HIF-1与线粒体呼吸链的关系及对细胞糖代谢的影响
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摘要
目 的
1.研究线粒体呼吸链和ATP、ROS等对HIF-1α的稳定积聚的影响。
2. 探讨缺氧时HIF-1对 Glut-1和GK及ATP产生的影响。
材料和方法
大鼠肝BRL细胞,以DMEM培养基加小牛血清培养,分别分为正常对照组(21%O2)(常氧组),缺氧组(1%O2),各常氧和缺氧加阻断剂组(碘乙酸、鱼藤酮、抗霉素A、叠氮钠、2,4-二硝基苯酚),常氧和缺氧分别加ATP、H2O2、谷胱甘肽组,常氧加吲哚美辛组、缺氧组和缺氧加吲哚美辛组,缺氧4h。收获细胞,分别行Western Blot 检测各组的HIF-1α、Glut-1和GK蛋白;荧光检测二乙酸二氯荧光素测定各组的细胞内ROS产生;高效液相色谱法检测ATP和能荷;免疫荧光法检测细胞内HIF-1α的表达和核转位。RT-PCR检测各组的Glut-1和GK的mRNA含量。酶学方法检测GK的活性。
结 果
分别于常氧(21%O2)和缺氧(1% O2)条件下以由低到高不同浓度鱼藤酮、抗霉素和叠氮钠阻断线粒体呼吸链复合体I、III和IV 4h,Western Blotting检测HIF-1α蛋白,结果表明,缺氧时随各阻断剂浓度的增高,细胞内HIF-1α蛋白量逐渐减少到基本无表达;常氧情况下阻断线粒体呼吸链复合体I、III和IV,与未阻断一样,无HIF-1α蛋白稳定积聚。
与常氧(21%O2)对照组相比,缺氧(1%O2)4h时细胞ATP和能荷明显降低;常氧下采用碘乙酸阻断糖酵解或分别以鱼藤酮、抗霉素和叠氮钠阻断呼吸链复合体I 、III 、IV 4h,细胞ATP和能荷较对照组明显降低;缺氧时采用碘乙酸阻断糖酵解或分别以鱼藤酮、抗霉素和叠氮钠阻断呼吸链复合体I 、III 、IV 4h,细胞ATP和能荷均较单纯缺氧明显降低。
常氧情况下细胞有一定的ROS产生,缺氧4h时细胞的ROS产生减少,缺氧并应用碘乙酸阻断糖酵解或分别以鱼藤酮、抗霉素和叠氮钠阻断呼吸链复合体I 、III 、IV 4h,细胞ROS产生更加减少。
Western Blot 检测,BRL细胞常氧下HIF-1α仅微量表达,检测呈阴性,缺氧4h时其表达明显增加,缺氧同时以不同的浓度碘乙酸阻断糖酵解,BRL细胞的HIF-1α产
生稳定与单纯缺氧相比较无明显改变。在常氧(21%O2)和缺氧(1% O2)条件下应用2,4-二硝基苯酚使氧化磷酸化解藕联,BRL细胞ATP和ROS产生减少。常氧(21%O2)时应用2,4-二硝基苯酚低浓度时对HIF-1α无影响,高浓度时可诱导HIF-1α的稳定积聚。在缺氧(1% O2)条件下低剂量应用2,4-二硝基苯酚,使HIF-1α产生被抑制,高剂量时HIF-1α的产生增加。在常氧(21%O2)和缺氧(1% O2)条件下分别在细胞培养液中加入H2O2、ATP和谷胱甘肽对HIF-1α产生无明显影响。
免疫荧光检测表明,常氧(21%O2)时细胞有微量HIF-1α表达并核转位。缺氧(1% O2)时HIF-1α的稳定积聚增加,并大量核转位;缺氧(1% O2)时应用吲哚美辛抑制HIF-1α稳定积聚,细胞内HIF-1α含量和核转位明显减少。
RT-PCR检测表明,单纯缺氧(1% O2)BRL细胞内Glut-1和GK的mRNA较常氧(21% O2)时明显增加。Western Blot检测表明,单纯缺氧(1% O2)BRL细胞内Glut-1和GK蛋白量较常氧(21% O2)时明显增加。缺氧(1% O2)时以吲哚美辛抑制HIF-1α稳定积聚后Glut-1和GK的mRNA增加受抑制。缺氧(1% O2)时以吲哚美辛抑制HIF-1α稳定积聚4h后,Glut-1和GK蛋白增加受到抑制。缺氧(1% O2)后GK活性明显增强。缺氧(1% O2)时以吲哚美辛抑制HIF-1α稳定积聚4h后GK的活性较单纯缺氧对照组降低。缺氧(1% O2)时BRL细胞ATP产生减少,缺氧(1% O2)时以吲哚美辛抑制HIF-1α稳定积聚4h时ATP 较对照组降低。
结 论
在大鼠肝细胞(BRL),细胞单纯缺氧时HIF-1α积聚和稳定反应需要具有完整功能的线粒体呼吸链的支持。
在大鼠肝细胞(BRL),缺氧时ATP和ROS的量改变不是HIF-1α积聚和稳定的原因,缺氧时此两者的改变可能只是伴随状态。
缺氧时HIF-1可以诱导Glut-1和GK的转录和表达。抑制HIF-1α的稳定积聚时,可以减少缺氧时其调控的Glut-1和GK的表达,并抑制GK在缺氧时增强的活性,影响糖酵解和细胞能量的产生。
Hypoxia-inducible factor 1(HIF-1) is a heterodimeric transcription factor. HIF-1 protein consists of two subunits, HIF-1αand HIF-1 β,both subunits belong to a family of basic helix-loop-helix transcription factors containing a PER-ARNT-SIM homology domain. HIF-1αis the subunit regulated by cellular O2 tension. HIF-1 regulates transcriptional activation of genes (more than 60), including erythropoietin, vascular endothelial growth factor, glycolytic enzymes, and glucose transporters. Under normoxic conditions HIF-1α protein undergoes rapid degradation by a proteasome. Hypoxia increases HIF-1α protein stability by inhibiting its degradation. It is a master regulator of oxygen homeostasis in cell and tissue , and plays a critical roles in physiological and pathological process. There are many research on the regulation of HIF-1α in hypoxia, but the detail of the regulation in HIF-1α is still not clear. The mechanisms of sensing low oxygen and transducing this signal to activate HIF-1α are not well understood.
