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Am J Physiol Heart Circ Physiol 292: H758-H766, 2007. First published November 3, 2006; doi:10.1152/ajpheart.00718.2006
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Oxygen Sensing: Life and Death of a Cell

Insulin-induced activation of hypoxia-inducible factor-1 requires generation of reactive oxygen species by NADPH oxidase

Sudipta Biswas,1 Manveen Kaur Gupta,1 Debasis Chattopadhyay,2 and Chinmay K. Mukhopadhyay1

1Special Centre for Molecular Medicine, Jawaharlal Nehru University; and 2National Centre for Plant Genome Research, New Delhi, India

Submitted 6 July 2006 ; accepted in final form 27 October 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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 H2O2 in DPI-pretreated cells not only confirmed the role of ROS but also identified H2O2 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.

reduced nicotinamide adenine dinucleotide phosphate oxidase; gene expression

REACTIVE OXYGEN SPECIES (ROS) are originally thought to have only deleterious biological effects on organisms by virtue of their potential to cause oxidative damages to cellular components. However, it has been appreciated later that ROS play a very significant role in normal biological processes, including signaling pathways regulating various transcription factors like activator protein-1 (18, 19), NF-{kappa}B (4, 19), p53 (22), and subsequent gene expression. In the growing list of transcription factors influenced by cellular ROS generation, a recent addition is the hypoxia-induced oxygen sensing transcription factor hypoxia-inducible factor (HIF)-1. The HIF-1 is found to be consisting of a dimer of subunits HIF-1{alpha} and HIF-1beta. In normoxia, the hydroxylation of HIF-1{alpha} at the proline residues (402 and 564) is catalyzed by proline hydroxylases using molecular oxygen as the substrate (13–15). The tumor suppressor von Hippel-Lindau protein specifically binds with hydroxylated HIF-1{alpha} and promotes subsequent ubiquitinization and proteolysis (27), whereas HIF-1beta is found to be constitutive. In response to hypoxia, HIF-1{alpha} is not hydroxylated at the proline residues and therefore is stabilized. The stabilized HIF-1{alpha} translocates into the nucleus and binds to HIF-1beta and thus forms HIF-1 complex and binds to hypoxia response element (HRE, 5'-RCGTG-3') to increase the transcription of target genes like VEGF, GLUT1, erythropoietin (Epo), and others (13). For the last several years there have been contradictory reports on the participation of mitochondria-generated ROS in hypoxia-induced activation of HIF-1 (17). Initially, it was reported that mitochondria produced a burst of ROS in response to hypoxia and that this burst is necessary and sufficient to activate HIF-1 (6). The involvement of ROS, as well as any conventional signaling pathways, was the subject of concern because of the observation that, during hypoxia, decline in oxygen levels directly decreases the activity of the prolyl hydroxylases, thereby preventing hydroxylation of the HIF-1{alpha} protein (7). A series of articles published recently, however, reconfirms the participation of mitochondrial ROS in HIF-1 activation by hypoxia (5, 11, 26) using a p38 MAP kinase pathway (7), although exactly how ROS activates HIF-1 still remains to be understood.

Besides hypoxia, HIF-1 is also activated in normoxic conditions by several important physiological stimuli like insulin (35, 38), IGF-I/II (38), EGF (30), PDGF, and angiotensin II (31). Incidentally, insulin is known to increase the intracellular ROS generation to regulate the posttranslational translocation of GLUT4 (23, 24). Insulin plays a crucial role in regulating the metabolic pathways associated with energy storage and utilization. It regulates expression of GLUT1 and phosphoglycerate kinase-1 and several other genes like VEGF, Epo, and endothelin-1 (13). Insulin increases the transcription of many of these target genes by activating HIF-1 (38). We also have demonstrated that insulin regulates iron homeostasis gene ceruloplasmin (Cp) by activating HIF-1 (35). The current study is aimed to investigate the role of insulin-induced ROS generation in HIF-1 activation and subsequent gene expression. Here we demonstrated that insulin-stimulated ROS generation by NADPH oxidase is essential for the activation of HIF-1 in insulin-sensitive hepatic cells HepG2 and cardiac myoblasts H9c2. To understand the mechanism, we found that, in HepG2 cells, the NADPH oxidase-generated ROS regulates phosphorylation of insulin receptor kinases as well as phosphatidylinositol 3-kinase (PI3-kinase) activity, essential for HIF-1 activation and subsequent expression of genes like VEGF, GLUT1, and Cp.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. Human recombinant insulin (Invitrogen), apocynin, diphenyleneiodonium chloride (DPI), 2',7'-dichlorodihydrofluorescein (DCF) diacetate (DCF-DA) were purchased from (Calbiochem). Monoclonal anti-phosphotyrosine (4G10) and polyclonal antibodies to the insulin receptor beta-subunit (IR-beta), insulin receptor substrate-1 (IRS-1), were from Upstate Biotechnology. Antibodies to phosphorylated Akt (Ser473) and Akt protein (not isoform specific) were purchased from New England Biolabs. All cell culture reagents and other reagents were obtained from Sigma, unless otherwise mentioned.

