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Dorrance H. Hamilton Research Laboratories, Division of Endocrinology, Diabetes and Metabolic Diseases, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107,1 Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia 303222
Received 3 September 2003/ Returned for modification 8 October 2003/ Accepted 26 November 2003
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ABSTRACT |
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INTRODUCTION |
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Insulin signaling is initiated by the phosphorylation of specific tyrosyl residues of the cell surface insulin receptor, which activates its exogenous kinase activity and promotes the phosphorylation of IRS proteins on specific tyrosine residues (42). Much of insulin's downstream signaling to metabolic events involves the activation of phosphatidylinositol (PI) 3'-kinase activity by the docking of its p85 subunit to tyrosine-phosphorylated IRS-1 and IRS-2, which is linked to a network of distal signaling responses, including activation of protein kinase Akt and the stimulation of glucose uptake and other metabolic responses in specific cell types (23, 45). In parallel, insulin also activates cellular growth pathways mediated by Erk mitogen-activated protein kinases (MAPKs). In addition to these signaling events, our investigators have recently demonstrated that insulin stimulation of adipose and hepatoma cells leads to the rapid generation of cellular reactive oxygen species that facilitates both early and distal signal transduction through the insulin action pathway (34, 35). The effect of insulin-stimulated oxidant species is mediated at least in part via the oxidative inhibition of cellular protein-tyrosine phosphatases (PTPases), which depend on a reduced thiol moiety for catalytic activity and are normally inhibitory to insulin signal transduction (19, 37). PTP1B, in particular, has been considered to be a major candidate PTPase for the regulation of insulin action in a variety of studies, including knockout mouse models (13, 18, 25).
The molecular components of the insulin-responsive oxidant-generating activity in cell types that are targets for insulin action have not been identified. Previous work has demonstrated that the catalytic activity of the insulin-responsive system has enzymatic characteristics consistent with an NAD(P)H oxidase activity, catalyzing the reduction of molecular oxygen to generate superoxide, which undergoes subsequent dismutation to generate H2O2 (26-28, 38). The prototypic NAD(P)H oxidase complex from phagocytic cells has been extensively characterized. It consists of six subunits, including two plasma membrane-associated proteins, gp91phox and p22phox, which comprise flavocytochrome b558, and four cytosolic factors, p47phox, p67phox, p40phox, and rac (1). The catalytic gp91phox subunit (also now known as Nox2) contains C-terminal homology to known flavoprotein dehydrogenases and consensus sequences comprising a putative NAD(P)H binding site (9, 39). Nox2 has recently been used in cloning studies to identify a small family of five homologous Nox [NAD(P)H oxidase] enzymes which are variably expressed in different tissues (8, 30).
To initiate studies into the molecular mechanism of the insulin-induced oxidant species in insulin-sensitive cells, in the present work we first identified that Nox4 was a prominent NAD(P)H oxidase catalytic subunit homolog expressed in adipose cells. Overexpression of wild-type Nox4 and Nox4 constructs lacking cofactor binding domains for NAD(P)H or FAD/NAD(P)H using adenovirus-mediated gene delivery in differentiated 3T3-L1 adipocytes revealed that the deletion constructs function as dominant negatives and suppress insulin-induced cellular oxidant generation and insulin signaling, including tyrosine phosphorylation of the insulin receptor and its substrate protein IRS-1, and attenuate the activation of downstream signaling kinases as well as glucose uptake. In parallel studies, reduction of Nox4 protein mass by transfection of specific small interfering RNA (siRNA) constructs also resulted in inhibition of the insulin action cascade. Overexpression of Nox4 also significantly reversed the inhibition of insulin-stimulated insulin receptor tyrosine phosphorylation induced by coexpression of PTP1B by inhibiting the catalytic activity of PTP1B. These data suggest that Nox4 provides a novel link between the insulin receptor and the generation of cellular reactive oxygen species that enhance insulin signal transduction via the oxidative inhibition of cellular PTPases, including PTP1B.
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MATERIALS AND METHODS |
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Cell culture. 3T3-L1 preadipocytes were cultured in Dulbecco's modified Eagle's medium containing 25 mM glucose (DMEM) plus 10% fetal calf serum in a 5% CO2 atmosphere and were differentiated to adipocytes as previously described (35). Briefly, confluent cells were placed in differentiation medium (DMEM containing 10% fetal bovine serum, 100 nM insulin, 0.25 µM dexamethasone, and 500 µM isobutylmethylxanthine) for 2 days. The medium was then changed to DMEM containing 10% fetal bovine serum and 100 nM insulin. After an additional 6 days of incubation, the cells were used in the indicated experiments.
