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

Open AccessPublished:May 24, 2002DOI:https://doi.org/10.1074/jbc.M204152200
      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 (
      • Treins C.
      • Giorgetti-Peraldi S.
      • Murdaca J.
      • Van Obberghen E.
      ). 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.
      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
      Insulin controls glucose and lipid metabolism, regulates protein synthesis, and promotes cell growth and differentiation. Following ligand binding, the insulin receptor tyrosine kinase is activated, leading to receptor autophosphorylation and the subsequent phosphorylation of intracellular proteins including insulin receptor substrates 1 and 2 and Shc. These initial events stimulate multiple signaling cascades that mediate cellular responses to the hormone (
      • Withers D.J.
      • White M.
      ). Among the substrates of the insulin receptor, insulin receptor substrates 1 and 2 are involved mainly in the activation of the PI3K1 pathway, whereas Shc participates in the activation of the Ras/MAPK cascade. The Ras/MAPK and PI3K pathways have been implicated in insulin-induced gene transcription (
      • Saltiel A.R.
      • Pessin J.E.
      ,
      • Van Obberghen E.
      • Baron V.
      • Delahaye L.
      • Emanuelli B.
      • Filippa N.
      • Giorgetti-Peraldi S.
      • Lebrun P.
      • Mothe-Satney I.
      • Peraldi P.
      • Rocchi S.
      • Sawka-Verhelle D.
      • Tartare-Deckert S.
      • Giudicelli J.
      ). The activated MAPK phosphorylates transcription factors such as p62TCF involved in the transcription of genes that are implicated in proliferation and differentiation in response to insulin (
      • Saltiel A.R.
      • Kahn C.R.
      ). In contrast, insulin regulates the expression of genes involved in glucose metabolism through a PI3K-dependent pathway. Thus, insulin inhibits the transcription of genes encoding PEPCK, the rate-limiting enzyme in gluconeogenesis, and glucose-6-phosphatase through a PI3K pathway (
      • Sutherland C.
      • O'Brien R.M.
      • Granner D.K.
      ,
      • Dickens M.
      • Svitek C.A.
      • Culbert A.A.
      • O'Brien R.M.
      • Tavare J.M.
      ). Furthermore, a PI3K-dependent pathway is involved in the regulation of gene expression of lipogenic enzymes by insulin such as FAS (fatty-acid synthase) (
      • Wang D.
      • Sul H.S.
      ). Finally, insulin also regulates the expression of genes implicated in the angiogenic response such as erythropoietin (EPO) and vascular endothelial growth factor (VEGF), but the molecular details of this action are lacking (
      • Masuda S.
      • Chikuma M.
      • Sasaki R.
      ,
      • Lu M.
      • Amano S.
      • Miyamoto K.
      • Garland R.
      • Keough K.
      • Qin W.
      • Adamis A.P.
      ).
      VEGF is a key angiogenic factor involved in a wide variety of biological processes including embryonic development, wound healing, tumor progression, and metastasis. VEGF has emerged as a major mediator of intraocular neovascularization and as such plays a key role in the etiology of diabetic retinopathy (
      • Duh E.
      • Aiello L.P.
      ). Indeed, it has been observed that intraocular VEGF levels are increased in diabetic patients suffering from proliferative retinopathy (
      • Funatsu H.
      • Yamashita H.
      • Noma H.
      • Shimizu E.
      • Yamashita T.
      • Hori S.
      ). VEGF expression is mainly regulated by tissue oxygen content (
      • Ikeda E.
      • Achen M.G.
      • Breier G.
      • Risau W.
      ,
      • Levy A.P.
      • Levy N.S.
      • Wegner S.
      • Goldberg M.A.
      ) but also by growth factors and cytokines, including platelet-derived growth factor, epidermal growth factor, insulin, insulin-like growth factor-I, tumor necrosis factor α, and transforming growth factor β (
      • Wang D.
      • Huang H.J.
      • Kazlauskas A.
      • Cavenee W.K.
      ,
      • Maity A.
      • Pore N.
      • Lee J.
      • Solomon D.
      • O'Rourke D.M.
      ,
      • Goldman C.K.
      • Kim J.
      • Wong W.L.
      • King V.
      • Brock T.
      • Gillespie G.Y.
      ,
      • Miele C.
      • Rochford J.J.
      • Filippa N.
      • Giorgetti-Peraldi S.
      • Van Obberghen E.
      ,
      • Punglia R.S., Lu, M.
      • Hsu J.
      • Kuroki M.
      • Tolentino M.J.
