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

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.

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 (2). Among the substrates of the insulin receptor, insulin receptor substrates 1 and 2 are involved mainly in the activation of the PI3K 1 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 (3,4). The activated MAPK phosphorylates transcription factors such as p62 TCF involved in the transcription of genes that are implicated in proliferation and differentiation in response to insulin (5). 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 ratelimiting enzyme in gluconeogenesis, and glucose-6-phosphatase through a PI3K pathway (6,7). Furthermore, a PI3K-dependent pathway is involved in the regulation of gene expression of lipogenic enzymes by insulin such as FAS (fattyacid synthase) (8). 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 (9,10).
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 (11). Indeed, it has been observed that intraocular VEGF levels are increased in diabetic patients suffering from proliferative retinopathy (12). VEGF expression is mainly regulated by tissue oxygen content (13,14) but also by growth factors and cytokines, including platelet-derived growth factor, epidermal growth factor, insulin, insulin-like growth factor-I, tumor necrosis factor ␣, and transforming growth factor ␤ (15)(16)(17)(18)(19)(20). Hypoxia stimulates VEGF expression through at least three mechanisms including increased gene transcription, regulation at the translational level, and mRNA stabilization (14,21). The transcriptional regulation of VEGF is mediated by the transcription factor hypoxia-inducible factor 1 (HIF-1) (22)(23)(24). HIF-1 is a basic helix-loop-helix transcription factor, which is composed of two subunits, HIF-1␣ and HIF-1␤. HIF-1␤, also known as the arylhydrocarbon nuclear translocator, is constitutively expressed, whereas HIF-1␣ expression is increased upon hypoxia. In normoxia, HIF-1␣ is rapidly ubiquitinated by the von Hippel-Lindau tumor suppressor E3 ligase complex and subjected to proteasomal degradation (25). Under hypoxic conditions, HIF-1␣ is not degraded and accumulates to form an active complex with HIF-1␤. 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 factorbinding protein-1). It has been shown that insulin increases VEGF expression through a PI3K-dependent pathway in fibroblasts overexpressing insulin receptors (18). However, the identity and regulation of the transcription factor involved in this process remain unknown.
Here we report that insulin stimulates HIF-1␣ subunit accumulation, HIF-1 activation, and VEGF expression. Our results show that insulin regulates HIF-1␣ expression through a translation-dependent pathway. Moreover, insulin-induced HIF-1 regulation and VEGF expression require a PI3K/TORdependent pathway.
All of the chemical reagents were purchased from Sigma. U0126 was purchased from Promega Inc. (Madison, WI). Oligonucleotides and culture media were purchased from Invitrogen.
DNA Plasmids-pGL2 basic P12 VEGF promoter corresponds to nucleotides from Ϫ1005 to Ϫ906 of the human VEGF promoter cloned upstream of a luciferase-coding sequence (27). The construct PKB-myr (constitutively active form of PKB) was a gift from B. Hemmings (F. Miescher Institute, Basel, Switzerland) (28). pCEP-MEK* is a constitutively active form of MEK that was obtained by deleting the region encompassing amino acids 32-52 and by mutating the two serine residues 218 and 222 to aspartic acid (29).
Transfection-ARPE-19 cells were transiently transfected using the FuGENE 6 transfection reagent (Roche Diagnostics) according to manufacturer's instructions. Sixteen hours after the addition of DNA, F12/ Dulbecco's modified Eagle's medium with 10% (v/v) fetal calf serum was changed, and before being stimulated, the cells were incubated for an additional 16 h with F12/Dulbecco's modified Eagle's medium with 0.2% (w/v) bovine serum albumin.
RNA Isolation and Northern Blot Analysis-Trizol reagent (Invitrogen) was used to extract total cellular RNA from confluent cells grown in 100-mm tissue culture plates according to the manufacturer's instructions. Cells were serum-deprived overnight in medium containing 0.2% (w/v) bovine serum albumin, and cells were pretreated with or not with inhibitors for 30 min and stimulated for the indicated lengths of time. RNA was extracted, and 10 g of total RNA were denatured in formamide and formaldehyde and separated by electrophoresis in formaldehyde-containing agarose gels. RNA was transferred to Hybond-N membranes (Amersham Biosciences) and cross-linked to the membrane by UV radiation. Human VEGF cDNA fragment or a PCR product encoding amino acids 330 -528 of human HIF-1␣ was used as a probe (30). The probes were labeled with [␥-32 P]dCTP by random priming using the Rediprime kit (Amersham Biosciences) and purified with the Probequant kit (Amersham Biosciences). The hybridizations were performed at 42°C in NorthernMax hybridization buffer (Ambion, Inc, Austin, TX). The membranes were washed in 1ϫ SSC, 0.5% (w/v) SDS, and radioactivity was quantitated using a Storm 840 (Amersham Biosciences).