Cell lines and culture conditions. Human hepatocarcinoma HepG2 cells and cardiac myoblasts H9c2 cells (from ATCC) were cultured in DMEM, supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine. Cells at 50–60% confluence were used in all experiments and were treated with appropriate concentrations of insulin after serum deprivation. Cells were maintained in a humidified atmosphere containing 5% CO2-95% air at 37°C (Forma-Scientific).

Intracellular ROS measurement in cell culture. Fluoresence microscopy and spectrophotometry were used to measure intracellular ROS with DCF-DA as the probe. Cells incubated with medium alone or cells treated with various compounds to induce ROS production as mentioned in respective experiments were incubated with 5 µM DCF-DA in DMEM for 30 min at 37°C in the dark. The cells were washed in PBS, trypsinized, and resuspended in 3 ml PBS, and the intensity of fluorescence was immediately read in a fluorescence spectrophotometer at 500 nm for excitation and at 530 nm for emission (6). Similarly, ROS production was also checked by fluorescence microscopy using the probe DCF-DA.

Immunoblot analysis. After the treatment, as indicated in different experiments, cells were lysed in buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 1% Triton X-100, 100 mM sodium fluoride, 1 mM EGTA, 1 mM EDTA, 2 mM sodium vanadate (NaV), 10 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, and a protease inhibitor mixture (Roche Applied Science). The lysates were briefly sonicated and centrifuged at 13,000 g for 10 min; 40 µg of protein of the cleared supernatant were resolved by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membrane. PVDF membranes were subjected to immunoblotting with either monoclonal antibody for phosphotyrosine (4G10) to detect IR-beta and IRS tyrosine phosphorylation; polyclonal antibody to detect phospho-Akt; or additional antibodies to detect total protein levels of the IR-beta, IRS-1, and Akt, where indicated. After incubation with horseradish peroxidase-conjugated secondary antibodies, proteins were visualized by enhanced chemiluminescence, according to the manufacturer's protocol (Amersham Biosciences).

RNA blot analysis. RNA was isolated from serum-deprived HepG2 cells using Tripure reagent (Roche Applied Science). Total RNA (20 µg) was denatured in formamide/formaldehyde, electrophoresed through 1% agarose gel containing 6% formaldehyde, and then blotted onto nylon membranes (Schleicher and Schuell). After being cross-linked by ultraviolet irradiation (Stratalinker, Stratagene), the membranes were hybridized to a 0.65-kb EcoRI fragment of VEGF165 (35), 646-base pair (bp) BstXI/BamHI restriction fragment (positions 984–1,629 of the open reading frame) of a human Cp cDNA as described (35), or 0.6 kb-fragment of GLUT1 (generated by RT-PCR, cloned into pcDNA3 and confirmed by sequencing) as described before (38) and labeled by random priming with [{alpha}-32P]dCTP using a New England Biolab kit.

Preparation of nuclear extracts. Nuclear extracts were prepared from HepG2 cells as described before (35). Briefly, 1 x 108 cells were washed with ice-cold phosphate-buffered saline and then with a solution containing (in mM) 10 Tris·HCl (pH 7.8), 1.5 MgCl2, and 10 KCl, supplemented with a protease inhibitor mixture containing 0.5 DTT and 0.4 phenylmethylsulfonyl fluoride and 2 µg/ml each of leupeptin, pepstatin, and aprotinin. After incubation on ice for 10 min, the cells were lysed by 10 strokes with a Dounce homogenizer, and the nuclei were pelleted. The pellet was resuspended in a solution containing (in mM) 420 KCl, 20 Tris·HCl (pH 7.8), and 1.5 MgCl2 and 20% glycerol, supplemented with the protease mixture described above and incubated at 4°C with gentle agitation. The nuclear extract was centrifuged at 10,000 g for 10 min, and the supernatant was dialyzed twice against a solution containing (in mM) 20 Tris·HCl (pH 7.8), 100 KCl, and 0.2 EDTA and 20% glycerol. Protein concentration was determined by using the Bio-Rad reagent with bovine serum albumin as standard.

