Published online before print December 4, 2008, doi: 10.1161/ATVBAHA.108.174219
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© 2009 American Heart Association, Inc.
Cell Biology/Signaling |
Nox4 Acts as a Switch Between Differentiation and Proliferation in Preadipocytes
From the Institut für Kardiovaskuläre Physiologie, Goethe-Universität, Frankfurt am Main, Germany.
Correspondence to Ralf P. Brandes, Institut für Kardiovaskuläre Physiologie, Goethe-Universität, Theodor-Stern-Kai 7, D-60596 Frankfurt am Main, Germany. E-mail r.brandes{at}em.uni-frankfurt.de
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Abstract |
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Objective— Insulin promotes differentiation of preadipocytes into adipocytes. Insulin also stimulates reactive oxygen species (ROS) production, and the NADPH oxidases Nox1 and Nox4 are important sources of ROS. We determined in human and mouse preadipocytes whether Nox proteins contribute to ROS formation and differentiation in response to insulin.
Methods and Results— The expression of Nox1 and Nox4 was increased during insulin-induced differentiation, and insulin increased ROS production. SiRNA against Nox4 but not Nox1 inhibited insulin-induced differentiation and ROS production but promoted proliferation. Nox4 overexpression yielded the opposite effect. As observed by siRNA and overexpression, Nox4 controlled the expression of MAP kinase phosphatase-1 (MKP-1), which reduces insulin-induced ERK1/2 activation. Consequently, downregulation of Nox4 promoted ERK1/2 signaling: Proliferation was increased and through phosphorylation of the inhibitory site serine612, ERK1/2 inhibited the activation of the insulin-receptor substrate-1 (IRS-1) and thereby prevented differentiation in response to insulin. Inhibition of ERK1/2 or overexpression of MPK-1 promoted insulin-induced differentiation. Accordingly, insulin-induced proliferation was enhanced by siRNA against MKP-1, whereas inhibition of ERK1/2 or overexpression of MKP-1 attenuated proliferation.
Conclusions— Nox4 acts as a switch from insulin-induced proliferation to differentiation by controlling MKP-1 expression, which limits ERK1/2 signaling.
Key Words: oxidative stress • superoxide • NADPH oxidase • differentiation
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Introduction |
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Insulin is a major adipogen. It stimulates the uptake of glucose, which is then converted into triglycerides and stored in fat droplets leading to adipocyte maturation; insulin also promotes the differentiation of preadipocytes in the stroma of the adipose tissue into adipocytes.1 Although paracrine autacoids as well as transcription factors and signaling cascades have been identified to be involved in adipocyte differentiation and insulin signaling, these complex processes are still incompletely understood.2 Insulin is also known to acutely increase the reactive oxygen species (ROS) production in adipocytes,3 but the enzymes responsible for ROS formation in these cells have not been extensively characterized. Moreover, it is unknown whether ROS are required for insulin-induced differentiation.
Besides mitochondria, the Nox family of NADPH oxidases is considered the most important source of ROS in the body.4 It is accepted that Nox1- and Nox2-dependent ROS formation requires activation of the proteins by cytosolic activators, whereas Nox4 is constitutively active and independent of activator proteins.4,5 It is therefore assumed that Nox1 and -2 mediate short-term effects, whereas Nox4 is responsible for long-lasting events such as controlling cell cycle progression and proliferation.6
We hypothesize that insulin elicits a prolonged increase in ROS formation, which is mediated by an increase in the expression of constitutively active Nox4. Moreover, we investigated whether or not this induction of Nox4 is required for long-term processes in response to insulin, such as proliferation and differentiation, and determined the underlying mechanisms.
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Methods |
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For a detailed Material and Methods section please see the supplemental materials (available online at http://atvb.ahajournals.org).