RT-PCR and Northern blot analysis for NAD(P)H catalytic subunit homologs. Reverse transcription-PCR (RT-PCR) was performed as described by Cheng et al. (8). For Northern analysis, total RNA was isolated from differentiated 3T3-L1 adipocytes using the TRIzol reagent (Invitrogen). Human HepG2 hepatoma cells, human HL-60 promyelocytic leukemia cells, and murine MMC (SV40-transformed glomerular mesangial cells) were used as positive controls for Nox1, Nox2, and Nox4, respectively. RNA was separated electrophoretically on formaldehyde-agarose gels, transferred to Hybond-N+ membrane (Amersham Biosciences), and hybridized with C-terminal cDNA probes of murine Nox1, Nox2, and Nox4 according to the manufacturer's recommendations (NorthernMax; Ambion, Austin, Tex.). cDNA probes were synthesized using the RT-PCR method (Invitrogen) and sequenced before probing to Hybond-N+ membranes to confirm their sequence specificity. Primers used for cDNA probe synthesis were Nox1 (forward, 5'-AAG TGG CTG TAC TGG TTG GG-3'; reverse, 5'-CCA CAT AAG AAA ACC CCC ACC G-3'; cDNA length, 411 bp), Nox2 (forward, 5'-CCA GTG AAG ATG TGT TCA GCT A-3'; reverse, 5'-AGG GTG TTC ACT TGC AAT GGT C-3'; cDNA length, 406 bp), and Nox4 (forward, 5'-GAA GCC CAT TTG AGG AGT CA-3'; reverse, 5'-GGG TCC ACA GCA GAA AAC TC-3'; cDNA length, 409 bp).
Generation of recombinant wild-type and Nox4 deletion constructs in adenoviral vectors.
The human wild-type Nox4 cDNA was cloned as described previously (8). cDNA constructs for dominant-negative Nox4 lacking NAD(P)H or FAD-NAD(P)H binding domains were generated by removing C-terminal sequences from the wild-type Nox4 cDNA encoding 578 amino acids (GenBank accession number AF254621) for 
NAD(P)H (including start codon through 1,150 bp; amino acids 1 to 383) or for FAD-NAD(P)H (start codon through 938 bp; amino acids 1 to 312) (Fig. 1). These cDNA constructs were introduced into defective Ad-5 adenoviruses using the Adeno-X expression system, which is driven by the human cytomegalovirus immediate-early promoter-enhancer and also contains a bovine growth hormone polyadenylation signal, according to the instructions provided by the manufacturer (BD Biosciences). The presence of the intact cDNA inserts was characterized in recombinant adenovirus DNA purified from transfected 293 cells by PCR. Recombinant virus was recovered from a large-scale preparation of adenovirus from 293 cells, and titers were determined using the Adeno-X rapid titer kit (BD Biosciences). A recombinant adenovirus encoding bacterial ß-galactosidase was used as control.
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Detection of the transduced Nox4 deletion constructs was performed by RT-PCR, since the available antibody to Nox4 was directed at a C-terminal protein epitope that is lacking in both of the recombinant constructs. Using reagents provided by Ambion, oligo(dT)-directed RT of cellular RNA was followed by 35 cycles of PCR with a reverse primer (5'-AATGATGGTGACTGGC-3') and a forward primer (5'-TCTCAGTGAATTACAGT-3') to generate a Nox4 fragment of 550 bp.
Transfection of siRNAs into 3T3-L1 adipocytes. The following siRNAs were designed to target the murine Nox4 cDNA sequences: siRNA 1 (5'-AACGAAGGGGTTAAACACCTC-3'); siRNA 3 (5'-AAAAGCAAGACTCTACACATC-3'); scrambled, control siRNA (5'-CAGTCGCGTTTGCGACTGG-3') (Dharmacon, Inc.). Either 20 nmol of scrambled or siRNA 1 or 3, or 10 nmol each of siRNA 1 and siRNA 3 was electroporated into differentiated 3T3-L1 adipocytes as described elsewhere (6). Briefly, cells were electroporated with siRNA and reseeded in six-well plates; 48 h later, cells were starved for 4 h and stimulated without or with insulin (100 nM) for 5 min prior to lysis with ice-cold 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, 10 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail (Sigma). For immunoprecipitation of Nox4 protein, lysates were briefly sonicated and cleared by centrifugation at 13,000 x g for 10 min, followed by immunoprecipitation using the polyclonal Nox4 antibody, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transfer onto polyvinylidene difluoride (PVDF) membranes, and Western blotting with Nox4-specific antibody using standard methods.
The Nox4 antibody was generated from a His-tagged recombinant fragment of human Nox4 encoding the C-terminal amino acids 320 to 428, which has 95% homology with murine Nox4 in that region. After purification on a nickel-nitrilotriacetic acid column (Qiagen), a polyclonal antibody was generated in rabbits. The rabbit antiserum was purified on a protein A column and also passed through a bacterial lysate column (Pierce) to remove nonspecific reactivity with Escherichia coli antigens.