Electrophoretic mobility shift assay. Sequences of the sense strands of the oligonucleotide probes used for EMSA were as follows: 5'-TCT GTA CGT GAC CAC ACT CAC CTC-3' (Cp-HRE) and 5'-GCC CTA CGT GCT GTC TCA CAC AGC-3' (Epo-HRE). The sense and antisense strands were annealed, gel purified, and end-labeled with [{gamma}32P]ATP using T4-polynucleotide kinase (Promega). Unincorporated nucleotide was removed by gel filtration using G-25 Sephadex columns (Quick Spin TE, Roche Applied Science). To measure DNA protein interaction, 1 x 105 counts/min of oligonucleotide probe were incubated with 5 µg of nuclear extract and 0.5 µg of sonicated, denatured salmon sperm DNA (Invitrogen) in 10 mM Tris·HCl (pH 7.8), 50 mM NaCl, 1 mM MgCl2, 1 mM EDTA, 5 mM DTT, and 5% glycerol for 20 min at 4°C in a total volume of 20 µl. The reaction mixture was subjected to electrophoresis (200 V in 0.3x Tris-buffered EDTA solution at 4°C) using 5% nondenaturing polyacrylamide gels. Dried gels were subjected to autoradiography for up to 24 h.

Construction of vectors containing Cp-HRE. Cp promoter/enhancer construct containing hypoxia response element (Cp-HRE) between –3,639 and –3,544 nt from Cp translation initiation site was ligated into the SacI and XhoI sites of the pGL3prom vector (Promega) upstream of the SV40 promoter and luciferase gene as described before (28, 35). Site-directed mutagenesis of the Cp-HRE in this construct (from TACGTG to TAAAAG) was done by megaprimer method (28, 35). All constructs were verified by sequencing.

Transient transfection of cells and reporter gene assays. To measure transcriptional efficiency of chimeric Cp-HRE construct, HepG2 or H9c2 cells at ~50% confluence in 35-mm dishes were transiently transfected for 6 h with a reporter plasmid (2 µg) using Lipofectamin 2000 (Invitrogen). To monitor transfection efficiency, a reporter gene construct (0.25 µg) containing beta-galactosidase behind an SV40 promoter was cotransfected as described earlier. Transfected cells were allowed to recover for 6 h in DMEM with 10% fetal bovine serum and, after serum deprivation, were incubated with insulin or other treatments in serum-free medium for 16 h as described elsewhere (35). Luciferase (Promega) and beta-galactosidase (Invitrogen) activities in cell extracts were determined as described in the manufacturer's protocol.

Statistical analysis. All experiments have been performed at least three times with similar results, and representative experiments are shown. Results from estimation of ROS and reporter experiments are expressed as means ± SE (n = 3 individual experiments).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Insulin-stimulated ROS regulates HIF-1 activation. To check that insulin induces ROS in HepG2 cell and characterize it, serum-deprived cells were incubated with DCF-DA, a sensitive oxidant indicator dye. Following stimulation with different concentrations of insulin, DCF fluorescence was estimated after 10 min. Figure 1A shows that insulin treatment increased ROS generation compared with untreated cells by a concentration-dependent manner, and maximum increase of ~100% was found by 10 nM insulin. The generation of ROS was optimal at ~10 min, and, after 30 min of stimulation, the fluorescence by ROS generation was almost undetectable (data not shown). Pretreatment of the cells with 5 µM DPI or apocynin (300 µM), inhibitors of NADPH oxidase before insulin treatment, completely blocked the increase in DCF fluorescence, indicating the involvement of NADPH oxidase in ROS generation by insulin (Fig. 1B). A similar result was also found by the pretreatment of antioxidant PDTC, whereas a specific scavenger of superoxide radical SOD failed to show any effect, indicating that either superoxide is not involved in the process or the addition of SOD from outside the cell could not block intracellular ROS generation. Similar effects of DPI and apocynin were also obtained by fluorescence microscopy (Fig. 1C). Cardiac myoblasts H9c2 also showed similar results in response to insulin, although induction of ROS was found faster than in HepG2 cells (data not shown).


Figure 1
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  		Fig. 1. Generation of reactive oxygen species (ROS) by insulin (Ins) in HepG2 cells. A: serum-deprived HepG2 cells were incubated with 2',7'-dichlorodihydrofluorescein (DCF) diacetate (DCF-DA) for 30 min and then treated with different concentrations of Ins for 10 min and washed with 1x PBS twice, and extracts were made and analyzed to estimate fluorescence. The experiment was repeated 3 times. B: HepG2 cells were treated with diphenyleneiodonium chloride (DPI; 5 µM), apocynin (300 µM), pyrrolidine dithiocarbamate (PDTC; 100 µM), and SOD (100 U/ml) for 30 min before Ins (10 nM) treatment, and, after 10 min, DCF fluorescence was measured. Experiments were repeated at least 3 times. Results are expressed as means ± SE (n = 3 replicate wells). C: HepG2 cells were plated onto a glass slip on a 12-well plate and, after 24 h, changed to the serum-free condition, followed by 1 h of DPI and apocynin treatment. DCF-DA (10 µM) was added 30 min before the Ins treatment (10 nM). After 10 min, cells were washed twice with 1x PBS, and images were captured with fluorescence microscope.