      • Keough K.
      • Levy A.P.
      • Levy N.S.
      • Goldberg M.A.
      • D'Amato R.J.
      • Adamis A.P.
      ,
      • Ryuto M.
      • Ono M.
      • Izumi H.
      • Yoshida S.
      • Weich H.A.
      • Kohno K.
      • Kuwano M.
      ). Hypoxia stimulates VEGF expression through at least three mechanisms including increased gene transcription, regulation at the translational level, and mRNA stabilization (
      • Levy A.P.
      • Levy N.S.
      • Wegner S.
      • Goldberg M.A.
      ,
      • Stein I.
      • Neeman M.
      • Shweiki D.
      • Itin A.
      • Keshet E.
      ). The transcriptional regulation of VEGF is mediated by the transcription factor hypoxia-inducible factor 1 (HIF-1) (
      • Wang G.L.
      • Semenza G.L.
      ,
      • Semenza G.L.
      ,
      • Semenza G.L.
      ). HIF-1 is a basic helix-loop-helix transcription factor, which is composed of two subunits, HIF-1α and HIF-1β. HIF-1β, also known as the arylhydrocarbon nuclear translocator, is constitutively expressed, whereas HIF-1α expression is increased upon hypoxia. In normoxia, HIF-1α is rapidly ubiquitinated by the von Hippel-Lindau tumor suppressor E3 ligase complex and subjected to proteasomal degradation (
      • Jaakkola P.
      • Mole D.R.
      • Tian Y.M.
      • Wilson M.I.
      • Gielbert J.
      • Gaskell S.J.
      • Kriegsheim A.
      • Hebestreit H.F.
      • Mukherji M.
      • Schofield C.J.
      • Maxwell P.H.
      • Pugh C.W.
      • Ratcliffe P.J.
      ). Under hypoxic conditions, HIF-1α is not degraded and accumulates to form an active complex with HIF-1β. HIF-1 regulates the transcription of numerous genes involved in vascular development (VEGF, EPO, and heme oxygenase 1), in glucose and energy metabolism (glucose transporters and glycolytic enzymes), in iron metabolism (transferrin), and in cell proliferation and viability (insulin-like growth factor-2 and insulin-like growth factor-binding protein-1). It has been shown that insulin increases VEGF expression through a PI3K-dependent pathway in fibroblasts overexpressing insulin receptors (
      • Miele C.
      • Rochford J.J.
      • Filippa N.
      • Giorgetti-Peraldi S.
      • Van Obberghen E.
      ). However, the identity and regulation of the transcription factor involved in this process remain unknown.
      Here we report that insulin stimulates HIF-1α subunit accumulation, HIF-1 activation, and VEGF expression. Our results show that insulin regulates HIF-1α expression through a translation-dependent pathway. Moreover, insulin-induced HIF-1 regulation and VEGF expression require a PI3K/TOR-dependent pathway.

      DISCUSSION

      Diabetic retinopathy is the major cause of blindness in Western countries. VEGF is involved in the pathogenesis of both background and proliferative retinopathy. Intraocular VEGF is increased in eyes from patients with blood-retinal barrier breakdown and neovascularization. Clinical studies have demonstrated that long term insulin therapy reduces the risk of diabetic retinopathy progression. However, it has also been observed that intensive insulin therapy leads to a transient worsening of retinopathy characterized by a blood-retinal barrier breakdown (
      • Agardh C.D.
      • Eckert B.
      • Agardh E.
      ,
      • Brinchmann-Hansen O.
      • Dahl-Jorgensen K.
      • Sandvik L.
      • Hanssen K.F.
      ,
      • Moskalets E.
      • Galstyan G.
      • Starostina E.
      • Antsiferov M.
      • Chantelau E.
      ). It has been proposed that the worsening of retinopathy could be attributed to chronic hyperinsulinemia induced by insulin treatment. Indeed, it has been shown that insulin stimulates VEGF expression, which in turn would stimulate neovascularization (
      • Poulaki V.
      • Qin W.
      • Joussen A.M.
      • Hurlbut P.
      • Weigand S.J.
      • Rudge J.
      • Yancopoulos G.D.
      • Adamis A.P.
      ,
      • Zelzer E.
      • Levy Y.
      • Kahana C.
      • Shilo B.Z.
      • Rubinstein M.
      • Cohen B.
