丙型肝炎病毒核心蛋白下调AMP激活蛋白激酶导致肝细胞糖代谢紊乱  

HCV core protein down-regulates AMP-activated protein kinase and induces the glucose metabolism disorders in hepatocytes

作　　者：孙丽杰[1] 赵勇华[1] 李树臣[1] 于建武[1] 康鹏[1] 刘伟[2] 

机构地区：[1]哈尔滨医科大学附属第二医院感染病科,150086 [2]哈尔滨医科大学附属第二医院科研实验中心,150086

出　　处：《肝脏》2013年第6期374-377,397,共5页Chinese Hepatology

基　　金：黑龙江省教育厅科学技术研究项目(12531292)

摘　　要：目的探讨HCV核心蛋白对AMP激活蛋白激酶(AMPK)表达及肝细胞葡萄糖代谢的影响。方法表达HCV核心蛋白的质粒转染L02人肝细胞。分别应用液体闪烁计数仪、实时荧光定量-PCR、Western印迹检测AMPK活性、mRNA及蛋白的表达。应用放射同位素法和葡萄糖氧化酶法测定肝细胞葡萄糖摄取率和糖异生。采用6-磷酸葡萄糖脱氢酶偶联比色法和乳酸脱氢酶偶联比色法测定葡萄糖激酶(GK)和磷酸烯醇丙酮酸羧激酶(PEPCK)活性。RT-PCR检测AMPK下游调节糖代谢的基因的葡萄糖转运蛋白(GLUT)1、GLUT2、PEPCK、葡萄糖-6-磷酸酶(G6Pase)和GK mRNA水平。结果与转染空载体的L02人肝细胞相比,表达HCV核心蛋白L02人肝细胞AMPKα2活性[(0.2±0.05)比(1.0±0.3),t=6.443,P<0.01]、AMPKα2 mRNA[(0.3±0.05)比(1.0±0.3),t=5.638,P<0.01]及磷酸化-AMPK蛋白的表达水平[(0.2±0.05)比(0.6±0.2),t=4.753,P<0.01]明显下降,PEPCK活性[mIU/min/mg蛋白:(35.80±8.70)比(25.50±5.80),t=2.413,P<0.05]明显升高,而GK活性[mIU/min/mg蛋白:(1.70±0.35)比(3.55±0.84),t=4.929,P<0.01]明显降低,葡萄糖摄取率[cpm:(5000±800)比(20000±5000),t=7.256,P<0.01]及其调节基因GLUT2mRNA水平[(0.5±0.1)比(1±0.3),t=3.873,P<0.01]明显下降;糖异生[(2.5±0.5)比(1.0±0.2),t=6.823,P<0.01]及其调节基因PEPCK[(2.8±0.7)比(1.0±0.3),t=5.789,P<0.01]、G6Pase mRNA水平[(2.5±0.5)比(1.0±0.2),t=6.822,P<0.01]明显升高;GK mRNA水平[(0.6±0.1)比(0.9±0.3),t=2.324,P<0.05]明显下降。结论HCV核心蛋白下调AMPK活性及表达,改变其下游调节糖代谢相关基因表达,降低葡萄糖摄取能力,增加糖异生,导致肝细胞葡萄糖代谢紊乱。Objective The aim of this study was to investigate the effect of HCV core protein on expression of AMP- activated protein kinase （AMPK） and glucose metabolism of hepatocytes. Methods HCV core protein expression plasmid was transfected into L02 cells. The activity, levels of mRNA and protein of AMPK were detected by a scintillation counter, real time-PCR （RT-PCR） and western blot, respectively. Glucose uptaking rate by hepatocytes and gluconeogenesis were detected using radioactive isotope and glucose oxidase methods. Glucose-6-phosphate dehydrogenase couple chromatometry and lactate dehydrogenase couple chromatometry were used to determine the activity of glucokinase （GK） and phosphoenolpyruvate carboxykinase （PEPCK）. The mRNA levels of AMPK downstream glucose-metabolism genes [glucose transporter （GLUT） 1, GLUT 2, glucose 6-phosphatase （G6Pase） and GK] were measured by RT-PCR. Results In L02 cells expression HCV core protein, the activity （0.2 ± 0.05 vs. 1.0 ± 0.3, t= 6. 443, P〈0.01）, levels of mRNA （0.3± 0.05 vs. 1.0 ± 0.3, t = 5. 638, P〈0.01）, protein of phospho-AMPK （0.2 ± 0.05 vs. 0.6±0.2, t = 4. 753, P〈 0.01） were significantly decreased; The activity of PEPCK （mIU/min/mg protein： 35.80 ± 8.70 vs. 25.50±5.80, t = 2.413, P〈0.05） was significantly increased; The activity of GK （mIU/min/mg protein： 1.70 ± 0.35 vs. 3.55 ± 0.84, t =4, 929, P〈0. 01） was significantly decreased; Glucose uptaking rate （count per minute, cpm： 5000± 800 vs. 20000± 5000, t =7.256, P〈0.0I） and its regulatorgeneGLUT2 mRNAlevels （0.5-＋0.1 vs. 1＋0.3, t =3.873, P〈0.01） were significantly decreased; Gluconeogenesis （2.5 ± 0.5 vs. 1.0 ± 0.2, t = 6. 823, P〈0.01 ） and its regulator genes PEPCK （2.8±0.7 vs. 1.0±0.3, t=5.789, P〈0.01） and G6Pase mRNA levels（2.5＋0.5 vs. 1.0±0.2, t=6.822, P〈0.01） were significantly increased; The gene GK levels（0. 6 ±0. 1 vs. 0. 9 ± 0. 3, t = 2. 324, P〈0. 05） was significantly decreased. Conclus

关 键 词：丙型肝炎病毒 核心蛋白 AMP激活蛋白激酶 AMPK葡萄糖摄取 糖异生 

分 类 号：R363[医药卫生—病理学]