Cell Culture
Human preadipocytes were isolated from the stroma vascular fraction of liposuctions by collagenase digestion.7 3T3-L1 fibroblasts were obtained from ATCC. Differentiation was induced by standard protocol. Unless otherwise indicated, experiments were conducted at day 10 (3T3-L1) and 12 (human preadipocytes) after confluence. For transfection, Gene-Eraser was used for siRNAs and Gene-Jammer was used for plasmids (Stratagene). Overexpression of Nox4 was achieved by lentiviral transduction. Differentiation was determined by AdipoRed staining and quantified by fluorescence-activated-cell sorter (FACS) or microplate fluorimeter measurements. Proliferation was determined by cell counting or BrdU incorporation.
Immunoblotting and Immunprecipitation
Extractions for Western blots and immunoprecipitations were performed with Nonidet (1%) lysis buffer containing protease and phosphatase inhibitors. The anti-Nox4 antibody was kindly provided by A.M. Shah (King’s College, London, UK).8
H2O2 Production
H2O2 was measured by a Amplex red/Horseradish peroxide assay.
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Results |
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Insulin-Induced Differentiation Depends on ROS
Insulin induced the differentiation of 3T3-L1 cells into adipocytes as observed by fat droplet formation (Figure 1A), downregulation of the preadipocyte marker pref-1 (Figure 1B) as well as upregulation of the fatty-acid binding protein FABP4/aP2 (+425±11%, P<0.05), an important marker protein of mature adipocytes.9 Accumulation of fat droplets was considered to be the final step of differentiation succeeding a previous occurrence of changes in the gene expression.10 Therefore, in this study only cells with fat droplets were interpreted as fully differentiated. Insulin also induced a marked increase in ROS formation, which was apparent 4 hours after the beginning of insulin stimulation and still detectable 2 days after the onset of stimulation (Figure 1C). To test whether ROS are involved in differentiation, we stimulated 3T3-L1 cells and human preadipocytes with H2O2 (30 µmol/L, every other day) which resulted in differentiation even in the absence of insulin (Figure 1D). Accordingly, supplementation of catalase or overexpression of cytosolic catalase blocked the insulin-induced differentiation of preadipocytes (catalase overexpression: Figure 1E, catalase supplementation: solvent versus catalase with insulin 100 versus 49±11%* mean±SEM, n=3, *P<0.05). This effect was also reflected by the fact that catalase prevented the insulin-induced downregulation of pref-1 mRNA (% downregulation of pref-1 in response to insulin: control condition: –75.1±10.2; catalase –17±36.77,* P<0.05 versus solvent; n=3). Similarly, the nonspecific NADPH oxidase inhibitor diphenylene iodonium (10 µmol/L) blocked insulin-induced differentiation and pref-1 mRNA downregulation (data not shown).
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Insulin-Induced ROS Formation Is Mediated by an Induction of Nox4
Quantitative RT-PCR was performed to identify the NADPH oxidase homologue responsible for the sustained increase of ROS formation in response to insulin. 3T3-L1 fibroblasts expressed Nox1, Nox2, as well as Nox4, which is the predominant homologue on the mRNA level (Figure 2A). Nox3 and Nox5 were undetectable in 3T3-L1 and human preadipocytes. Duox1 and 2 expression was detected at very low level only (ct values of 34 and 35, respectively). In the course of insulin-induced differentiation, the expression of Nox1 and Nox4 increased, whereas Nox2 mRNA expression remained unchanged (Figure 2A). Accordingly, Nox4 protein expression was observed to be increased in differentiated as compared to undifferentiated 3T3-L1 cells (Figure 2B).
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We next determined the contribution of Nox1 and Nox4 to insulin-stimulated ROS production. A Nox4 siRNA was developed which decreased Nox4 protein expression by 
70% (Figure 2C). Using this tool, it was observed that the constitutive ROS production in differentiating preadipocytes was predominantly mediated by Nox4: whereas siRNA against Nox1 had only a small, yet significant effect, Nox4 siRNA largely attenuated ROS formation at day 12 of differentiation (Nox1–33±20%, Nox4–73±27%, mean±SEM, n=5, P<0.05). Subsequently, the effect of Nox4 siRNA on the induced ROS production after 48 hours of insulin stimulation was determined. Nox4 siRNA prevented the insulin-induced mRNA expression of Nox4 and blocked the insulin-induced increase in ROS production (Figure 2D).