Assay of intracellular H2O2 in 3T3-L1 adipocytes. Intracellular generation of H2O2 was visualized as described previously (2, 5, 35). After wild-type and dominant-negative Nox4 encoding recombinant adenovirus transduction into 3T3-L1 adipocytes, cells were stimulated with insulin (100 nM) for 5 min. Following stimulation, 3T3-L1 adipocytes were washed with minimal essential medium (lacking phenol red) and then incubated in the dark for 10 min with CM-DCF-DA. The fluorescence of CM-DCF-DA was measured by using a Bio-Rad confocal microscope at an excitation wavelength of 488 nm and emission at 515 to 540 nm. To avoid photooxidation of the indicator dye, the fluorescence image was collected by a single rapid scan with identical parameters for all samples. Where indicated, the fluorescence intensity was quantitated from sampled images using Scion Image software (Scion Corporation, Frederick, Md.).
Immunoblotting. After wild-type and dominant-negative Nox4-encoding or wild-type and dominant-negative PTP1B-encoding recombinant adenovirus transduction or siRNA transfection into 3T3-L1 adipocytes, cells were stimulated with insulin for 5 min and then 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, 10 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail (Sigma). The lysates were briefly sonicated and centrifuged at 13,000 x g for 10 min, and 75 µg of protein of the cleared supernatant was resolved by SDS-PAGE and transferred to PVDF membrane using a semidry Western blotting apparatus (AP Biotech). PVDF membranes were subjected to immunoblotting with either monoclonal antibody for phosphotyrosine (4G10) to detect insulin receptor ß-subunit and IRS tyrosine phosphorylation, polyclonal antibody to detect phospho-Akt, and monoclonal antibody to detect phospho-Erk1/2 MAPK, or additional antibodies to detect total protein levels of the insulin receptor ß-subunit, IRS-1, Akt, and ERK1/2 MAPK where indicated. Following incubation with horseradish peroxidase-conjugated secondary antibodies, proteins were visualized by ECL according to the instructions provided by the manufacturer. The immunoblotting signals were quantitated using an ImageStation 440 (Kodak). Protein was measured by the method of Bradford (7).
Immunoprecipitation of IRS-1 to assess p85 (PI 3'-kinase subunit) binding. Cell treatments and preparation of lysates from 3T3-L1 adipocytes were performed as described above. Briefly, cells were serum starved overnight in DMEM containing 0.5% BSA. After stimulation with insulin as indicated, cells were snap-frozen with liquid nitrogen and lysed in ice-cold lysis buffer. The lysates were briefly sonicated and centrifuged at 15,000 x g for 10 min. After normalizing the protein concentration, cleared lysates were incubated with IRS-1 antibody overnight at 4°C. The samples were then incubated for 2 h with a 50% (vol/vol) slurry of protein A-agarose at 4°C, pelleted by centrifugation in a microcentrifuge, and washed three times with ice-cold lysis buffer. SDS gel sample buffer was then added, and the samples were heated to boiling, processed by SDS gel electrophoresis, and transferred to PVDF membranes. Membranes were probed overnight with antibody to the p85 noncatalytic subunit of PI 3'-kinase. Following incubation with horseradish peroxidase-conjugated secondary antibodies, bound antibody was detected using ECL. To reprobe the blot, the membrane was incubated for 30 min at 50°C in stripping buffer (62.5 mM Tris-HCl [pH 6.8], 100 mM 2-mercaptoethanol, and 2% SDS) and then washed several times with TBST (20 mM Tris-HCl [pH 7.6], 137 mM NaCl, and 0.1% Tween 20). The PVDF membrane was reprobed with the IRS-1 antibody, and bound antibody was detected as described above.
Glucose uptake in 3T3-L1 adipocytes. Adenovirus-transduced or siRNA-transfected 3T3-L1 adipocytes were starved for serum for 4 h, washed with KRPH buffer (5 mM Na2HPO4, 20 mM HEPES [pH 7.4], 1 mM MgSO4, 1 mM CaCl2, 136 mM NaCl, 4.7 mM KCl, 0.2% [wt/vol] BSA), and treated with 100 nM insulin in KRPH buffer where indicated. Following 11 min of incubation with insulin, glucose uptake was assessed by the addition of 100 µM 2-deoxy-D-glucose containing 0.5 µCi of [3H]2-deoxy-D-glucose as described previously (14). The reaction was stopped 4 min later by washing the cells three times with ice-cold phosphate-buffered saline. The cells were then solubilized in 0.05% (wt/vol) SDS at 37°C for 30 min, and aliquots were subjected to scintillation counting. Nonspecific uptake (<10% of the total) was determined in the presence of cytochalasin B (50 µM) and was subtracted from the total uptake.
Specific activity of PTP1B. Under strictly anaerobic conditions to preserve the endogenous level of catalytic activity and avoid air oxidation (47), PTP1B was immunoprecipitated from lysates of transduced 3T3-L1 adipocytes (375 µg of protein) with a monoclonal antibody directed at a C-terminal epitope that preserves its enzymatic activity (Ab-2) followed by adsorption to Tris-acryl protein G. PTPase activity was measured by the hydrolysis of para-nitrophenyl phosphate in the anaerobic chamber in washed immunoprecipitates as our investigators have reported previously (35). The reaction was terminated by the addition of 50 µl of 1 M NaOH, and the absorption was determined at 410 nm.