 
We have previously shown that insulin activates HIF-1 to increase Cp expression by interacting with Cp-HRE (35). To find the optimal concentration of insulin to activate HIF-1, we transfected Cp-HRE-driven luciferase chimera in to HepG2 cells and treated with increasing concentration of insulin. A maximum induction of ~3.3-fold luciferase activity was detected by 10 nM of insulin, whereas mutated HRE failed to show any considerable induction (Fig. 2A). To find out whether insulin-stimulated ROS generation is involved in HIF-1 activation, HepG2 cells were pretreated with DPI (5 µM) or apocynin (300 µM) before insulin treatment (10 nM) and, after 8 h, nuclear extracts were prepared for EMSA. Insulin-induced DNA binding complex of HIF-1 was completely blocked either by DPI or apocynin pretreatment (Fig. 2B). The identity of the HIF-1-DNA complex was confirmed by supershift analysis using HIF-1{alpha} antibody (Fig. 2C). Similarly, when serum-deprived H9c2 cells were pretreated with antioxidant PDTC (100 µM), insulin-stimulated Epo-HRE-HIF-1 complex formation was also completely blocked (Fig. 2D). The involvement of ROS in insulin-stimulated HIF-1 activation was further confirmed by HRE-driven reporter luciferase assay. Pretreatment of antioxidants, like DPI, apocynin, or PDTC, were found to block insulin-stimulated ROS generation and to completely block the insulin-dependent increase in luciferase reporter activity in HepG2 (Fig. 2E) and H9c2 (data not shown) cells. The SOD, which failed to block insulin-dependent ROS generation, also failed to inhibit the increased insulin-stimulated expression of luciferase gene (Fig. 2E). Generation of reactive nitrogen species, particularly, nitric oxide, was reported as a mediator of HIF-1 activation (33). The nitric oxide had also been reported to attenuate aortic smooth muscle cell motility stimulated by insulin by decreasing H2O2 levels (39). To find out whether nitric oxide has any influence on insulin-stimulated HIF-1 activation initially, we examined nitric oxide generation in insulin-stimulated HepG2 cells. No induced generation of nitric oxide was detected by insulin stimulation (data not shown). Similarly, a specific inhibitor of nitric oxide synthase, N{omega}-nitro-L-arginine methyl ester, did not inhibit insulin-mediated HIF-1-DNA complex formation (data not shown), suggesting that nitric oxide has no influence in insulin-stimulated HIF-1 activation.


Figure 2
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  		Fig. 2. Influence of ROS in Ins-induced hypoxia-inducible factor-1 (HIF-1) activation. A: wild-type and mutant ceruloplasmin (Cp)-hypoxia response element (HRE)-luciferase constructs were transfected into HepG2 cells. After recovery and serum deprivation, cells were treated with Ins (0–30 nM). Luciferase activity was detected in cell lysate after 16 h. Results were normalized with beta-galactosidase activity. B: to check the effect of NADPH-oxidase inhibitors on HIF-1-HRE binding, EMSA was performed. Serum-deprived HepG2 cells were untreated, treated for 8 h with Ins (10 nM), pretreated with DPI (5 µM) and apocynin (300 µM) for 30 min before Ins treatment, and treated with only DPI and apocynin. Nuclear extracts were mixed with 32P-labeled, double-stranded 24-mer probe containing the Cp-HRE. Probe-bound complexes were resolved by 5% nondenaturing PAGE and visualized by autoradiography. The positions of the HIF-1 and nonspecific (NS) complexes are indicated by arrows. C: supershift analysis was performed using HIF-1{alpha} antibody (2 µg, Novus Biologicals). D: H9c2 cells were treated with PDTC (100 µM) 30 min before Ins (10 nM) treatment, and EMSA was performed with nuclear extracts mixed with the erythropoietin (Epo)-HRE. E: HepG2 cells were transiently transfected with Cp-HRE and beta-galactosidase. After recovery, the transfected cells were treated with DPI (5 µM), apocynin (300 µM), PDTC (100 µM), and SOD (100 U/ml) 30 min before Ins (10 nM) treatment. After 16 h, luciferase activity in cell lysates was measured and normalized for beta-galactosidase activity. mHRE, mutant HRE; Ab, antibody.