Mitochondrion is the major organella that consuming oxygen in cell, and oxidative phosphorylation and energy production were taken place in it. The involvement of mitochondria in the regulation of HIF-1α has been postulated, and some models based on the role of the mitochondrial electron transport chain in hypoxia response were set up. But the results are paradoxical: some have proposed hypoxia response based on the production of ROS by the electron transport chain in mitochondria; other thinks that the mitochondria was not involved in the hypoxia response; and some other various result. This study was undertaken to determine the consequences of inhibition on electron transport chain in hypoxic induction of HIF-1 protein, the role of ROS and ATP in the hypoxia response, and the effect of inhibition of HIF-1αon Glut-1 and GK in cultured BRL cells.
Objective:
To investigate the effect of inhibition on mitochondria respiratory chain on HIF-1α, and observe the role of ATP and ROS in the progress; research the effect of inhibiting expression of HIF-1αon the Glut-1 and GK expression ,and the production of ATP of cell.
Material and Methods
Cell Culture and Reagents A rat liver cell line BRL (Institute of cell biology Shanghai) was maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin solution.
Group and Treatment Cells were pretreated with or without iodoacetic acid, rotenone , antimycin A, sodium azide , 2,4- dinitrophenol and indomethacin exposed to normoxia or hypoxia (1% O2, 5% CO2, 94% N2) for 1-4h in plexiglass modular chambers.
Western Blotting Analysis Cells were harvested in cold PBS,RIPA to extracts protein.Whole extracts were prepared, and 20μg of protein was subjected to electrophoresis. It was separated in SDS-6% polyacrylamide gel and transferred to PVDF membranes using standard procedures.
Measurement of ROS Intracellular ROS generation was assessed using 2',7'- dichlorofluorescein diacetate (Molecular Probe).
HPLC(high performance liquid chromatography) assay cell ATP, ADP, AMP.
Immunofluorescence examine the expression of cytoplasmic HIF-1αand its nuclear translocation in cell.
RT-PCR test the mRNA of Glut-1 and GK.
Enzyme method measure the activity of GK.
Results
Blocking of electron transfer at complex I, III and IV by rotenone , antimycin A, sodium azide resulted in inhibition of the accumulation of HIF-1αesposed to the hypoxia. The accumulation of HIF-1αin hypoxia did not involved in inhibition of glycolysis by iodoacetic acid both in nomorxia and hypoxia. In normoxia the all inhibitors mentioned above do not affect the expression of HIF-1α.