Nuclear Extract Preparation and Electrophoretic Mobility Shift Assay-The nuclear extracts were prepared as described previously (1). Sense and antisense oligonucleotides corresponding to the HIF-1 binding site in the human EPO gene, 5Ј-GATCGCCCTACGTGCTGTCTCA-3Ј, were used (31). The oligonucleotides were annealed, and the doublestranded oligonucleotide (10 pmol) was labeled with T4 polynucleotide kinase and [␥-32 P]dATP. The probe was purified with the Probequant kit.
Binding reactions were performed as described previously (32). The reactions contained 10 g of nuclear extract and 0.1 g of denatured salmon sperm DNA (Sigma) in 10 mM Tris-HCl, pH 7.5, 50 mM KCl, 50 mM NaCl, 1 mM MgCl 2 , 1 mM EDTA, 5 mM dithiothreitol, and 5% (v/v) glycerol. After preincubation for 5 min at room temperature, the probe (2.5 ϫ 10 5 cpm) was added, and the incubation was continued for an additional 15 min, after which the reaction mixtures were loaded onto 5% nondenaturing polyacrylamide gels. Electrophoresis was performed at 185 V in 0.25ϫ Tris borate EDTA (22.25 mM Tris-HCl, 22.25 mM boric acid, and 1.25 mM EDTA) at 4°C. The gels were vacuum-dried, and radioactivity was determined on a Storm 840.
HIF-1 DNA Binding Assay-The nuclear extracts were prepared using a nuclear extract kit (Active Motif Europe, Rixensart, Belgium) according to manufacturer's instructions. HIF-1 binding to the hypoxia response element was assessed using Trans-AM HIF transcription factor assay kit (Active Motif Europe). In this assay, an oligonucleotide containing the HIF-1 binding site from the EPO gene is attached to a 96-well plate. The active form of HIF-1 contained in cell extracts specifically binds to this oligonucleotide and can be revealed by incubation with antibodies using enzyme-linked immunosorbent assay technology with absorbance reading. 10 g of nuclear extract were analyzed for HIF-1 binding to the oligonucleotide according to the manufacturer's instructions. The specificity for this assay was monitored by competition with free wild type or mutated oligonucleotide according to the manufacturer's instructions.
Luciferase Assays-To assay the transcriptional activity of HIF-1, we used the pGL2 basic P12 VEGF promoter vector, which contains a HIF-1 binding site (from Ϫ975 to Ϫ968) downstream from the luciferase gene (27). ARPE cells in 12-wells plates were transiently co-transfected with the reporter plasmid and with Rous sarcoma virus-␤-galactosidase as a control for transfection efficiency. The cells were stimulated for 16 h, and luciferase assays were performed as described in the protocols and applications guide (Promega). The luciferase activity was measured using a chemiluminometer Wallac 1420 (PerkinElmer Life Sciences). The ␤-galactosidase activity was performed as described in the Promega's protocols and applications guide. Cells lysates were incubated with a 2ϫ assay buffer (200 mM sodium phosphate buffer, pH 7.8, 2 mM MgCl 2 , 100 mM ␤-mercaptoethanol, 1.33 mg/ml o-nitrophenyl ␤-D-galactopyranoside). The absorbance at 420 nm was measured with a spectrophotometer Wallac 1420.