 
H2O2 reverses DPI-mediated inhibition of HIF-1 activation. To confirm the role of ROS as well as to identify the oxidant generated by insulin, serum-deprived HepG2 cells were pretreated with 5 µM of DPI and, subsequently, H2O2 was added along with insulin. The addition of 30 µM H2O2 along with insulin was able to restore the HIF-1 activation even in presence of DPI as detected by EMSA (Fig. 3A). A similar result was also obtained with transfected HRE-luciferase construct (Fig. 3B). The role of H2O2 in the HIF-1 activation was further confirmed by greater stimulation of HIF-1 activity, when suboptimal concentration of insulin and H2O2 were added together. Cp-HRE-luciferase construct was transfected in subconfluent HepG2 cells, and the addition of 2 nM insulin was found to increase ~1.7-fold luciferase activity. Addition of H2O2 (10–100 µM) with 2 nM of insulin increased the HIF-1-mediated luciferase activity by >2 nM insulin treatment (Fig. 3C), whereas only H2O2 had no effect on HIF-1 activation (data not shown).


Figure 3
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  		Fig. 3. Restoration of Ins-induced HIF-1 activation by H2O2 after DPI treatment. A: serum-deprived HepG2 cells were treated with 5 µM of DPI. After 30 min, Ins (10 nM) or 30 µM H2O2 plus Ins was added. After 8 h, nuclear extracts were prepared, and EMSAs were performed with 32P-labeled Cp-HRE probe. B: HepG2 cells were transiently transfected with Cp-HRE-luciferase chimera and beta-galactosidase. After recovery and serum deprivation, cells were treated with only media, Ins (10 nM), DPI (5 µM, 30 min before Ins treatment) plus Ins, H2O2 (30 µM) plus Ins in DPI-pretreated cells, DPI (5 µM) alone, H2O2 (30 µM) alone, or DPI (5 µM) plus H2O2 (30 µM). After 16 h of treatment, cell extracts were prepared, luciferase activity was measured, and transfection efficiency was normalized with beta-galactosiadase activity. C: in Cp-HRE-luciferase-transfected, serum-deprived HepG2 cells, Ins (2 nM) and different concentrations of H2O2 (0–100 µM) were added simultaneously. After 16 h, luciferase activity was estimated from cell extracts and normalized with cotransfected beta-galactosidase activity.

 
Role of NADPH oxidase on the activation of HIF-1 by insulin. The inhibition of HIF-1 activation by specific inhibitors of NADPH oxidase, DPI and apocynin, strongly indicates the role of NADPH oxidase in this process. To further confirm that, we checked whether the treatment of NADPH compliments the activation of HIF-1 in presence of a suboptimal concentration of insulin. We initially tested the H2O2 generation by NADPH in the presence of a suboptimal concentration of insulin. The generation of H2O2 by 3 nM insulin was ~50% less than the maximum generation found by insulin (10 nM). While 25 µM NADPH was added along with 3 nM insulin, the generation of H2O2 was found comparable with 10 nM insulin-stimulated H2O2 generation (Fig. 4A). When Cp-HRE-luciferase chimera was transfected into subconfluent HepG2 cells and was treated with either insulin (3 nM) or NADPH (25 µM) or both together, the increase in luciferase activity was higher with simultaneous addition of insulin and NADPH compared with insulin alone (Fig. 4B). The addition of only NADPH did not show any increase in luciferase activity. When NADH (25 µM) was added simultaneously with insulin (3 nM), no further induction of luciferase activity, than with only insulin (3 nM), was detected (data not shown). Since DPI also can block mitochondrial NADH oxidase activity, the possibility of participation of NADH oxidase-induced ROS generation was ruled out by this experiment.


Figure 4
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  		Fig. 4. Influence of NADPH on Ins-induced HIF-1 activation. A: HepG2 cells were incubated with DCF-DA (10 µM) 30 min before Ins (3 nM) treatment. Cells were washed with 1x PBS, and DCF fluorescence was measured after 10 min of Ins treatment. B: luciferase activities were determined in Cp-HRE-luciferase chimera-transfected HepG2 cells after various treatments like media alone, Ins (3 nM), NADPH (25 µM), and NADPH plus Ins for 16 h. Results were normalized with beta-galactosidase activity. C: after transfection of Cp-HRE-luciferase chimera, serum-deprived HepG2 cells were initially treated with 0.1 mM MnCl2 for 1 h, washed with 1x PBS, and incubated with 10 nM Ins in serum-free DMEM. As indicated, in some systems, 5 µM DPI was added 30 min before the addition of Ins. After 16 h, luciferase activity was estimated in cell extracts. Final results are documented after normalization of beta-galactosidase activity. C, inset: in a similar system, DCF-DA (10 µM) was added 30 min before the addition of Ins, and DCF fluorescence was estimated after 10 min as a measure of ROS generation. Cont, control.