Nox4 Mediates Differentiation but Impairs Proliferation in Response to Insulin
SiRNA was used to test the contribution of Nox1 and Nox4 to insulin-induced differentiation. SiRNA directed against Nox4 but not Nox1 siRNA blocked the insulin-induced accumulation of fat droplets in 3T3-L1 cells (Figure 3A). SiNox4 also prevented the induction of aP2 in the course of differentiation (scrambled siRNA 2±1.4; Nox1 siRNA; 6.9±5; Nox4 siRNA 0.9±0.7; fold induction determined by the 2–
ct method; mean±SEM, n=3, P<0.05 scrambled siRNA versus Nox4 siRNA). Intriguingly, overexpression of Nox4 in human preadipocytes promoted the accumulation of fat droplets even in the absence of insulin, suggesting that Nox4 is a direct mediator of insulin-induced differentiation (Figure 3B). Pref-1 is not only a marker for preadipocytes but also maintains these cells in an undifferentiated state. We therefore determined the effect of Nox4 on pref-1 expression. Insulin-induced differentiation was associated with a marked reduction in pref-1 mRNA expression and this effect was almost completely prevented by siRNA directed against Nox4 (Figure 3C). Likewise, lentiviral-mediated overexpression of Nox4 induced a drastic downregulation of pref-1 even in the absence of insulin (Figure 3D). These observations suggest that pref-1 is an effector of Nox4.
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Differentiation and proliferation represent dichotomous aspects of cellular function and proliferation is frequently associated with dedifferentiation. As insulin is known to stimulate proliferation of fibroblasts, the role of Nox4 in this process was determined. Insulin stimulated the proliferation of 3T3-L1 cells, and, importantly, this effect was potentiated by siRNA directed against Nox4 whereas Nox1 siRNA was without effect (BrdU incorporation: Figure 3E, determination of the increase in total cell number in response to insulin: scrambled siRNA 15.5±3.4%, Nox4 siRNA 33.2±9.4%*, P<0.05). Accordingly, we observed a reduction of proliferation in human preadipocytes after overexpression of Nox4 (Figure 3F). These observations indicate that Nox4 shifts the balance from insulin-induced proliferation to differentiation. Interestingly, overexpression of catalase enhanced the insulin-induced proliferation of 3T3-L1, suggesting that the effects of Nox4 are mediated by alterations in ROS level (Figure 1F).
Nox4 Attenuates MAP Kinase Signaling by Inducing MKP-1
Proliferation in response to insulin is mediated by the Ras-Raf-MEK-ERK1/2 pathway. Indeed, the basal and insulin-stimulated phosphorylation or ERK1/2 was enhanced when Nox4 was downregulated by siRNA, whereas it was attenuated when Nox4 was overexpressed in human preadipocytes (Figure 4A). As activation of ERK1/2 is limited by the MAP kinase phosphatase-1 (MKP-1), we tested the effect of Nox4 on this protein. MKP-1 expression paralleled that of Nox4: whereas downregulation of Nox4 attenuated MKP-1 expression, overexpression of the NADPH oxidase in human preadipocytes increased it. Importantly, Nox4 had no effect on the expression of other important phosphatases such as PP2A and PTP1B (Figure 4B). Moreover, we observed that Nox4 overexpression did not inhibit MKP-1 activity (Schröder K, unpublished observation, 2008) and siRNA directed against Nox1 did not alter MKP-1 expression or insulin-induced ERK1/2 activation in 3T3-L1 cells (data not shown). These observations identify Nox4, by controlling MKP-1 expression, as a negative regulator of ERK1/2 signaling.