Statistical analyses. Quantitative data are presented as the mean ± standard error of the mean for three to five experiments. Statistical analysis was based on Student's t test for comparison of two groups and one-way analysis of variance for multiple group comparisons. A P value less than 0.05 was used to determine statistical significance.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For comparison with the studies on the effects of Nox4, 3T3-L1 adipocytes were also transduced with recombinant wild-type human PTP1B and a site-directed catalytically inactive mutant enzyme (Cys215
er) using the same adenovirus system (12). By Western blot analysis, the level of PTP1B protein in the transduced 3T3-L1 adipocytes was increased 10- to 15-fold compared with cells transduced with the ß-galactosidase-encoding adenovirus (Fig. 3C).
Effect of expression of recombinant Nox4 and PTP1B on the insulin-induced oxidant signal. Insulin-stimulated generation of cellular reactive oxygen species was measured in the transduced 3T3-L1 adipocytes after loading with the sensitive fluorescent oxidant indicator dye CM-DCF-DA (35). At 72 h postadenoviral infection, adipocytes were stimulated with 100 nM insulin for 5 min, and in control cells a rapid increase in intracellular DCF fluorescence was detected within 1 min using confocal microscopy (Fig. 4). Compared to controls (either uninfected or those infected with recombinant ß-galactosidase), insulin-induced DCF fluorescence was increased by 21% (P = 0.02) in cells overexpressing wild-type Nox4. Importantly, insulin-induced oxidant generation was sharply attenuated by 72 to 75% in cells transduced with either of the Nox4 deletion constructs (P < 0.001). These findings are consistent with the deletion constructs acting in a dominant-negative fashion to inhibit insulin-induced oxidant generation, and they provide strong evidence that the NAD(P)H oxidase system in adipocytes involves the catalytic activity of Nox4. In parallel, overexpression of wild-type PTP1B, but not the catalytically inactive mutant, suppressed insulin-stimulated oxidant generation to a similar extent as the Nox4 deletion constructs, further suggesting that the post-insulin receptor signal transduction cascade is largely coupled to the generation of the oxidant signal (Fig. 4).
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Following stimulation with insulin, the siRNA-transfected cells with reduced endogenous Nox4 expression demonstrated a 49 to 64% (P = 0.01) decrease in insulin receptor tyrosyl phosphorylation compared to the control cells transfected with the scrambled siRNA controls (Fig. 5D). Thus, cellular reduction of Nox4 protein abundance significantly affected the initiation of the insulin signaling cascade to a similar degree as the dominant-negative effects of the transduced Nox4 deletion constructs (Fig. 5A).
Effect of expression of recombinant Nox4 constructs and wild-type PTP1B on PI 3'-kinase p85 subunit binding to IRS-1. We next assessed whether the effects of Nox4 affected downstream insulin signaling to the PI3'-kinase pathway in the 3T3-L1 cells (Fig. 6). Overexpression of wild-type Nox4 itself did not significantly affect the insulin-stimulated association between the p85 subunit of PI 3'-kinase and IRS-1. However, in cells overexpressing the Nox4 FAD-NAD(P)H deletion construct or wild-type PTP1B, the stimulation of the p85 subunit association with IRS-1 was reduced by 30 to 33% compared to control (P < 0.001).
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2-fold with each of the recombinant Nox4 deletion constructs (Fig. 9A). Following insulin stimulation, glucose uptake was increased by 3.9- and 3.0-fold over the basal level in the control, untransduced cells and in the ß-galactosidase-transduced controls, respectively. In the wild-type Nox4-overexpressing cells, the level of insulin-stimulated glucose uptake was increased by 22% over the level in control cells (P = 0.05), but due to the increase in basal uptake the degree of stimulation was reduced to 2.4-fold. In 3T3-L1 adipocytes overexpressing the dominant-negative Nox4 FAD-NAD(P)H construct, the level of insulin-stimulated glucose transport was decreased by 32% compared to the wild-type overexpressing cells (P = 0.001), and the level of insulin stimulation was markedly reduced to only 1.7-fold compared to controls. Changes in insulin-stimulated glucose uptake with the Nox4 NAD(P)H construct were at an intermediate level.
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Role of Nox4 in the modulation of insulin receptor tyrosine phosphorylation by PTP1B.
In order to determine if Nox4 interacted with PTP1B in its effect on insulin signal transduction, we cotransduced two recombinant adenoviruses to increase the level of expression of PTP1B and wild-type Nox4 or the dominant-negative FAD-NAD(P)H construct (Fig. 5C). As described above and shown in Fig. 5A, overexpression of PTP1B alone reduced the insulin-stimulated autophosphorylation of the insulin receptor by 67%. Cooverexpression of Nox4 along with PTP1B significantly diminished the effect of PTP1B alone on insulin receptor ß-subunit tyrosine phosphorylation by 46%. When cooverexpressed, the FAD-NAD(P)H construct had no significant effect on the reduction of insulin-stimulated receptor tyrosine phosphorylation by PTP1B overexpression (Fig. 5C).
