| 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...
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