Low concentration of 2,4- dinitrophenol inhibits the accumulation of HIF-1αin hypoxia,high concentration of 2,4- dinitrophenol results in accumulation of HIF-1α.
In hypoxia the production of ROS is decreased, and the inhibitors decrease the trend.The addition of extrinsic ATP,H2O2 and glutathione can not changed the HIF-1αaccumulation in hypoxia.
In
1.研究线粒体呼吸链和ATP、ROS等对HIF-1α的稳定积聚的影响。
2. 探讨缺氧时HIF-1对 Glut-1和GK及ATP产生的影响。
材料和方法
大鼠肝BRL细胞,以DMEM培养基加小牛血清培养,分别分为正常对照组(21%O2)(常氧组),缺氧组(1%O2),各常氧和缺氧加阻断剂组(碘乙酸、鱼藤酮、抗霉素A、叠氮钠、2,4-二硝基苯酚),常氧和缺氧分别加ATP、H2O2、谷胱甘肽组,常氧加吲哚美辛组、缺氧组和缺氧加吲哚美辛组,缺氧4h。收获细胞,分别行Western Blot 检测各组的HIF-1α、Glut-1和GK蛋白;荧光检测二乙酸二氯荧光素测定各组的细胞内ROS产生;高效液相色谱法检测ATP和能荷;免疫荧光法检测细胞内HIF-1α的表达和核转位。RT-PCR检测各组的Glut-1和GK的mRNA含量。酶学方法检测GK的活性。
结 果
分别于常氧(21%O2)和缺氧(1% O2)条件下以由低到高不同浓度鱼藤酮、抗霉素和叠氮钠阻断线粒体呼吸链复合体I、III和IV 4h,Western Blotting检测HIF-1α蛋白,结果表明,缺氧时随各阻断剂浓度的增高,细胞内HIF-1α蛋白量逐渐减少到基本无表达;常氧情况下阻断线粒体呼吸链复合体I、III和IV,与未阻断一样,无HIF-1α蛋白稳定积聚。
与常氧(21%O2)对照组相比,缺氧(1%O2)4h时细胞ATP和能荷明显降低;常氧下采用碘乙酸阻断糖酵解或分别以鱼藤酮、抗霉素和叠氮钠阻断呼吸链复合体I 、III 、IV 4h,细胞ATP和能荷较对照组明显降低;缺氧时采用碘乙酸阻断糖酵解或分别以鱼藤酮、抗霉素和叠氮钠阻断呼吸链复合体I 、III 、IV 4h,细胞ATP和能荷均较单纯缺氧明显降低。
常氧情况下细胞有一定的ROS产生,缺氧4h时细胞的ROS产生减少,缺氧并应用碘乙酸阻断糖酵解或分别以鱼藤酮、抗霉素和叠氮钠阻断呼吸链复合体I 、III 、IV 4h,细胞ROS产生更加减少。
Western Blot 检测,BRL细胞常氧下HIF-1α仅微量表达,检测呈阴性,缺氧4h时其表达明显增加,缺氧同时以不同的浓度碘乙酸阻断糖酵解,BRL细胞的HIF-1α产
生稳定与单纯缺氧相比较无明显改变。在常氧(21%O2)和缺氧(1% O2)条件下应用2,4-二硝基苯酚使氧化磷酸化解藕联,BRL细胞ATP和ROS产生减少。常氧(21%O2)时应用2,4-二硝基苯酚低浓度时对HIF-1α无影响,高浓度时可诱导HIF-1α的稳定积聚。在缺氧(1% O2)条件下低剂量应用2,4-二硝基苯酚,使HIF-1α产生被抑制,高剂量时HIF-1α的产生增加。在常氧(21%O2)和缺氧(1% O2)条件下分别在细胞培养液中加入H2O2、ATP和谷胱甘肽对HIF-1α产生无明显影响。
免疫荧光检测表明,常氧(21%O2)时细胞有微量HIF-1α表达并核转位。缺氧(1% O2)时HIF-1α的稳定积聚增加,并大量核转位;缺氧(1% O2)时应用吲哚美辛抑制HIF-1α稳定积聚,细胞内HIF-1α含量和核转位明显减少。
RT-PCR检测表明,单纯缺氧(1% O2)BRL细胞内Glut-1和GK的mRNA较常氧(21% O2)时明显增加。Western Blot检测表明,单纯缺氧(1% O2)BRL细胞内Glut-1和GK蛋白量较常氧(21% O2)时明显增加。缺氧(1% O2)时以吲哚美辛抑制HIF-1α稳定积聚后Glut-1和GK的mRNA增加受抑制。缺氧(1% O2)时以吲哚美辛抑制HIF-1α稳定积聚4h后,Glut-1和GK蛋白增加受到抑制。缺氧(1% O2)后GK活性明显增强。缺氧(1% O2)时以吲哚美辛抑制HIF-1α稳定积聚4h后GK的活性较单纯缺氧对照组降低。缺氧(1% O2)时BRL细胞ATP产生减少,缺氧(1% O2)时以吲哚美辛抑制HIF-1α稳定积聚4h时ATP 较对照组降低。
结 论
在大鼠肝细胞(BRL),细胞单纯缺氧时HIF-1α积聚和稳定反应需要具有完整功能的线粒体呼吸链的支持。
在大鼠肝细胞(BRL),缺氧时ATP和ROS的量改变不是HIF-1α积聚和稳定的原因,缺氧时此两者的改变可能只是伴随状态。
缺氧时HIF-1可以诱导Glut-1和GK的转录和表达。抑制HIF-1α的稳定积聚时,可以减少缺氧时其调控的Glut-1和GK的表达,并抑制GK在缺氧时增强的活性,影响糖酵解和细胞能量的产生。
Hypoxia-inducible factor 1(HIF-1) is a heterodimeric transcription factor. HIF-1 protein consists of two subunits, HIF-1αand HIF-1 β,both subunits belong to a family of basic helix-loop-helix transcription factors containing a PER-ARNT-SIM homology domain. HIF-1αis the subunit regulated by cellular O2 tension. HIF-1 regulates transcriptional activation of genes (more than 60), including erythropoietin, vascular endothelial growth factor, glycolytic enzymes, and glucose transporters. Under normoxic conditions HIF-1α protein undergoes rapid degradation by a proteasome. Hypoxia increases HIF-1α protein stability by inhibiting its degradation. It is a master regulator of oxygen homeostasis in cell and tissue , and plays a critical roles in physiological and pathological process. There are many research on the regulation of HIF-1α in hypoxia, but the detail of the regulation in HIF-1α is still not clear. The mechanisms of sensing low oxygen and transducing this signal to activate HIF-1α are not well understood.