In control experiments, neither overexpression of catalytically inactive PTP1B (mPTP1B) alone nor cotransduction of Nox4 along with mPTP1B affected insulin receptor autophosphorylation (Fig. 5C), as observed in our studies described above (Fig. 5A). In addition, cotransduction the FAD-NAD(P)H deletion construct along with mPTP1B inhibited insulin receptor tyrosine phosphorylation by 40%, suggesting that the underlying interaction between the dominant-negative Nox4 homolog and the endogenous active PTP1B was retained, similar to the experiments shown in Fig. 5A.
Role of Nox4 in the modulation of PTP1B catalytic activity.
To determine whether Nox4 overexpression affected the activity of PTP1B, we transduced 3T3-L1 cells with active PTP1B with and without cotransduction with active Nox4 and measured PTP1B activity specifically in immunoprecipitates using anaerobic conditions to avoid artifactual enzyme oxidation and inhibition by air exposure (Fig. 10). As our investigators reported previously (35), insulin stimulation inhibited the endogenous activity of PTP1B in 3T3-L1 cells by 50%, and viral infection with the recombinant virus encoding ß-galactosidase had no nonspecific effects in control studies. In the absence of insulin stimulation, transduction of recombinant human PTP1B increased the activity in the immunoprecipitates by 52%, which was reduced by 53% in the cells that were cotransduced with the Nox4 enzyme. In the cells overexpressing PTP1B without or with cotransduction of Nox4, insulin stimulation reduced PTPase activity in the PTP1B immunoprecipitates by 16 and 23%, respectively. These findings reveal a clear association between the activity of PTP1B as isolated from the intact cells and the activity of Nox4.
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DISCUSSION |
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In the present work, we have initiated a molecular approach to the identification of the insulin-stimulated cellular NAD(P)H oxidase activity expressed in the insulin-sensitive 3T3-L1 adipocyte model system. The identification of a small subfamily consisting of five mammalian NAD(P)H oxidase catalytic subunits (Nox1 to Nox5) homologous to the catalytic subunit of the respiratory burst oxidase, an NAD(P)H-dependent, superoxide-generating enzyme present in phagocytes known as gp91phox or Nox2 has facilitated our testing of the potential involvement of individual candidate enzymes as effectors of the insulin-stimulated cellular production of H2O2 (8). Each of the Nox family gp91phox homologs encodes a protein of 65 kDa and contains five to six conserved predicted transmembrane 
-helices containing putative heme binding regions and a flavoprotein homology domain containing predicted binding sites for both FAD and NAD(P)H. The Nox homologs exhibit unique patterns of cellular expression: Nox1 is predominantly expressed in colon and vascular smooth muscle cells; Nox3 is expressed primarily in fetal tissues; Nox4 is expressed in kidney, placenta, and glioblastoma cells; and Nox5 is expressed in spleen and uterus (8). Since we found that Nox4 mRNA expression was relatively abundant in adipose tissue, 3T3-L1 adipocytes, and HepG2 hepatoma cells (unpublished data), in the present work we evaluated whether Nox4 played a role in cellular insulin-induced oxidant production and insulin signaling. Indeed, using Nox4 deletion constructs that function in a dominant-negative fashion as well as confirmatory studies using siRNA loading to reduce Nox4 abundance, our data indicate clearly that Nox4 is involved in the generation of H2O2 by insulin stimulation of differentiated 3T3-L1 adipocytes.
In addition to effects on insulin-stimulated H2O2 generation, manipulation of Nox4 impacts significantly on both early and late events in insulin signal transduction, including insulin-stimulated insulin receptor and IRS-1 tyrosine phosphorylation, and the activation of downstream serine kinases and glucose uptake. Thus, the effects of Nox4 on insulin signaling are likely to be mediated by its effects on cellular oxidant generation, which our investigators have previously shown to modulate autophosphorylation of the insulin receptor and its major postreceptor substrates upstream as well as downstream signaling through the activation of cellular glucose transport (34, 35). Furthermore, we and others have shown that an endogenous oxidant signal is integral to the cellular activation of Akt via the upstream stimulation of PI 3'-kinase (34, 43), which we have demonstrated in the present work is also attenuated by interference with Nox4 abundance or function.
