目的:研究氧化应激在肝癌细胞(HepG2细胞)中对糖异生关键基因G6PC (glucose-6-phosphatase,G-6-pase)和GLUT4 (glucose transport protein 4)的影响,并揭示其潜在的调控机制。方法:利用双氧水(H2O2)处理肝癌细胞导致其细胞内氧化应激水...目的:研究氧化应激在肝癌细胞(HepG2细胞)中对糖异生关键基因G6PC (glucose-6-phosphatase,G-6-pase)和GLUT4 (glucose transport protein 4)的影响,并揭示其潜在的调控机制。方法:利用双氧水(H2O2)处理肝癌细胞导致其细胞内氧化应激水平升高,然后通过qRT-PCR检测G6PC基因和GLUT4基因在不同时间和不同浓度药物处理后的转录表达情况,再通过Western Blot技术检测G6PC和GLUT4在不同时间和不同浓度药物处理后的蛋白表达变化。最后,通过免疫荧光实验对比双氧水处理前后G6PC和GLUT4的蛋白表达情况以及转录因子FOXO1(Forkhead box O 1)的蛋白定位情况,从而揭示氧化应激在肝癌细胞中调控糖异生的作用机制。结果:(1) qRT-PCR实验结果表明G6PC基因和GLUT4基因的转录表达与H2O2处理的时间和浓度均呈正相关;(2)Western Blot实验结果表明G6PC和GLUT4的蛋白表达与H2O2处理的时间和浓度均呈负相关;(3) H2O2处理后,免疫荧光结果显示G6PC和GLUT4蛋白整体荧光亮度较对照组减弱,但G6PC和GLUT4在核内的荧光亮度较对照组稍强;(4) H2O2处理后,免疫荧光结果显示转录因子FOXO1在核内的荧光亮度较对照组减弱及胞质荧光亮度较对照组增强。结论:氧化应激能够诱导转录因子FOXO1从细胞质转入细胞核,从而导致HepG2肝癌细胞糖异生基因(G6PC和GLUT4)的转录表达升高。展开更多
Prostaglandin E-2(PGE(2)) is a well-known mediator of beta-cell dysfunction in both type 1 and type 2 diabetes.We recently reported that down-regulation of the Akt pathway activity is implicated in PGE(2)-induced panc...Prostaglandin E-2(PGE(2)) is a well-known mediator of beta-cell dysfunction in both type 1 and type 2 diabetes.We recently reported that down-regulation of the Akt pathway activity is implicated in PGE(2)-induced pancreatic beta-cell dysfunction.The aim of this study was to further dissect the signaling pathway of this process in pancreatic beta-cell line HIT-T15 cells and primary mouse islets.We found that PGE(2) time-dependently increased the c-Jun N-terminal kinase(JNK) pathway activity.JNK inhibition by the JNK-specific inhibitor SP600125 reversed PGE(2)-inhibited glucose-stimulated insulin secretion(GSIS).PGE(2) induced dephosphorylation of Akt and FOXO1, leading to nuclear localization and transactivation of FOXO1.Activation of FOXO1 induced nuclear exclusion but had no obvious effect on the whole-cell protein level of pancreatic and duodenal homeobox 1(PDX1).However, these effects were all attenuated by JNK inhibition.Furthermore, adenovirus-mediated overexpression of dominant-negative(DN)FOXO1 abolished whereas constitutively active(CA)-FOXO1 mimicked the effects of PGE(2) on GSIS in isolated mouse islets.In addition, we demonstrated that DN-JNK1 but not DN-JNK2 or CA-Akt abolished the PGE(2)-induced AP-1 luciferase reporter activity, whereas DN-JNK1 and CA-Akt but not DN-JNK2 reversed the effect of PGE(2) on FOXO1 transcriptional activity, and overexpression of DN-JNK1 rescued PGE(2)-impaired GSIS in mouse islets.Our results revealed that activation of the JNK is involved in PGE(2)induced beta-cell dysfunction.PGE(2)-mediated JNK1 activation, through dephosphorylation of Akt and FOXO1, leads to nuclear accumulation of FOXO1 and nucleocytoplasmic shuttling of PDX1, finally resulting in defective GSIS in pancreatic beta-cells.展开更多