Insulin Stimulates HIF-1␣ Accumulation in Human Retinal
Epithelial Cells-We have previously shown that insulin induces VEGF expression in cell culture and in intact animals (1). To study the effect of the hormone on the transcription factor HIF-1, we first investigated the insulin effect on HIF-1␣ protein expression. ARPE-19 cells were treated for 4 h with insulin or with cobalt chloride (CoCl 2 ) as a positive control. Whole cell lysates and nuclear extracts were analyzed by Western blotting using an antibody to HIF-1␣ (Fig. 1, A and B). The divalent metal CoCl 2 is known to induce HIF-1␣ expression, HIF-1 DNA binding activity, and transactivation of HIF-1 target genes (33)(34)(35)(36). Indeed, the CoCl 2 treatment of cells led to an accumulation of HIF-1␣ in ARPE-19 cells. Insulin induces HIF-1␣ expression in both whole cell lysates and in nuclear extracts. As observed in Fig. 1, C and D, HIF-1␣ expression was induced by 0.1 nM insulin, and the maximal induction was seen at 100 nM. The expression of HIF-1␣ was transiently detectable within 1 h and maximal within 6 h and then returned to basal levels within 24 h of treatment. These results show that the incubation of ARPE-19 cells with insulin results in a time-and concentration-dependent elevation of HIF-1␣ protein levels.
Insulin Activates the Transcription Factor HIF-1-We next determined whether insulin-induced HIF-1␣ accumulation was correlated with an activation of the transcription factor HIF-1.
To do this, we measured the ability of HIF-1 to bind to DNA and to transactivate a HIF-1-dependent reporter gene. ARPE-19 cells were treated for 4 h with insulin or CoCl 2 , and the nuclear extracts were isolated. A double-stranded oligonucleotide containing the HIF-1 binding site present in the EPO gene was used as a probe in an electrophoretic mobility shift assay ( Fig. 2A). Both insulin and CoCl 2 induced a shift of the labeled probe. This binding was verified by an enzyme-linked immunosorbent assay test using an immobilized oligonucleo-tide to assess HIF-1 DNA binding activity (Fig. 2B). HIF-1 DNA binding activity was stimulated after 4 h of insulin or CoCl 2 treatment. The specificity of HIF-1 binding was tested by competition with free oligonucleotides. The binding was specific, because it was abolished in the presence of an excess of wild type oligonucleotide, whereas the mutated oligonucleotide had no effect. We next measured the transcriptional activity of HIF-1 using a SV40 promoter-luciferase unit downstream of a 99-bp hypoxia response element (pGL2 basic P12 VEGF promoter) relative to co-transfected Rous sarcoma virus-␤-galactosidase (Fig. 2C). After 16 h of insulin or CoCl 2 treatment, the luciferase activity in cell extracts was determined and normalized to the ␤-galactosidase activity. Insulin and CoCl 2 induced a statistically significant 1.55-and 3.5-fold increase (Ϯ0.0864 and Ϯ0.375, respectively) in luciferase activity, respectively. Therefore, the accumulation of HIF-1␣ subunit induced by insulin can be correlated with the activation of the transcription factor HIF-1.
Insulin Induces HIF-1␣ through a Translation-dependent Pathway-To obtain a better understanding of the processes involved in HIF-1␣ accumulation in response to insulin treatment, we investigated the effect of insulin on the amount of HIF-1␣ mRNA. ARPE-19 cells were stimulated with insulin or CoCl 2 for 6 h, RNA was extracted, and Northern blot analysis was performed using a HIF-1␣ cDNA probe (Fig. 3A). We found that insulin or CoCl 2 treatment did not modify HIF-1␣ mRNA expression, suggesting that insulin does not regulate HIF-1␣ mRNA transcription. HIF-1␣ has been shown to be degraded through the proteasome pathway during normoxia. Therefore, to study the effect of insulin on HIF-1␣ degradation, we looked at the effect of a specific inhibitor of proteasome, MG132 (Fig.  3B). ARPE-19 cells were incubated with insulin in the absence or presence of MG132. After 4 h, insulin was removed, and the level of HIF-1␣ protein was evaluated by Western blot using a HIF-1␣ antibody. As expected, in the absence of MG132, insulin induced an accumulation of HIF-1␣. Within 15 min after the removal of insulin, a decrease in HIF-1␣ protein could be seen, and HIF-1␣ was undetectable 30 min after withdrawal of the hormone. We observed that the inhibition of the proteasome by MG132 led to a high level of expression of HIF-1␣, even in the absence of insulin stimulation. This elevated the expression of HIF-1␣ in the presence of the proteasome inhibitor, preventing us from detecting any effect of insulin on HIF-1␣ degradation.