The Nox4 deletion constructs lacking NAD(P)H or FAD/NAD(P)H cofactor binding domains appear to act in a dominant-negative fashion, inhibiting the generation of cellular H2O2 and insulin signaling, perhaps by interfering with coupling of the insulin receptor signal to endogenous Nox4. Besides the identification of a role for Nox4 as a component of the oxidant-generating system in 3T3-L1 cells reported here, little is known about additional components of this signaling complex that may have a regulatory role in insulin-sensitive cell types. The well-characterized NAD(P)H oxidase complex from phagocytic cells has a multisubunit structure, including plasma membrane and cytosolic components whose complex formation is highly regulated and tightly coupled to enzymatic superoxide production (1). Thus, overexpression of Nox4 with the deleted cofactor domains may inhibit the formation of a functional NAD(P)H oxidase complex in the 3T3-L1 system. A more detailed characterization of additional components of adipocyte Nox interaction proteins will help elucidate these regulatory mechanisms. Several groups have recently reported the cloning of Nox-interacting subunit proteins in nonphagocytic cells that have dramatic effects on the regulation of superoxide production by Nox1 (e.g., reference 4). These, or related proteins, may have a role in the regulation of Nox4 function in insulin and growth factor signaling. Also, small GTPases or heterotrimeric G-proteins may be involved in cellular oxidant production: rac has been shown to be a regulatory component of the phagocytic NAD(P)H oxidase complex (1), and evidence for the involvement of Gi2 in the adipocyte oxidant-generating system responsive to insulin has been reported (29).
Several cellular targets of H2O2 produced by growth factor stimulation have been identified, and our understanding of their role in signal transduction is beginning to unfold (24, 46). One of the major effects of oxidants that mimic insulin action involves the inhibition of thiol-dependent cellular enzymes, including PTPases, that modulate various steps in signal transduction from the insulin receptor (34, 35) as well as several other growth factor receptor-induced tyrosine phosphorylation cascades (31, 37). PTP1B is an intracellular PTPase that has been implicated in the regulation of insulin signaling in cellular and transgenic mouse studies (reviewed in references 18 and 44). Consistent with these reports, we found that PTP1B overexpression consistently reduces insulin-stimulated tyrosine phosphorylation of its receptor and IRS-1 (Fig. 5A to C). In addition, the effect of PTP1B on insulin signal transduction as well as the catalytic activity of PTP1B itself as isolated from the 3T3-L1 adipocytes following adenovirus-mediated gene delivery was markedly diminished by overexpression of Nox4 (Fig. 5C and 9). These results provide an important mechanistic link between the insulin-stimulated oxidant-generating machinery and PTP1B as a target of oxidative inhibition. Additional signaling molecules that are potential targets of oxidative inhibition via the Nox4 pathway are other key mediators of growth factor signaling, including, for example, the tyrosine phosphatase TCPTP (17) and the 3'-PI(3,4,5)-PO4 phosphatase PTEN (32).
Further work will be needed to identify additional proteins that may have a role in the Nox4 signaling complex and to define the mechanism of coupling oxidant generation via Nox4 to the insulin receptor signaling cascade. This active area of research holds promise to define on a molecular basis a novel regulatory system for insulin action that was initially identified by observations made more than 30 years ago.
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ACKNOWLEDGMENTS |
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This work was supported by National Institutes of Health grant RO1 DK-43396 and a mentor-based postdoctoral fellowship grant from the American Diabetes Association to B. J. Goldstein. K. Mahadev is supported by a postdoctoral fellowship training grant award from the National Institute of Diabetes and Digestive and Kidney Diseases.
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FOOTNOTES |
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REFERENCES |
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2. Bae, Y. S., S. W. Kang, M. S. Seo, I. C. Baines, E. Tekle, P. B. Chock, and S. G. Rhee. 1997. Epidermal growth factor (EGF)-induced generation of hydrogen peroxide. Role in EGF receptor-mediated tyrosine phosphorylation. J. Biol. Chem. 272:217-221.
3. Bae, Y. S., J. Y. Sung, O. S. Kim, Y. J. Kim, K. C. Hur, A. Kazlauskas, and S. G. Rhee. 2000. Platelet-derived growth factor-induced H2O2 production requires the activation of phosphatidylinositol 3-kinase. J. Biol. Chem. 275:10527-10531.
4. Banfi, B., R. A. Clark, K. Steger, and K. H. Krause. 2003. Two novel proteins activate superoxide generation by the NADPH oxidase NOX1. J. Biol. Chem. 278:3510-3513.
5. Barrett, W. C., J. P. DeGnore, Y. F. Keng, Z. Y. Zhang, M. B. Yim, and P. B. Chock. 1999. Roles of superoxide radical anion in signal transduction mediated by reversible regulation of protein-tyrosine phosphatase 1B. J. Biol. Chem. 274:34543-34546.
6. Bose, A., A. Guilherme, S. I. Robida, S. M. Nicoloro, Q. L. Zhou, Z. Y. Jiang, D. P. Pomerleau, and M. P. Czech. 2002. Glucose transporter recycling in response to insulin is facilitated by myosin Myo1c. Nature 420:821-824.[CrossRef][Medline]
7. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.[CrossRef][Medline]
8. Cheng, G., Z. Cao, X. Xu, E. G. Van Meir, and J. D. Lambeth. 2001. Homologs of gp91phox: cloning and tissue expression of Nox3, Nox4, and Nox5. Gene 269:131-140.[CrossRef][Medline]
9. Cross, A. R., J. Rae, and J. T. Curnutte. 1995. Cytochrome b-245 of the neutrophil superoxide-generating system contains two nonidentical hemes. Potentiometric studies of a mutant form of gp91phox. J. Biol. Chem. 270:17075-17077.