 
It was reported earlier that manganese acts as a cofactor for NADPH-dependent generation of H2O2 in vitro (20) and that supplementation of manganese leads to increased H2O2 generation in the presence of insulin in the isolated plasma membrane of fat cells. To prove further that H2O2 generation by NADPH oxidase plays an essential role in HIF-1 activation, we initially checked whether supplementation of MnCl2 increases the potentiality of H2O2 generation by insulin in HepG2 cells. We observed that treatment of serum-free HepG2 cells with MnCl2 before insulin indeed increased insulin-induced H2O2 generation by ~60–70% compared with only insulin treatment (Fig. 4C, inset). When we repeated the same treatment in Cp-HRE-luciferase chimera transfected HepG2 cells, about 60% increase in luciferase activity in MnCl2 pretreated cells were detected in presence of insulin (Fig. 4C). The blocking of increased generation of H2O2 and luciferase activity by DPI confirmed the role of NADPH oxidase.

Insulin-induced H2O2 modulates receptor kinase activity to activate HIF-1. Protein tyrosine phosphatases (PTPases) are known targets for H2O2 generated by insulin and other growth factors by a mechanism that involved oxidation of the catalytic PTPase thiol residue (8). Inactivation of PTPase leads to activation of receptor-linked protein tyrosine kinases (pY) and subsequent signaling pathways. We hypothesized that blocking of insulin-stimulated H2O2 generation by DPI would keep specific PTPase active so that insulin receptor-linked tyrosine kinases could not be phosphorylated and so that subsequent signaling pathways and target proteins (like HIF-1{alpha}) would not be activated. In that case, treatment with a specific inhibitor of PTPase could reverse the result. To investigate whether insulin-stimulated H2O2 generation is activating HIF-1 by involving similar mechanism, a specific inhibitor of tyrosine phosphatase NaV was added in DPI-treated HepG2 cells to check whether NaV compensates the lack of H2O2 generation and reverses activation of HIF-1. Fig. 5A shows that NaV can restore the DPI-mediated inhibition of HIF-1 activation in the presence of insulin, whereas Ser-Thr phosphatase inhibitor sodium fluoride failed to do so. To further confirm the role of H2O2 as a PTPase blocker, increasing concentrations of NaV were added along with suboptimal concentration of insulin (2 nM), which generates lower levels of H2O2 than the maximal dose of insulin (10 nM). The results showed that treatment of NaV simultaneous with insulin (2 nM) activated HIF-1 more than twofold in a concentration-dependent manner compared with only insulin (2 nM) (Fig. 5B). To confirm that insulin receptor-linked tyrosine kinases are regulated by ROS, the tyrosine phosphorylation of the insulin receptor and IRS-1/2 were then tested by measuring the insulin-stimulated autophosphorylation of its receptor and its high Mr substrate proteins in the HepG2 cells in the presence and absence of DPI or PDTC. Western blot analysis with anti-phosphotyrosine antibody (Fig. 6) revealed that the treatment of the cells with DPI before insulin treatment reduced the tyrosine phosphorylation of IRS and IR-beta significantly after 5 min of insulin treatment. Similar findings were detected when cells were pretreated with PDTC (100 µM) (data not shown). These findings strongly suggest that insulin-stimulated tyrosine phosphorylation receptor and related proteins are regulated by NADPH-linked ROS generation and are also responsible for subsequent activation of HIF-1.


Figure 5
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  		Fig. 5. Sodium vanadate (NaV) compliments Ins (I)-induced HIF-1 activation in the presence of DPI. A: subconfluent HepG2 cells were transfected with Cp-HRE-luciferase construct. In DPI (5 µM)-pretreated cells, either NaV (30 µM) or NaF (30 µM) was added 30 min before Ins. After 16 h, luciferase activity was measured and normalized against beta-galactosidase activity. B: Cp-HRE-luciferase construct was transfected in HepG2 cells, serum-deprived, and treated with NaV (0–30 µM) 30 min before Ins (3 nM). After 16 h, luciferase activity was determined in cell extracts and normalized for beta-galactosidase activity.

 

Figure 6
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  		Fig. 6. Effect of DPI on Ins-induced tyrosine phosphorylation of the Ins receptor beta-subunit (IR-beta) and insulin receptor substrate (IRS) proteins. Representative anti-phosphotyrosine (4G10) and anti-IR-beta and anti-IRS-1 immunoblots of HepG2 cell lysates were performed following stimulation with 10 nM Ins for 5 min with and without pretreatment of 5 µM DPI for 30 min. The migration positions of the tyrosine-phosphorylated high Mr IRS proteins (~185 kDa) and the IR-beta (95 kDa) are indicated as phosphotyrosine (pY) IRS-1/2 and pY IR-beta, respectively. The migration positions of IRS-1 and IR-beta subunit protein bands are indicated.