To analyze the insulin effect on HIF-1␣ synthesis, we performed a time course of HIF-1␣ disappearance in the presence of the protein translation inhibitor, cycloheximide (Fig. 3C). To this end, ARPE-19 cells were treated with insulin or CoCl 2 for 4 h, and cycloheximide was added for 15-60 min. In cells exposed to CoCl 2 , HIF-1␣ level remained constant over 60 min despite the lack of ongoing protein synthesis. This observation is consistent with previous studies showing that CoCl 2 had no effect on HIF-1␣ synthesis but blocked its degradation. In ARPE-19 cells treated with insulin, the addition of cycloheximide led to a decrease in HIF-1␣ expression starting at 15 min. After 60 min, HIF-1␣ was no longer detectable. Together, these results suggest that insulin increases HIF-1␣ protein levels through a translation-dependent pathway.
Constitutively Active PKB Induces HIF-1␣ Expression-We investigated the role of MEK-and PKB-dependent pathways in the regulation of HIF-1␣ in ARPE-19 cells. To this end, we transfected ARPE-19 cells with pcDNA 3 as a control with a constitutively active form of MEK (MEK*) or with a PKB-myr. The transfected cells were treated or not treated with insulin (100 nM), and whole cell lysates were prepared and analyzed by Western blotting using antibody to HIF-1␣ (Fig. 4). The expression of MEK* did not affect the amount of HIF-1␣ protein compared with control conditions. However, the expression of PKBmyr is sufficient to increase the HIF-1␣ protein level in basal conditions. In addition, when PKB-myr is expressed, insulin treatment does not further increase the level of HIF-1␣ protein.
Insulin Stimulates HIF-1␣ Accumulation and VEGF Expression through a PI3K/TOR-dependent pathway-To further evaluate the contribution of the PI3K pathway to the regulation of HIF-1␣ protein levels, we used the pharmacological inhibitors of PI3K and of a downstream effector, TOR. ARPE-19 cells were treated for 4 h with CoCl 2 or with insulin in the absence or presence of the inhibitors of PI3K (LY294002), TOR (rapamycin), or MEK (U0126). Whole cell lysates and nuclear extracts were prepared and analyzed by Western blotting using antibodies to HIF-1␣, Shc, or CREB (Fig. 5A). In agreement with the results obtained in Fig. 4, insulin increased HIF-1␣ expression. This increase was not affected by pretreatment with the MEK inhibitor U0126. Rather, the inhibition of PI3K activation by LY249002 totally blocked the expression of HIF-1␣ in response to insulin in both total cell lysates and in nuclear extracts. In addition, the treatment with rapamycin decreased HIF-1␣ protein after insulin stimulation by half the level. We confirmed that insulin-induced activation of PI3K, MAPK, and TOR was blocked by treatment with the respective inhibitor (data not shown).
To determine whether these inhibitors blocked HIF-1 activity, we measured the DNA binding activity of HIF-1 using an enzyme-linked immunosorbent assay test. ARPE-19 cells were stimulated with insulin in the absence or presence of LY294002, rapamycin, or U0126 (Fig. 5B). Insulin induced a 3-fold increase in DNA binding activity of HIF-1. The inhibition of PI3K by LY294002 totally blocked HIF-1 activation, whereas the inhibition of TOR by rapamycin induced a 40% decrease in insulin-induced HIF-1 activation. As expected, MEK does not appear to be involved, because the inhibition of MEK by U0126 had no effect on HIF-1 activity in response to insulin. Thus, the extent of inhibition of HIF-1␣ expression by the inhibitors tightly correlated with their ability to block insulin-induced DNA binding activity of HIF-1.
To examine whether this correlation could be extended to the HIF-1 activation and VEGF mRNA expression in response to insulin, we analyzed the VEGF mRNA expression profile after treatment with these inhibitors. ARPE-19 cells were treated with CoCl 2 or with insulin in the absence or presence of LY294002, U0126, or rapamycin. RNA was extracted, and Northern blot analyses were performed using a VEGF 165 cDNA probe (Fig. 6). Insulin and CoCl 2 induced a 3-and 5-fold increase in VEGF mRNA expression, respectively. The inhibition of PI3K blocked the insulin-induced VEGF mRNA expression. Moreover, rapamycin treatment resulted in a 40% inhibition. In contrast, blocking MEK did not affect the ability of insulin to induce VEGF mRNA expression in these cells. In summary, the insulin-induced accumulation of the HIF-1␣ subunit appears to be dependent on the PI3K/TOR pathway. This accumulation leads to HIF-1 transcriptional activity and VEGF mRNA expression.