      ). However, the molecular mechanisms involved in insulin-induced VEGF expression remain unknown. In this study, we show that insulin stimulates VEGF expression through the activation of the transcription factor HIF-1. This activation is regulated by a PI3K-dependent signaling pathway involving TOR. Moreover, in contrast to hypoxia, which is a major activator of HIF-1, insulin does not regulate HIF-1α through the inhibition of its degradation but through a translation-dependent mechanism.
      In ARPE-19 cells, insulin stimulates the accumulation of the regulated subunit HIF-1α. An increase in HIF-1α expression is directly correlated with the activity of the transcription factor HIF-1. Indeed, we show that insulin induces increased HIF-1α protein levels, augmented HIF-1 DNA binding activity, and stimulation of HIF-1-mediated reporter gene transcription. In normoxic conditions, HIF-1α is maintained at low levels by a degradation process involving the ubiquitin-proteasome system (
      • Salceda S.
      • Caro J.
      ,
      • Huang L.E., Gu, J.
      • Schau M.
      • Bunn H.F.
      ). Hypoxia rapidly increases the amount of HIF-1α by inhibiting its proteasome-dependent degradation. Surprisingly, insulin does not seem to act on HIF-1α degradation. A comparison of the half-life of HIF-1α after the removal of insulin or in presence of both insulin and cycloheximide, a translation inhibitor, shows that insulin does not stabilize the HIF-1α protein. Furthermore, insulin does not affect the transcription of HIF-1α mRNA, suggesting that it regulates HIF-1α translation. Nevertheless, we cannot exclude the possibility that insulin regulates the translation of a protein, which inhibits HIF-1α degradation. It is of interest to note that a recent study shows that heregulin, which activates the tyrosine kinase receptor HER2, stimulates HIF-1α synthesis (
      • Laughner E.
      • Taghavi P.
      • Chiles K.
      • Mahon P.C.
      • Semenza G.L.
      ), similar to our results concerning insulin action.
      In ARPE-19 cells, we found that the insulin effect on HIF-1α expression, HIF-1 activation, and VEGF expression are dependent on the PI3K·PKB·TOR pathway. In contrast, the MEK pathway does not appear to be required for insulin action on HIF-1. Both the MAPK and PI3K pathways have been implicated in HIF-1 regulation. The p42 and p44 MAPKs activate HIF-1 by promoting the phosphorylation of HIF-1α in response to hypoxia and its accumulation in response to advanced glycation end products or mersalyl (
      • Treins C.
      • Giorgetti-Peraldi S.
      • Murdaca J.
      • Van Obberghen E.
      ,
      • Richard D.E.
      • Berra E.
      • Gothie E.
      • Roux D.
      • Pouyssegur J.
      ,
      • Minet E.
      • Arnould T.
      • Michel G.
      • Roland I.
      • Mottet D.
      • Raes M.
      • Remacle J.
      • Michiels C.
      ,
      • Agani F.
      • Semenza G.L.
      ). PI3K-dependent pathways have been implicated in HIF-1 and VEGF expression in transformed cells (
      • Jiang B.H.
      • Zheng J.Z.
      • Aoki M.
      • Vogt P.K.
      ,
      • Zhong H.
      • Chiles K.
      • Feldser D.
      • Laughner E.
      • Hanrahan C.
      • Georgescu M.M.
      • Simons J.W.
      • Semenza G.L.
      ,
      • Blancher C.
      • Moore J.W.
      • Robertson N.
      • Harris A.L.
      ,
      • Jiang B.H.
      • Jiang G.
      • Zheng J.Z., Lu, Z.
      • Hunter T.
      • Vogt P.K.
      ). Moreover, the activation of PKB or inactivation of the tumor suppressor gene encoding phosphatase and tensin homolog deleted on chromosome 10, which dephosphorylates the PI3K reaction products phosphatidylinositol 3,4-biphosphate and phosphatidylinositol 3,4,5-triphosphate, increases HIF-1α protein levels and HIF-1-dependent reporter gene expression (
      • Laughner E.
      • Taghavi P.
      • Chiles K.
      • Mahon P.C.
      • Semenza G.L.
      ,
      • Zhong H.
      • Chiles K.
      • Feldser D.
      • Laughner E.
      • Hanrahan C.
      • Georgescu M.M.
      • Simons J.W.
      • Semenza G.L.
      ,
      • Jiang B.H.
      • Jiang G.
      • Zheng J.Z., Lu, Z.
      • Hunter T.
      • Vogt P.K.
      ,
      • Zundel W.
      • Schindler C.
      • Haas-Kogan D.
      • Koong A.
      • Kaper F.
      • Chen E.
      • Gottschalk A.R.
      • Ryan H.E.
      • Johnson R.S.