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Nox4 Facilitates Insulin Receptor Substrate-1–Mediated Insulin Signaling
On insulin-binding, the insulin receptor tyrosine phosphorylates insulin receptor substrate-1 (IRS-1), which then leads to differentiation via PI-3 kinase, Akt, and protein kinase C isoforms. Downregulation of Nox4 attenuated the insulin-induced tyrosine phosphorylation of IRS-1, whereas Nox4 overexpression in human preadipocytes enhanced it. Another phosphorylation site of IRS-1 is Ser612, which is known to hamper IRS-1 tyrosine phosphorylation. IRS-1 phosphorylation on Ser612 occurs by ERK1/2. Importantly, Nox4 attenuated the insulin-dependent phosphorylation at this site (Figure 4A). These data indicate that Nox4 promotes insulin-signaling through IRS-1 by attenuating the ERK1/2-mediated phosphorylation at the inhibitory serine site of this protein.
MKP-1 Is a Mediator of the Effects of Nox4
According to the above mentioned results, MKP-1 and ERK1/2 should essentially determine the balance between insulin-induced differentiation and proliferation. Inhibition of extracellular signal regulated kinase (ERK) 1/2 activation by PD98059 (10 µmol/L) or overexpression of MKP-1 enhanced insulin-induced differentiation (Figure 5A and 5B). Overexpression of MKP-1 prevented insulin-induced ERK1/2 phosphorylation in 3T3-L1 cells (insulin-induced increase in ERK1/2 phosphorylation: GFP control 5±0.1; MKP-1 plasmid: 1.2±0.1; n=5, P<0.05). Accordingly, PD98059 or overexpression of MKP-1 both inhibited insulin-induced proliferation (Figure 5C and 5D). To test the contribution of endogenous MKP-1 in this process siRNA was used. Indeed, MKP-1 siRNA more than tripled insulin-induced proliferation (Figure 5E). This demonstrates that endogenous MKP-1, which is controlled by Nox4, is central in attenuating insulin-induced proliferation. Unfortunately, because of the pronounced effect of MKP-1 siRNA on proliferation, the effect of this tool on the slow process of differentiation, which requires 12 days, could not be determined: the substantial changes in cell number interfered with the speed of the differentiation process.
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Discussion |
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In this study we provide evidence that Nox4 acts as a switch from proliferation to differentiation. Nox4, through an indirect effect, facilitates the tyrosine phosphorylation of IRS-1, which is required for the insulin-induced differentiation. This process appears to involve MKP-1: Nox4 controls the expression of MKP-1 and thereby limits the contribution of the proliferative Ras–Raf-ERK1/2 pathway to insulin signaling. ERK1/2 phosphorylates IRS-1 on serine-residues and thereby prevents IRS-1 tyrosine phosphorylation. The Nox4-dependent induction of MKP-1 prevents this effect and therefore promotes insulin-induced differentiation but attenuated insulin-induced proliferation.
Nox4 expression has been demonstrated to be regulated, and differentiating factors such as transforming growth factor (TGF) β1 are known to induce this NADPH oxidase, as also observed in the present study. Interestingly, high glucose also appears to induce Nox4 (Schröder K. et al, unpublished observation, 2008). Thus, it is appealing to speculate that Nox4 promotes the development of obesity in scenarios of combined hyperglycemia and hyperinsulinemia, like type II diabetes.