Mitochondrion is the major organella that consuming oxygen in cell, and oxidative phosphorylation and energy production were taken place in it. The involvement of mitochondria in the regulation of HIF-1α has been postulated, and some models based on the role of the mitochondrial electron transport chain in hypoxia response were set up. But the results are paradoxical: some have proposed hypoxia response based on the production of ROS by the electron transport chain in mitochondria; other thinks that the mitochondria was not involved in the hypoxia response; and some other various result. This study was undertaken to determine the consequences of inhibition on electron transport chain in hypoxic induction of HIF-1 protein, the role of ROS and ATP in the hypoxia response, and the effect of inhibition of HIF-1αon Glut-1 and GK in cultured BRL cells.
Objective:
To investigate the effect of inhibition on mitochondria respiratory chain on HIF-1α, and observe the role of ATP and ROS in the progress; research the effect of inhibiting expression of HIF-1αon the Glut-1 and GK expression ,and the production of ATP of cell.
Material and Methods
Cell Culture and Reagents A rat liver cell line BRL (Institute of cell biology Shanghai) was maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin solution.
Group and Treatment Cells were pretreated with or without iodoacetic acid, rotenone , antimycin A, sodium azide , 2,4- dinitrophenol and indomethacin exposed to normoxia or hypoxia (1% O2, 5% CO2, 94% N2) for 1-4h in plexiglass modular chambers.
Western Blotting Analysis Cells were harvested in cold PBS,RIPA to extracts protein.Whole extracts were prepared, and 20μg of protein was subjected to electrophoresis. It was separated in SDS-6% polyacrylamide gel and transferred to PVDF membranes using standard procedures.
Measurement of ROS Intracellular ROS generation was assessed using 2',7'- dichlorofluorescein diacetate (Molecular Probe).
HPLC(high performance liquid chromatography) assay cell ATP, ADP, AMP.
Immunofluorescence examine the expression of cytoplasmic HIF-1αand its nuclear translocation in cell.
RT-PCR test the mRNA of Glut-1 and GK.
Enzyme method measure the activity of GK.
Results
Blocking of electron transfer at complex I, III and IV by rotenone , antimycin A, sodium azide resulted in inhibition of the accumulation of HIF-1αesposed to the hypoxia. The accumulation of HIF-1αin hypoxia did not involved in inhibition of glycolysis by iodoacetic acid both in nomorxia and hypoxia. In normoxia the all inhibitors mentioned above do not affect the expression of HIF-1α.
