Insulin Stimulates Hypoxia-inducible Factor 1 through a Phosphatidylinositol 3-Kinase/Target of Rapamycin-dependent Signaling Pathway

doi:10.1074/jbc.M204152200

Insulin Stimulates Hypoxia-inducible Factor 1 through a Phosphatidylinositol 3-Kinase/Target of Rapamycin-dependent Signaling Pathway*

From the ^‡INSERM U145, Institut Fédératif de Recherche 50, Faculté de Médecine, Avenue de Valombrose, 06107 Nice Cedex 2, France and ^‖The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287-3914

Abstract

Hypoxia-inducible factor 1 (HIF-1) is a transcription factor involved in normal mammalian development and in the pathogenesis of several disease states. It consists of two subunits, HIF-1α, which is degraded during normoxia, and HIF-1β, which is constitutively expressed. Activated HIF-1 induces the expression of genes involved in angiogenesis, erythropoiesis, and glucose metabolism. We have previously reported that insulin stimulates vascular endothelial growth factor (VEGF) expression (1). In this study, we show that insulin activates HIF-1, leading to VEGF expression in retinal epithelial cells. Insulin activates HIF-1α protein expression in a dose-dependent manner with a maximum reached within 6 h. The expression of HIF-1α is correlated with the activation of HIF-1 DNA binding activity and the transactivation of a HIF-1-dependent reporter gene. Insulin does not appear to affect HIF-1α mRNA transcription but regulates HIF-1α protein expression through a translation-dependent pathway. The expression of an active form of protein kinase B and treatment of cells with specific inhibitors of phosphatidylinositol 3-kinase (PI3K), MAPK, and target of rapamycin (TOR) show that mainly PI3K and to a lesser extent TOR are required for insulin-induced HIF-1α expression. HIF-1 activity and VEGF expression are also dependent on PI3K- and TOR-dependent signaling. In conclusion, we show here that insulin regulates HIF-1 action through a PI3K/TOR-dependent pathway, resulting in increased VEGF expression.

Footnotes

↵* This work was supported in part by INSERM, the Association pour la Recherche contre le Cancer (Grant 5492), the University of Nice-Sophia Antipolis, Aventis Pharma Deutschland GmbH Grant 99206 (Frankfurt, Germany), and the European Community (QLG1-CT-1999-00674).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ Recipient of a fellowship from the Ministère de l'Enseignement Supérieur et de la Recherche (France).
↵¶ To whom correspondence should be addressed. Tel.: 33-4-93-81-54-47; Fax: 33-4-93-81-54-32; E-mail: peraldis@unice.fr.
Published, JBC Papers in Press, May 24, 2002, DOI 10.1074/jbc.M204152200
Abbreviations:

PI3K

phosphatidylinositol 3-kinase

ARPE

arising retinal pigment epithelial

EPO

erythropoietin

HIF-1

hypoxia-inducible factor 1

MAPK

mitogen-activated protein kinase

MEK

mitogen-activated protein kinase/extracellular signal-regulated kinase kinase

PKB

protein kinase B

CREB

cAMP-response element-binding protein

E3

ubiquitin-protein isopeptide ligase

PEPCK

phosphoenolpyruvate carboxykinase

TOR

target of rapamycin

VEGF

vascular endothelial growth factor

CoCl₂

cobalt chloride

4E-BP1

eukaryotic translation initiation factor 4E-binding protein

PKB-myr

constitutively active form of PKB
- Received April 29, 2002.
The American Society for Biochemistry and Molecular Biology, Inc.