To induce differentiation into adipocytes, 2 different protocols were used for human and mouse cells, which contain several compounds which interfere with signal transduction and potentially even NADPH oxidase expression. Ciglitazone, which was used for human preadipocyte differentiation, has been shown to downregulate expression of several NADPH oxidases and to reduce ROS production in endothelial cells.11 Rosiglitazone is known to downregulate p47phox in monocytes and macrophages, and thereby it attenuates the respiratory burst in these cells.12 In 3T3-L1 cells, dexamethasone was used instead of glitazones; dexamethasone also induces PPAR gamma.13 Moreover, we have previously observed that glucocorticoids downregulate p22phox in vascular smooth muscle cell (VSMC).14 On the other hand, high concentrations of glucocorticoids may even increase oxidative stress in endothelial cells, although the underlying mechanism is unclear.15 Finally, IBMX, which is given for 2 days in the course of differentiation, has been shown to downregulate p47phox phosphorylation in leukocytes.16 These observations could suggest that the process of differentiation requires a reduction in cellular ROS formation. This assumption, however, is in contrast to the fact that hydrogen peroxide or overexpression of Nox4 per se promotes differentiation. Moreover, it has consistently been demonstrated that the process of differentiation is associated with an increased ROS production: Serum-deprivation induced differentiation of smooth muscle cells17 as well as TGFβ1-induced differentiation of human fibroblasts into myofibroblasts18 were both associated with an increased ROS production. These observations might indicate that effects of the glucocorticoids and PPAR
agonists are unrelated to their actions on ROS production.
The main factor for differentiation into adipocytes and accumulation of fat droplets, however, is insulin. Priming for differentiation using the above mentioned protocols in the absence of insulin did not result in fat accumulation. Indeed, a robust link between insulin and increased cellular ROS production exists: First reports demonstrated that insulin acutely increases the hydrogen peroxide formation in membrane preparations of adipocytes in an NADPH-dependent manner.19 Using the DCHF assay, also an acute insulin-induced formation of peroxides was detected in intact 3T3-L1 fibroblasts. Interestingly, the insulin-induced ROS formation was prevented when cells were infected by a dominant negative Nox4 construct lacking parts of the cytosolic tail of the enzyme.20 On the basis of these observations, it might however be premature to conclude that the insulin-induced ROS formation is mediated by Nox4 as such a fragment can induce different types of unspecific actions.21,22
In the present study, insulin stimulated the cellular ROS production within 4 hours from the onset of exposure, and this effect was maintained for several days. We assume that ROS production is a consequence of an insulin-mediated induction of the protein. Indeed, such a short time from induction until ROS production was recently demonstrated using tetracycline-inducible Nox4 expression plasmids.23 We observed that insulin stimulated the mRNA expression of Nox1 as well as of Nox4, suggesting that either Nox1 or Nox4 contributes to the increased ROS production in response to insulin. Indeed, siRNA directed against Nox1 and even more prominent against Nox4 attenuated the basal ROS-production in 3T3-L1 cells and also prevented the insulin-induced prolonged increase in radical formation. These observations are compatible with previous reports in other cellular systems, suggesting that Nox4 mediates the basal constitutive ROS production in endothelial cells,24 smooth muscle cells,25 and renal mesangial cells,26 and that the basal ROS production correlates with the expression of Nox4.27 The mechanism of acute insulin-induced ROS production, which was not a focus of this article, however, still needs to be identified. Although siRNA against Nox1 slightly attenuated the basal ROS production in 3T3-L1 cells, differentiation and insulin signaling were completely unaffected by changing Nox1 expression, and therefore the exact contribution of this NADPH oxidase to the function of preadipocytes remains to be determined.
The observations of the present work suggest that insulin-stimulated differentiation involves the induction of Nox4. Recently an article appeared which suggested that Nox4 is downregulated in the course of differentiation.28 Despite this, the authors describe a dramatically higher ROS production in mature adipocytes as compared to undifferentiated 3T3-L1 cells. However, this contradicts the decrease in Nox4 expression described by the authors. Interestingly, in the same article it was reported that adipose tissue of obese but not lean mice contains high level of Nox4. As a consequence those observations rather fit into the model proposed in the present study than into a concept of downregulation of Nox4 during differentiation.
How Does Nox4 Control Differentiation?