FIG. 3. Insulin induces HIF-1␣ through a translation-dependent pathway.
A, ARPE-19 cells were stimulated with insulin (100 nM) or CoCl 2 (200 M) for 6 h. RNA was extracted and analyzed by Northern blotting using a probe corresponding to the HIF-1␣ cDNA. The blot was subsequently probed with 18 S rRNA as a control. B, ARPE-19 cells were treated or not for 4 h with the proteasome inhibitor MG132 (10 mM) and stimulated with insulin (100 nM). After 4 h, insulin was removed, and cells were incubated in Dulbecco's modified Eagle's medium containing 0.2% bovine serum albumin (BSA) for the indicated times. Whole cell lysates were prepared and analyzed by Western blotting using antibodies to HIF-1␣ or Shc. C, HIF-1␣ expression was induced by the exposure of ARPE-19 cells to insulin (100 nM) or CoCl 2 (200 M) for 4 h. Cycloheximide (CHX) was added to a final concentration of 10 g/ml, and the cells were harvested after being incubated for the indicated time in the presence of CHX and the inducer. Whole cell lysates were prepared and analyzed by Western blotting using antibodies to HIF-1␣ or Shc.

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 (37)(38)(39). 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 (40,41). 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-1␣ 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-1␣. An increase in HIF-1␣ expression is directly correlated with the activity of the transcription factor HIF-1. Indeed, we show that insulin induces increased HIF-1␣ protein levels, augmented HIF-1 DNA binding activity, and stimulation of HIF-1-mediated reporter gene transcription. In normoxic conditions, HIF-1␣ is maintained at low levels by a degradation process involving the ubiquitin-proteasome system (42,43). Hypoxia rapidly increases the amount of HIF-1␣ by inhibiting its proteasome-dependent degradation. Surprisingly, insulin does not seem to act on HIF-1␣ degradation. A comparison of the half-life of HIF-1␣ after the removal of insulin or in presence of both insulin and cycloheximide, a translation inhibitor, shows that insulin does not stabilize the HIF-1␣ protein. Furthermore, insulin does not affect the transcription of HIF-1␣ mRNA, suggesting that it regulates HIF-1␣ transla-tion. Nevertheless, we cannot exclude the possibility that insulin regulates the translation of a protein, which inhibits HIF-1␣ degradation. It is of interest to note that a recent study shows that heregulin, which activates the tyrosine kinase receptor HER2, stimulates HIF-1␣ synthesis (30), similar to our results concerning insulin action.
In ARPE-19 cells, we found that the insulin effect on HIF-1␣ expression, HIF-1 activation, and VEGF expression are dependent on the PI3K⅐PKB⅐TOR 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-1␣ in response to hypoxia and its accumulation in response to advanced glycation end products or mersalyl (1, 44 -46). PI3K-dependent pathways have been implicated in HIF-1 and VEGF expression in transformed cells (47)(48)(49)(50). 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 3,4,5triphosphate, increases HIF-1␣ protein levels and HIF-1-dependent reporter gene expression (30,48,50,51).
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-1␣ 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-1␣ by PKB has been excluded (51). However, PKB could be involved in the insulin regulation of HIF-1␣ 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 (52,53). 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 PI3K⅐PKB⅐TOR pathway (54). 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 (55,56). 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-1␣ 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 (1,57). Moreover, the co-treatment of retinal epithelial cells with both insulin and advanced glycation end products increases VEGF expression (1). The advanced glycation end products and insulin activate HIF-1 through distinct pathways, because advanced glycation endinduced HIF-1 activation is dependent on MAPK, whereas insulin-induced HIF-1 activation is dependent on PI3K. Hypoxia blocks HIF-1␣ 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 PI3K⅐PKB⅐TOR-dependent pathway. Ours results suggest that insulin similar to heregulin regulates HIF-1␣ synthesis. It remains to established whether such an effect on HIF-1␣ synthesis is a general feature of receptor tyrosine kinases.