文摘目的:研究氧化应激在肝癌细胞(HepG2细胞)中对糖异生关键基因G6PC (glucose-6-phosphatase,G-6-pase)和GLUT4 (glucose transport protein 4)的影响,并揭示其潜在的调控机制。方法:利用双氧水(H2O2)处理肝癌细胞导致其细胞内氧化应激水平升高,然后通过qRT-PCR检测G6PC基因和GLUT4基因在不同时间和不同浓度药物处理后的转录表达情况,再通过Western Blot技术检测G6PC和GLUT4在不同时间和不同浓度药物处理后的蛋白表达变化。最后,通过免疫荧光实验对比双氧水处理前后G6PC和GLUT4的蛋白表达情况以及转录因子FOXO1(Forkhead box O 1)的蛋白定位情况,从而揭示氧化应激在肝癌细胞中调控糖异生的作用机制。结果:(1) qRT-PCR实验结果表明G6PC基因和GLUT4基因的转录表达与H2O2处理的时间和浓度均呈正相关;(2)Western Blot实验结果表明G6PC和GLUT4的蛋白表达与H2O2处理的时间和浓度均呈负相关;(3) H2O2处理后,免疫荧光结果显示G6PC和GLUT4蛋白整体荧光亮度较对照组减弱,但G6PC和GLUT4在核内的荧光亮度较对照组稍强;(4) H2O2处理后,免疫荧光结果显示转录因子FOXO1在核内的荧光亮度较对照组减弱及胞质荧光亮度较对照组增强。结论:氧化应激能够诱导转录因子FOXO1从细胞质转入细胞核,从而导致HepG2肝癌细胞糖异生基因(G6PC和GLUT4)的转录表达升高。
文摘Prostaglandin E-2(PGE(2)) is a well-known mediator of beta-cell dysfunction in both type 1 and type 2 diabetes.We recently reported that down-regulation of the Akt pathway activity is implicated in PGE(2)-induced pancreatic beta-cell dysfunction.The aim of this study was to further dissect the signaling pathway of this process in pancreatic beta-cell line HIT-T15 cells and primary mouse islets.We found that PGE(2) time-dependently increased the c-Jun N-terminal kinase(JNK) pathway activity.JNK inhibition by the JNK-specific inhibitor SP600125 reversed PGE(2)-inhibited glucose-stimulated insulin secretion(GSIS).PGE(2) induced dephosphorylation of Akt and FOXO1, leading to nuclear localization and transactivation of FOXO1.Activation of FOXO1 induced nuclear exclusion but had no obvious effect on the whole-cell protein level of pancreatic and duodenal homeobox 1(PDX1).However, these effects were all attenuated by JNK inhibition.Furthermore, adenovirus-mediated overexpression of dominant-negative(DN)FOXO1 abolished whereas constitutively active(CA)-FOXO1 mimicked the effects of PGE(2) on GSIS in isolated mouse islets.In addition, we demonstrated that DN-JNK1 but not DN-JNK2 or CA-Akt abolished the PGE(2)-induced AP-1 luciferase reporter activity, whereas DN-JNK1 and CA-Akt but not DN-JNK2 reversed the effect of PGE(2) on FOXO1 transcriptional activity, and overexpression of DN-JNK1 rescued PGE(2)-impaired GSIS in mouse islets.Our results revealed that activation of the JNK is involved in PGE(2)induced beta-cell dysfunction.PGE(2)-mediated JNK1 activation, through dephosphorylation of Akt and FOXO1, leads to nuclear accumulation of FOXO1 and nucleocytoplasmic shuttling of PDX1, finally resulting in defective GSIS in pancreatic beta-cells.