 
Insulin-induced ROS regulates the PI3-kinase for activation of HIF-1. PI3-kinase was previously reported as a signaling mediator of insulin-induced activation of HIF-1 (36). To find out whether NADPH oxidase-linked ROS generation regulates PI3-kinase activity and subsequent HIF-1 activation, we initially confirmed the role of PI3-kinase in HIF-1 activation by using specific inhibitor LY-294002. EMSA was performed with Cp-HRE as a HIF-1 binding probe using nuclear extract after insulin treatment from LY-294002-pretreated HepG2 cells. A dose-dependent inhibition of HIF-1-HRE complex formation by the PI3-kinase inhibitor was observed (Fig. 7A). To find out the influence of insulin-generated oxidant on PI3-kinase activity, the downstream Akt phosphorylation level was tested in the presence and absence of DPI. Exposure of HepG2 to DPI before insulin treatment blocked ~80% activation of Akt phosphorylation as found by immunoblotting analysis with phospho-Akt antibody (Fig. 7B), suggesting the role of oxidant in insulin-stimulated signaling pathways to activate HIF-1.


Figure 7
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  		Fig. 7. Involvement of ROS-regulated phosphatidylinositol 3-kinase activity on Ins-stimulated HIF-1 activation. A: nuclear extracts were prepared from HepG2 cells treated with only media, LY-294002 (50 µM) alone, or Ins (10 nM) pretreated with LY-294002 (0–50 µM) for 8 h. An EMSA was performed with Cp-HRE probe. B: HepG2 cells were pretreated with 5 µM DPI for 30 min before stimulation with 10 nM Ins. After 5 min, cell lysates were prepared and immunoblotting was performed with a polyclonal antibody to detect phospho-Akt (Ser473) or a nonisoform-specific anti-Akt antibody.

 
HIF-1-dependent gene expression by insulin can be blocked by DPI. To finally confirm the influence of NADPH oxidase-generated ROS in insulin-induced HIF-1-mediated expression of genes, HepG2 cells were treated with DPI before insulin treatment and Northern blot analyses were performed using human VEGF165, GLUT1, and Cp gene-specific probes. These genes are known to be regulated by HIF-1 (35, 38). Figure 8 shows the increase in insulin-induced expressions of VEGF165, GLUT1, and Cp mRNA by 2.9-, 3.1-, and 2.3-fold, respectively. The pretreatment of DPI blocked the increased expressions of these genes, confirming the role of NADPH oxidase-generated ROS in insulin-induced HIF-1-mediated gene expression.


Figure 8
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  		Fig. 8. Effect of DPI on Ins-induced HIF-1-mediated gene expression. HepG2 cells were treated with media, Ins (10 nM), pretreated DPI (5 µM) plus Ins, and DPI (5 µM). After 16 h, total RNA was isolated and Northern blot analyses were performed with {alpha}-32P-labeled cDNAs of VEGF165 (top), GLUT1 (middle), and Cp (bottom). For VEGF165 and GLUT1, 28S ribosomal subunits were detected by ultraviolet irradiation as loading control. For Cp, the same blot was reprobed with beta-actin. The magnitude of expressions was quantitated densitometrically and normalized either by 28S subunit or beta-actin and expressed with respect to untreated controls.

 

