title: Insulin-induced activation of hypoxia-inducible factor-1 requires generation of reactive oxygen species by NADPH oxidase.
abstract: Hypoxia-inducible factor (HIF)-1 activation in response to hypoxia requires mitochondrial generation of reactive oxygen species (ROS). In contrast, the requirement of ROS for HIF-1 activation by growth factors like insulin remains unexplored. To explore that, insulin-sensitive hepatic cell HepG2 or cardiac muscle cell H9c2 cells were pretreated with NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) or apocynin and HIF-1 activation was tested by electrophoretic mobility shift and reporter gene assay. Antioxidants DPI or apocynin completely blocked insulin-stimulated HIF-1 activation. The restoration of HIF-1 activation by H(2)O(2) in DPI-pretreated cells not only confirmed the role of ROS but also identified H(2)O(2) as the responsible ROS. The role of NADPH oxidase was further confirmed by greater stimulation of HIF-1 during simultaneous treatment of suboptimal concentration of insulin along with NADPH but not by NADH. The role of oxidant generated by insulin is found to inhibit the protein tyrosine phosphatase as suggested by the following observations. First, tyrosine phosphatase-specific inhibitor sodium vanadate compensates DPI-inhibited HIF-1 activity. Second, sodium vanadate stimulates HIF-1 activation with suboptimal concentration of insulin. Third, DPI and pyrrolidene dithiocarbamate (PDTC) blocks insulin-receptor tyrosine kinase activation. The activity of phosphatidylinositol 3-kinase as evidenced by Akt phosphorylation, involved in HIF-1 activation, is also dependent on ROS generation by insulin. Finally, DPI pretreatment blocked insulin-stimulated expression of genes like VEGF, GLUT1, and ceruloplasmin. Overall, our data provide strong evidence for the essential role of NADPH oxidase-generated ROS in insulin-stimulated activation of HIF-1.
authors.affiliation: Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi 110-067, India.
authors.forename: Sudipta
authors.initials: S
authors.lastname: Biswas
authors.forename: Manveen Kaur
authors.initials: MK
authors.lastname: Gupta
authors.forename: Debasis
authors.initials: D
authors.lastname: Chattopadhyay
authors.forename: Chinmay K
authors.initials: CK
authors.lastname: Mukhopadhyay
mesh_terms: D000098:Acetophenones / Q000494:pharmacology; D000975:Antioxidants / Q000494:pharmacology; D045744:Cell Line, Tumor; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D051793:Hypoxia-Inducible Factor 1 / Q000235:genetics / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009861:Onium Compounds / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011392:Proline / Q000031:analogs & derivatives / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism* / Q000494:pharmacology; D011972:Receptor, Insulin / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013859:Thiocarbamates / Q000494:pharmacology; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D014638:Vanadates / Q000494:pharmacology
chemical_list: D000098:Acetophenones; D000975:Antioxidants; D004791:Enzyme Inhibitors; D051793:Hypoxia-Inducible Factor 1; D007328:Insulin; D009861:Onium Compounds; D017382:Reactive Oxygen Species; D013859:Thiocarbamates; C066229:prolinedithiocarbamate; D014638:Vanadates; C007517:diphenyleneiodonium; D004247:DNA; D011392:Proline; C056165:acetovanillone; D006861:Hydrogen Peroxide; D019255:NADPH Oxidases; D019869:Phosphatidylinositol 3-Kinases; D011972:Receptor, Insulin; D017027:Protein Tyrosine Phosphatases