10. Czech, M. P., J. C. Lawrence, Jr., and W. S. Lynn. 1974. Evidence for electron transfer reactions involved in the Cu2+- dependent thiol activation of fat cell glucose utilization. J Biol. Chem. 249:1001-1006.
11. Czech, M. P., J. C. Lawrence, Jr., and W. S. Lynn. 1974. Evidence for the involvement of sulfhydryl oxidation in the regulation of fat cell hexose transport by insulin. Proc. Natl. Acad. Sci. USA 71:4173-4177.
12. Dadke, S., and J. Chernoff. 2002. Interaction of protein tyrosine phosphatase (PTP) 1B with its substrates is influenced by two distinct binding domains. Biochem. J. 364:377-383.[CrossRef][Medline]
13. Elchebly, M., P. Payette, E. Michaliszyn, W. Cromlish, S. Collins, A. L. Loy, D. Normandin, A. Cheng, J. Himms-Hagen, C. C. Chan, C. Ramachandran, M. J. Gresser, M. L. Tremblay, and B. P. Kennedy. 1999. Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene. Science 283:1544-1548.
14. Fingar, D. C., S. F. Hausdorff, J. Blenis, and M. J. Birnbaum. 1993. Dissociation of pp70 ribosomal protein S6 kinase from insulin- stimulated glucose transport in 3T3-L1 adipocytes. J. Biol. Chem. 268:3005-3008.
15. Finkel, T. 2000. Redox-dependent signal transduction. FEBS Lett. 476:52-54.[CrossRef][Medline]
16. Flynn, J. T., M. Westbrooks, and K. A. Lucas. 1997. Establishment and characterization of a line of adipose-derived human microvascular endothelial cells (HADMEC-5) transformed by simian virus 40 large T antigen expression: application to endotoxin research. Shock 8:45-54.[Medline]
17. Galic, S., M. Klingler-Hoffmann, M. T. Fodero-Tavoletti, M. A. Puryer, T. C. Meng, N. K. Tonks, and T. Tiganis. 2003. Regulation of insulin receptor signaling by the protein tyrosine phosphatase TCPTP. Mol. Cell. Biol. 23:2096-2108.
18. Goldstein, B. J. 2001. Protein-tyrosine phosphatase 1B (PTP1B): a novel therapeutic target for type 2 diabetes mellitus, obesity and related states of insulin resistance. Curr. Drug Targets Immune Endocr. Metabol. Disorders 1:265-275.
19. Goldstein, B. J. 2002. Protein-tyrosine phosphatases: emerging targets for therapeutic intervention in type 2 diabetes and related states of insulin resistance. J. Clin. Endocrinol. Metab. 87:2474-2480.
20. Griendling, K. K., and M. Ushio-Fukai. 2000. Reactive oxygen species as mediators of angiotensin II signaling. Regul. Peptides 91:21-27.[CrossRef][Medline]
21. Herrlich, P., and F. D. Bohmer. 2000. Redox regulation of signal transduction in mammalian cells. Biochem. Pharmacol. 59:35-41.[CrossRef][Medline]
22. Junn, E., K. N. Lee, H. R. Ju, S. H. Han, J. Y. Im, H. S. Kang, T. H. Lee, Y. S. Bae, K. S. Ha, Z. W. Lee, S. G. Rhee, and I. Choi. 2000. Requirement of hydrogen peroxide generation in TGF-beta 1 signal transduction in human lung fibroblast cells: involvement of hydrogen peroxide and Ca2+ in TGF-beta 1-induced IL-6 expression. J. Immunol. 165:2190-2197.
23. Kido, Y., J. Nakae, and D. Accili. 2001. The insulin receptor and its cellular targets. J. Clin. Endocrinol. Metab. 86:972-979.
24. Kim, J. R., H. W. Yoon, K. S. Kwon, S. R. Lee, and S. G. Rhee. 2000. Identification of proteins containing cysteine residues that are sensitive to oxidation by hydrogen peroxide at neutral pH. Anal. Biochem. 283:214-221.[CrossRef][Medline]
25. Klaman, L. D., O. Boss, O. D. Peroni, J. K. Kim, J. L. Martino, J. M. Zabolotny, N. Moghal, M. Lubkin, Y. B. Kim, A. H. Sharpe, A. Stricker-Krongrad, G. I. Shulman, B. G. Neel, and B. B. Kahn. 2000. Increased energy expenditure, decreased adiposity, and tissue-specific insulin sensitivity in protein-tyrosine phosphatase 1B-deficient mice. Mol. Cell. Biol. 20:5479-5489.
26. Krieger-Brauer, H. I., and H. Kather. 1992. Human fat cells possess a plasma membrane-bound H2O2-generating system that is activated by insulin via a mechanism bypassing the receptor kinase. J. Clin. Investig. 89:1006-1013.
27. Krieger-Brauer, H. I., and H. Kather. 1995. Antagonistic effects of different members of the fibroblast and platelet-derived growth factor families on adipose conversion and NADPH-dependent H2O2 generation in 3T3-L1 cells. Biochem. J. 307:549-556.
