS)1、蛋白激酶B(Akt)、AMPK、CREB转录共激活因子2(TORC2)的磷酸化水平均明显增加(P＜0.01);而且,糖原合成酶激酶3β的磷酸化水平亦明显增加,而PEPCK的蛋白表达明显降低,差异均有统计学意义(P＜0.01).结论 KLF14过表达可能通过活化胰岛素InsR/IRS/Akt经典通路,从而改善肝细胞的胰岛素抵抗,且可能通过AMPK/TORC2/PEPCK信号通路抑制肝糖异生,并促进糖摄取和肝糖原的合成,从而改善糖代谢.
목적 관찰Kruppel양인자14 (KLF14)기인과표체대간세포이도소신호통로이급AMP활화적단백격매(AMPK)관건신호분자적영향,탐토KLF14영향당대사적분자궤제.방법 건립KLF14과표체적Hepa1-6간암세포모형,분위공재조、이도소조、KLF14전염조화이도소+KLF14처리조,채용[3H]-2-탈양-D-포도당섭취법검측간암세포대포도당적섭취솔.이Western인적검측KLF14과표체대이도소신호통로중각신호분자적린산화수평급린산희순식병동산격매(PEPCK)단백표체적영향.결과 이도소처리Hepa1-6세포후,KLF14전염조여미전염조상비,포도당섭취솔명현승고(P＜0.01);이도소수체(InsR)、이도소수체저물(IRS)1、단백격매B(Akt)、AMPK、CREB전록공격활인자2(TORC2)적린산화수평균명현증가(P＜0.01);이차,당원합성매격매3β적린산화수평역명현증가,이PEPCK적단백표체명현강저,차이균유통계학의의(P＜0.01).결론 KLF14과표체가능통과활화이도소InsR/IRS/Akt경전통로,종이개선간세포적이도소저항,차가능통과AMPK/TORC2/PEPCK신호통로억제간당이생,병촉진당섭취화간당원적합성,종이개선당대사.
Objective To observe the effect of Kruppel-like factor 14 (KLF14) gene overexpression on the key molecules in insulin and AMP-activated protein kinase (AMPK) signal pathways,and to investigate the molecular mechanism underlying KLF14-regulated glucose metabolism.Methods KLF14 gene was overexpressed in Hepa1-6 hepatocytes,which was divided into four groups:empty vector group (EV),insulin treatment group (INS),KLF14 transfection group,and insulin plus KLF14 group.Glucose uptake was determined by 2-deoxy-D-[3 H]-glucose uptake assay in hepatic cells.Protein phosphorylations of key signaling molecules and phosphoenolpyruvate carboxykinase (PEPCK) protein expression were detected by Western blot.Results Compared with EV group,the glucose uptake rate was increased significantly in groups with KLF14 transfection after Hepa1-6 hepatocytes were treated with insulin (P＜0.01).Furthermore,the phosphorylation of insulin receptor,insulin receptor substrate 1,protein kinase B (Akt),AMPK,and CREB-regulated transcription coactivator 2 (TORC2) were significantly enhanced after KLF14 transfection and insulin treatment (P＜0.01).In addition,the phosphorylation of glycogen synthase kinase 3β protein was also elevated while PEPCK protein expression was decreased (P＜0.01).Conclusions KLF14 gene overexpression may play a role in improving hepatic insulin resistance by activating the classical pathway of insulin.Additionally,it may ameliorate glucose metabolism via inhibiting gluconeogenesis,promoting glucose uptake,and glycogen synthesis.