   DISCUSSION
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 MATERIALS AND METHODS
 RESULTS
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Several recent studies confirmed the involvement of mitochondria-generated ROS in hypoxia-induced activation of HIF-1 (5, 6, 11, 26). In contrast, the participation of ROS in HIF-1 activation by insulin or several other growth factors during normoxia remained unknown. The current study reveals that the activation of HIF-1 by insulin involves NADPH oxidase-linked generation of ROS, particularly, H2O2. To prove the involvement of ROS in insulin-stimulated activation of HIF-1, the following approaches were taken: 1) inhibition by NADPH oxidase inhibitors DPI, apocynin, as well as ubiquitious antioxidant PDTC; 2) restoration of HIF-1 activation in the presence of NADPH oxidase inhibitor DPI by the addition of external H2O2; and 3) maximal activation of HIF-1 by suboptimal concentration of insulin and the addition of H2O2. In contrast to mitochondrial generation of ROS for HIF-1 activation by hypoxia, we have found the flavoprotein NADPH oxidase-generated ROS is involved in response to insulin. Primarily, we used NADPH oxidase-specific inhibitor DPI and apocynin for blocking HIF-1 activation. Since DPI not only inhibits NADPH oxidase but also blocks mitochondrial NADH oxidase, to rule out the possibility of the participation of the later, we took several biochemical approaches. When the suboptimal concentration of insulin responsible to generate a lower amount of ROS than maximal was supplemented with external NADPH, the increased activation of HIF-1 was found (Fig. 4B), but an addition of NADH failed to do so. Krieger-Brauer and colleagues (20) reported in a series of in vitro biochemical studies that the elaboration of cellular H2O2 during the process of insulin signal transduction is generated by a novel plasma membrane-bound Mn2+-dependent NADPH oxidase that is coupled to G{alpha}i2 in adipocytes. Our finding of increased NADPH-dependent H2O2 generation by Mn2+ and insulin in HepG2 cells (Fig. 4C, inset) is consistent with their finding with a purified in vitro system. As a result of increased generation of ROS, the activation of HIF-1 was found to be greater by Mn2+ and insulin cotreatment, providing further evidence of membrane-bound NADPH oxidase-mediated ROS as an essential component for the process (Fig. 4C). So far it is also not very clear from any studies on how insulin activates NADPH oxidase in living cells, although the specific isoform involved in the process has been identified as NADPH oxidase 4 (Nox-4) in adipocytes, and HepG2 cells were shown to contain a greater amount of Nox-4 than other subunits of NADPH oxidase (27). The identification of the subunit responsible for ROS generation in response to insulin still remains an open question in HepG2 or H9c2 cells and needs further study.

As a mechanism we found that insulin-induced NADPH oxidase-linked generation of ROS activated insulin receptor tyrosine kinase by apparently inhibiting tyrosine phosphatase. Similar findings were reported previously for insulin in 3T3 adipocytes (24). In the present work we have demonstrated that the loss of the capacity of H2O2 generation by DPI in insulin-stimulated cells can be compensated by the addition of PTPase inhibitor NaV, indicating the role of H2O2 as a transient inhibitor of PTPase. PTPase is known to be inactivated by H2O2 in vivo (3) and thus is a widely known target for insulin-resistance diabetes (16). This model of insulin-stimulated ROS-mediated inactivation of PTPases and subsequent activation of receptor tyrosine kinases is consistent with other growth factors like EGF-mediated PTPase inactivation (21). If extrapolated, our result may also explain the mechanism of HIF-1 activation by other growth factors that are capable of ROS generation.

Insulin-stimulated H2O2 generation thus starts a signaling cascade to activate PI3-kinase (Fig. 7A), which was also reported before to be involved in HIF-1 activation by insulin (36). Now, in this study, we demonstrated that the insulin-induced PI3-kinase is also dependent on NADPH oxidase-mediated ROS generation as the downstream phosphorylation of Akt is blocked by DPI and PDTC (Fig. 7B). The PI3-kinase-Akt pathway has been shown to regulate HIF-1 activity in response to oncogenic signals and growth factors (1, 9), but how phosphorylation of Akt influences HIF-1 activation is not clear so far.

ROS were recognized for several years to have a regulatory role on insulin signal transduction (8), mainly on the translocation of GLUT4, but were never shown to take part in the process of gene regulation like transcription. Thus our finding of involvement of ROS in insulin-induced HIF-1 activation and subsequent expression of genes indicates that ROS may be crucial for general insulin action in responsive cells. The exposure of cells to exogenous H2O2 has been shown to modulate cellular insulin responsiveness differentially, depending to some extent on the concentration of oxidant, the duration of exposure, and cell type (2, 12). The exposure of 3T3-L1 adipocytes for several hours to micromolar concentration of H2O2 disrupts insulin-induced subcellular redistribution of IRS-1 and PI3-kinase between the cytosol and the low-density microsomal fraction and impairs the insulin-stimulated translocation of GLUT4 (32, 37), which may explain our observation that the addition of H2O2 alone cannot activate the HIF-1.

Insulin-induced activation of HIF-1 results in the induction of many genes, including VEGF, which plays an important role in developing several pathogenic conditions including angiogenesis and diabetic retinopathy (10, 29, 34). Since inhibition of increased VEGF expression is one of the probable ways to control these pathogenic situations, our finding of inhibition of VEGF synthesis in response to insulin by antioxidants, particularly those that can block NADPH oxidase activity, may be useful in this regard.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by a research grant from the Department of Biotechnology, New Delhi, India (to C. K. Mukhopadhyay).


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. K. Mukhopadhyay, Special Centre for Molecular Medicine, Jawaharlal Nehru Univ., New Delhi 110-067, India (e-mail: ckm2300{at}mail.jnu.ac.in)

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.


   REFERENCES
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