28. Krieger-Brauer, H. I., and H. Kather. 1995. The stimulus-sensitive H2O2-generating system present in human fat-cell plasma membranes is multireceptor-linked and under antagonistic control by hormones and cytokines. Biochem. J. 307:543-548.
29. Krieger-Brauer, H. I., P. K. Medda, and H. Kather. 1997. Insulin-induced activation of NADPH-dependent H2O2 generation in human adipocyte plasma membranes is mediated by Gi2. J. Biol. Chem. 272:10135-10143.
30. Lambeth, J. D. 2002. Nox/Duox family of nicotinamide adenine dinucleotide (phosphate) oxidases. Curr. Opin. Hematol. 9:11-17.[CrossRef][Medline]
31. Lee, S. R., K. S. Kwon, S. R. Kim, and S. G. Rhee. 1998. Reversible inactivation of protein-tyrosine phosphatase 1b in A431 cells stimulated with epidermal growth factor. J. Biol. Chem. 273:15366-15372.
32. Lee, S. R., K. S. Yang, J. Kwon, C. Lee, W. Jeong, and S. G. Rhee. 2002. Reversible inactivation of the tumor suppressor PTEN by H2O2. J. Biol. Chem. 277:20336-20342.
33. Livingston, J. N., P. A. Gurny, and D. H. Lockwood. 1977. Insulin-like effects of polyamines in fat cells. Mediation by H2O2 formation. J. Biol. Chem. 252:560-562.
34. Mahadev, K., X. Wu, A. Zilbering, L. Zhu, J. T. R. Lawrence, and B. J. Goldstein. 2001. Hydrogen peroxide generated during cellular insulin stimulation is integral to activation of the distal insulin signaling cascade in 3T3-L1 adipocytes. J. Biol. Chem. 276:48662-48669.
35. Mahadev, K., A. Zilbering, L. Zhu, and B. J. Goldstein. 2001. Insulin-stimulated hydrogen peroxide reversibly inhibits protein-tyrosine phosphatase 1B in vivo and enhances the early insulin action cascade. J. Biol. Chem. 276:21938-21942.
36. May, J. M., and C. de Haen. 1979. Insulin-stimulated intracellular hydrogen peroxide production in rat epididymal fat cells. J. Biol. Chem. 254:2214-2220.
37. Meng, T. C., T. Fukada, and N. K. Tonks. 2002. Reversible oxidation and inactivation of protein tyrosine phosphatases in vivo. Mol. Cell 9:387-399.[CrossRef][Medline]
38. Mukherjee, S. P., and W. S. Lynn. 1977. Reduced nicotinamide adenine dinucleotide phosphate oxidase in adipocyte plasma membrane and its activation by insulin. Possible role in the hormone's effects on adenylate cyclase and the hexose monophosphate shunt. Arch. Biochem. Biophys. 184:69-76.[CrossRef][Medline]
39. Nisimoto, Y., H. Otsuka-Murakami, and D. J. Lambeth. 1995. Reconstitution of flavin-depleted neutrophil flavocytochrome b558 with 8-mercapto-FAD and characterization of the flavin-reconstituted enzyme. J. Biol. Chem. 270:16428-16434.
40. Orlicky, D. J., and J. Schaack. 2001. Adenovirus transduction of 3T3-L1 cells. J. Lipid Res. 42:460-466.
41. Rhee, S. G., Y. S. Bae, S. R. Lee, and J. Kwon. 2000. Hydrogen peroxide: a key messenger that modulates protein phosphorylation through cysteine oxidation. Sci. STKE 2000:E1.
42. Saltiel, A. R., and C. R. Kahn. 2001. Insulin signalling and the regulation of glucose and lipid metabolism. Nature 414:799-806.[CrossRef][Medline]
43. Shaw, M., P. Cohen, and D. R. Alessi. 1998. The activation of protein kinase B by H2O2 or heat shock is mediated by phosphoinositide 3-kinase and not by mitogen-activated protein kinase-activated protein kinase-2. Biochem. J. 336:241-246.
44. Tonks, N. K. 2003. PTP1B: from the sidelines to the front lines! FEBS Lett. 546:140-148.[CrossRef][Medline]
45. White, M. F. 2002. IRS proteins and the common path to diabetes. Am. J. Physiol. Endocrinol. Metab. 283:E413-E422.
46. Woo, H. A., H. Z. Chae, S. C. Hwang, K. S. Yang, S. W. Kang, K. Kim, and S. G. Rhee. 2003. Reversing the inactivation of peroxiredoxins caused by cysteine sulfinic acid formation. Science 300:653-656.
47. Zhu, L., A. Zilbering, X. Wu, K. Mahadev, J. I. Joseph, S. Jabbour, W. Deeb, and B. J. Goldstein. 2001. Use of an anaerobic environment to preserve the endogenous activity of protein-tyrosine phosphatases isolated from intact cells. FASEB J. 15:1637-1639.
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