      • Jefferson A.B.
      • Stokoe D.
      • Giaccia A.J.
      ).
      TOR seems to be involved only partly in the insulin action on HIF-1 activity, because the inhibition of TOR does not completely abolish HIF-1α expression and HIF-1 activation. These results suggest that at least the two following pathways are involved in insulin-induced HIF-1 regulation, a PKB-dependent/TOR-independent pathway and a PKB/TOR-dependent pathway. The PKB-dependent/TOR-independent pathway remains unknown, because a direct phosphorylation of HIF-1α by PKB has been excluded (
      • Zundel W.
      • Schindler C.
      • Haas-Kogan D.
      • Koong A.
      • Kaper F.
      • Chen E.
      • Gottschalk A.R.
      • Ryan H.E.
      • Johnson R.S.
      • Jefferson A.B.
      • Stokoe D.
      • Giaccia A.J.
      ). However, PKB could be involved in the insulin regulation of HIF-1α translation. It has been previously shown that insulin stimulates protein synthesis by the activation of eIF2B (eukaryotic translation initiation factor 2B), an essential translation initiation factor, through a PI3K/PKB/glycogen synthase kinase-3 pathway (
      • Welsh G.I.
      • Stokes C.M.
      • Wang X.
      • Sakaue H.
      • Ogawa W.
      • Kasuga M.
      • Proud C.G.
      ,
      • Welsh G.I.
      • Miller C.M.
      • Loughlin A.J.
      • Price N.T.
      • Proud C.G.
      ). For the PKB/TOR-dependent pathway, it has been shown that TOR activity positively regulates translation. Insulin induces the phosphorylation of 4E-BP1 through a PI3K·PKB·TOR pathway (
      • Takata M.
      • Ogawa W.
      • Kitamura T.
      • Hino Y.
      • Kuroda S.
      • Kotani K.
      • Klip A.
      • Gingras A.C.
      • Sonenberg N.
      • Kasuga M.
      ). The phosphorylation of 4E-BP1 results in a decrease in its binding affinity for eukaryotic translation initiation factor 4E, an essential translation initiation factor. The subsequent dissociation of eIF-4E (eukaryotic translation initiation factor) from 4E-BP1 promotes cap structure-dependent translation initiation (
      • Sonenberg N.
      • Gingras A.C.
      ,
      • Lawrence J.C.
      • Fadden P.
      • Haystead T.A.
      • Lin T.A.
      ). We could hypothesize that insulin-activated TOR by phosphorylation of 4E-BP1 dissociates eukaryotic translation initiation factor 4E from 4E-BP1 and stimulates the translation initiation of the HIF-1α mRNA.
      In diabetes, several factors could be involved in the worsening of the diabetic retinopathy including (i) advanced glycation end products generated during hyperglycemia, (ii) hypoxia resulting from microvascular retinal occlusion, and (iii) hyperinsulinemia stimulating VEGF expression through the up-regulation of HIF-1 expression (
      • Treins C.
      • Giorgetti-Peraldi S.
      • Murdaca J.
      • Van Obberghen E.
      ,
      • Pe'er J.
      • Folberg R.
      • Itin A.
      • Gnessin H.
      • Hemo I.
      • Keshet E.
      ). Moreover, the co-treatment of retinal epithelial cells with both insulin and advanced glycation end products increases VEGF expression (
      • Treins C.
      • Giorgetti-Peraldi S.
      • Murdaca J.
      • Van Obberghen E.
      ). The advanced glycation end products and insulin activate HIF-1 through distinct pathways, because advanced glycation end-induced HIF-1 activation is dependent on MAPK, whereas insulin-induced HIF-1 activation is dependent on PI3K. Hypoxia blocks HIF-1α degradation, whereas growth factors acting through tyrosine kinase receptors would increase its synthesis. The combination of these different signals enhances the activation of the transcription factor leading to increased VEGF gene expression. The result would be an amplification of the angiogenic signal leading to further progression of diabetic retinopathy.
      In conclusion, we have shown that insulin activates the transcription factor HIF-1 through a PI3K·PKB·TOR-dependent pathway. Ours results suggest that insulin similar to heregulin regulates HIF-1α synthesis. It remains to established whether such an effect on HIF-1α synthesis is a general feature of receptor tyrosine kinases.

      Acknowledgments

      We thank P. Peraldi for critical reading of this paper. We also thank J. Plouet (Toulouse, France) for VEGF165 cDNA and B. Hemmings, S. Tartare-Deckert, and M. Montminy for the gift of antibodies.

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