Low concentration of 2,4- dinitrophenol inhibits the accumulation of HIF-1αin hypoxia,high concentration of 2,4- dinitrophenol results in accumulation of HIF-1α.
In hypoxia the production of ROS is decreased, and the inhibitors decrease the trend.The addition of extrinsic ATP,H2O2 and glutathione can not changed the HIF-1αaccumulation in hypoxia.
In
引文
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Fandrey J., Frede S., Jelkmann W., Role of hydrogen peroxide in hypoxia-induced erythropoietin production, Biochem. J. 1994;303(3) :507–510.
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Chandel NS, McClintock DS, Feliciano CE,et al Reactive Oxygen Species Generated at Mitochondrial Complex III Stabilize Hypoxia-inducible Factor-1a during Hypoxia A MECHANISM OF O2 SENSING. J Biol Chem,2000;275( 33):25130–25138.
Agani FH, Pichiule P, Chavez JC, LaManna JC.The role of mitochondria in the regulation of hypoxia-inducible factor 1 expression during hypoxia. J Biol Chem. 2000;275(46):35863-35867.
Srinivas V, Leshchinsky I, Sang N, et al.Oxygen sensing and HIF-1 activation does not require an active mitochondrial respiratory chain electron-transfer pathway. J Biol Chem. 2001;276(25):21995-21998..
Vaux EC, Metzen E, Yeates KM, et al. Regulation of hypoxia- inducible factor is preserved in the absence of a functioning mitochondrial respiratory chain, Blood, 2001;98 (1) :296–302.
Enomoto N, Koshikawa N, Gassmann M, et al.Hypoxic induction of hypoxia-inducible factor-1alpha and oxygen-regulated gene expression in mitochondrial DNA-depleted HeLa cells. Biochem Biophys Res Commun. 2002;297(2):346-352.
Mateo J, Garcia-Lecea M, Cadenas S, et al.Regulation of hypoxia-inducible factor-1alpha by nitric oxide through mitochondria-dependent and -independent pathways.Biochem J. 2003;376(Pt 2):537-544.
Haddad JJ.Recombinant human interleukin (IL)-1 beta-mediated regulation of hypoxia-inducible factor-1 alpha (HIF-1 alpha) stabilization, nuclear translocation and activation requires an antioxidant/reactive oxygen species (ROS)-sensitive mechanism. Eur Cytokine Netw. 2002;13(2):250-260.
Jones MK, Szabo IL , Kawanaka H,et al. von Hippel Lindau tumor suppressor and HIF-1_:new targets of NSAIDs inhibition of hypoxia-induced angiogenesis1. FASEB 2002; 16(1): 264-266.
Wang ZY,Gleichmann H,Glut2 in pancreatic islets crucial target molecule in diabets induced with mutiple low doses of streptozotocin in mice.Diabets,1998;47:50-56.
Gradin K, McGuire J, Wenger RH, et al.Functional interference between hypoxia and dioxin signal transduction pathways: competition for recruitment of the Arnt transcription factor.Mol Cell Biol. 1996 ;16(10):5221-5231.
Gradin K, McGuire J, Wenger RH, et al. Huang LE, Arany Z, Livingston DM, et al.Activation of hypoxia-inducible transcription factor depends primarily upon redox-sensitive stabilization of its alpha subunit.J Biol Chem. 1996;271(50):32253-32259.
Berra E, Benizri E, Ginouves A,et al. HIF prolyl-hydroxylase 2 is the key oxygen sensor setting low steady-state levels of HIF-1alpha in normoxia.EMBO J. 2003 ;22(16):4082-4090.
Shetty M,Ismail BN, Loeb JN,et al. Tnduction of Glut-1 mRNA in response to inhibition of oxidative phosphorylation. Am J Physiol,265:c1224-1229.
Stephens JM,Pekala PH.Transcriptional repression of the C/EBP-alpha and Glut4 genes in 3T3-L1 adipocytes by tumor necrosis factor-alpha. Regulations is coordinate and independent of protein synthesis .J Biol Chem,1992,267:1358.-13584.
Behrooz A,Ismail-Beigi F.Dual control of Glut1 glucose transpoter gene expression by hypoxia and by inhibition of oxidative phosphorylation.J Biol Chem, 1997,272: 5555-5562.
King MP, Attardi G. Isolation of human cell lines lacking mitochondrial DNA. Methods Enzymol. 1996;264:304-313.