肝脏线粒体烟酰胺腺嘌呤二核苷酸磷酸依赖性异柠檬酸脱氢酶参与葡萄糖代谢机制的研究

Effect and mechanism of liver mitochondrial nicotinamide adenine dinucleotide phosphate dependent isocitrate dehydrogenase on glucose metabolism

目的探讨肝细胞中线粒体烟酰胺腺嘌呤二核苷酸磷酸依赖性异柠檬酸脱氢酶(IDH2)对葡萄糖代谢机制的影响。方法分别用高糖或低糖处理小鼠正常肝细胞AML12细胞株,于24、48、72 h后检测IDH2 mRNA及蛋白表达。慢病毒转染法构建IDH2敲低(KD-IDH2组)、IDH2敲低对照(KD-Ctrl组)、IDH2过表达(OE-IDH2组)以及IDH2过表达对照(OE-Ctrl组)细胞株,分别用正常糖、高糖、低糖处理细胞,每组设置3个复孔。流式细胞学检测细胞内活性氧簇(ROS)生成及细胞凋亡。实时定量聚合酶链反应和Western blot法检测糖异生、糖摄取、糖酵解相关基因[己糖激酶1(HK1)、丙酮酸激酶(PKLR)]以及胰岛素、胰高糖素通路相关蛋白[磷酸肌醇3-激酶(PI3K)、磷酸化PI3K(p-PI3K)、蛋白激酶B(AKT)、磷酸化AKT(p-AKT)、蛋白激酶A(PKA)、磷酸化PKA(p-PKA)]的表达。在胰岛素、胰高糖素刺激下,检测低糖条件下肝细胞葡萄糖输出能力。通过多功能酶标仪检测2-NBDG摄取用于评估肝细胞糖摄取能力。两组间比较采用t检验。结果AML12细胞在高糖处理48 h后,IDH2 mRNA表达明显下降(P<0.01)。KD-IDH2组细胞IDH2 mRNA表达量为KD-Ctrl组的(35.67±10.60)%(P<0.01),OE-IDH2组IDH2 mRNA表达上调为OE-Ctrl的(4.59±0.77)倍(P<0.01)。KD-IDH2细胞在低糖条件下,葡萄糖输出能力明显减弱(P<0.01),糖摄取能力为KD-Ctrl组的(1.23±0.06)倍(P<0.01),AKT、PI3K蛋白活化程度(p-AKT/AKT、p-PI3K/PI3K)显著增加,PKA活化程度(p-PKA/PKA)降低(P<0.01),OE-IDH2组细胞则有相反表现。高糖处理使KD-IDH2细胞中糖酵解途径相关基因HK1、PKLR表达上调(P<0.05),细胞内ROS水平较高(P<0.05),同时细胞凋亡增加(P<0.01)。结论肝脏IDH2表达调控影响肝细胞内葡萄糖代谢,IDH2表达降低使细胞内胰岛素信号通路活化增加,且低糖条件下糖异生途径下调;而过表达IDH2通过增加肝细胞对胰高糖素的响应并降低对胰岛素的敏感性上调肝糖生成。

Objective To observe the effect of mitochondrial nicotinamide adenine dinucleotide phosphate dependent isocitrate dehydrogenase(IDH2)on glucose metabolism in mouse hepatocytes.Methods AML12 cells were respectively treated with high glucose(HG)and low glucose(LG)for 24,48 and 72 hours.mRNA and protein expression of IDH2 were detected.Lentiviral vectors were used to construct IDH2-knockdown cells(KD-IDH2)and related control cell line(KD-Ctrl),as well as IDH2-overexpression cells(OE-IDH2)and related control cells(OE-Ctrl).Cells were respectively treated with normal,high or low concentration of glucose,with 3 replications in each group.To assess intracellular reactive oxygen species(ROS)generation and cell apoptosis,flow cytometry was adopted for detection.Real-time quantitative reverse transcription polymerase chain reaction was used to detect mRNA expression levels of gluconeogenesis,glucose uptake,glycolysis-related genes[hexokinase 1(HK1),pyruvate kinase(PKLR)],and expressions of proteins in insulin and glucagon signaling pathway[phosphoinositide 3-kinase(PI3K),phospho-PI3K(p-PI3K),protein kinase B(AKT),phospho-AKT(p-AKT),protein kinase A(PKA),phospho-PKA(p-PKA)]were determined by Western blotting.After stimulation with insulin and glucagon,glucose output capacity of hepatocytes was detected under low glucose condition.Uptake of 2-NBDG was determined by a multifunctional microplate reader to evaluate capacity of glucose uptake in liver cells.Comparison between two groups was performed with Student′s t test.Results After 48 hours of high glucose treatment,expression of IDH2 in AML12 cells decreased significantly(P<0.01).In KD-IDH2 group,IDH2 mRNA expression was(35.67±10.60)%of its control(P<0.01),which in OE-IDH2 group was increased by(4.59±0.77)fold of the control(P<0.01).Under low glucose condition,reduced glucose output capacity(P<0.01),higher glucose uptake rate(1.23±0.06 unfold of KD-Ctrl,P<0.01),increased activation of proteins in AKT/PI3K signaling pathway and decreased activation of PKA(P<0.01)were significantly observed in KD-IDH2 cells,while OE-IDH2 cells had opposite performances.Treatment with high glucose upregulated the expressions of glycolytic pathway-related genes in KD-IDH2 cells,such as HK1 and PKLR(P<0.05).At the same time,elevated levels of intracellular ROS(P<0.05)and cell apoptosis rate(P<0.01)were also detected.Conclusions Regulation of liver IDH2 expression affected glucose metabolism in hepatocytes.Decreased expression of liver IDH2 enhanced sensitivity of hepatocytes to insulin and down-regulated gluconeogenesis in low glucose condition.Overexpression of IDH2 increased glucose production of hepatocytes by augmenting its response to glucagon and reducing its sensitivity to insulin.