The undifferentiated state of preadipocytes is maintained by the EGF-like protein pref-1, which is released from preadipocytes themselves. Pref-1 expression is completely lost in the course of adipocyte differentiation, and overexpression of pref-1 in preadipocytes completely prevents differentiation.29 Pref-1 activates the MEK/ERK-pathway to block the induction of PPAR-2, which is required for further differentiation.30 Interestingly, pref-1 mRNA itself is governed by transcription factors dependent on the MEK/ERK1/2 pathway such as EGR-1,31 suggesting a positive feedback mechanism to maintain preadipocytes in undifferentiated state. In the present study we observed that insulin-induced downregulation of pref-1 was prevented by Nox4 siRNA. Nox4 siRNA also blocked the insulin-induced differentiation embodied in the prevention of aP2 expression and fat accumulation. This might suggest that Nox4 contributes to the downregulation of pref-1, potentially by interfering with pathways controlling pref-1 expression such as the MEK/ERK-pathway. Indeed, we could observe that Nox4 overexpression not only promoted differentiation per se but also decreased the phosphorylation of ERK1/2. Conversely, downregulation of Nox4 increased the phosphorylation of ERK1/2. This effect was probably mediated by MKP-1, the expression of which was dependent on Nox4 in the present study. As MKP-1 dephosphorylates MAPKs, ERK1/2 phosphorylation inversely correlates with Nox4 expression.
It is generally assumed that ROS promote proliferation. Apparently ROS derived from Nox4 lack this property,32 and it is unknown whether this is a consequence of the specific localization of Nox4 in the cell or the constitutive way in which Nox4 generates ROS. With the identification of MKP-1 induction by Nox4, we however provide a possible explanation why Nox4 does not promote proliferation.
MKP-1 Is an Effector of Nox4
Despite its role in pref-1 expression, Nox4-dependent regulation of MKP-1 expression directly controls insulin receptor signaling toward differentiation. After ligand binding the insulin receptor phosphorylates IRS-1 through its intrinsic tyrosine kinase activity,33,34 which subsequently activates PKC isoforms and Akt to induce differentiation.35,36 Simultaneously, the insulin receptor activates Ras, with consecutive activation of MEK and ERK1/2, which is usually depressed by Nox4-mediated induction of MKP-1. ERK1/2 is known to directly phosphorylate IRS-1 on serine 612,37 and this phosphorylation attenuates the direct insulin-receptor tyrosine kinase-dependent tyrosine phosphorylation of IRS-1. Indeed, downregulation of Nox4 attenuated the insulin-induced IRS-1 tyrosine phosphorylation but increased the IRS-1 serine phosphorylation. As a consequence, Nox4 guides the insulin signal toward differentiation by promoting the IRS-1 pathway and by attenuating the ERK1/2-pathway. Loss of Nox4, however, would favor ERK1/2-signaling, which not only inhibits differentiation but also should promote proliferation as observed in the present study. Indeed, we observed that MKP-1 downregulation massively promotes proliferation, whereas overexpression of the protein promoted differentiation. Accordingly, it was observed that mice lacking MKP-1 are resistant to diet-induced obesity.38
Although we provide evidence for a Nox4-dependent induction of MKP-1 in the present study, the underlying molecular mechanisms still remain to be determined. MKP-1 is an immediate early gene product which is rapidly and strongly induced by H2O2.39 Therefore it is attractive to speculate that ROS generated by Nox4 directly control MKP-1 expression. A possible explanation for such a mechanism might be that ROS are known to inactivate phosphatases, and indeed it has been demonstrated that insulin-stimulated ROS production inhibits the phosphatase PTP1B.20,40
In summary, we have demonstrated that by controlling MKP-1 expression, Nox4 attenuates ERK1/2-signaling in preadipocytes. Through this mechanism, Nox4 tips the balance from insulin-induced proliferation toward differentiation.
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Acknowledgments |
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We thank Sina Bätz and Jasmin Geib for excellent technical assistance.
Sources of Funding
This study was supported by grants from the Deutsche Forschungsgemeinschaft, the excellence cluster cardio-pulmonary system (ECCPS), the Schauffler-Stiftung, the Goethe-University, and by the European Vascular Genomic Network, a Network of Excellence supported by the European Community’s sixth Framework Program (Contract LSHM-CT-2003-503254).
Disclosures
None.
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Footnotes |
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Received July 17, 2008; revision accepted November 23, 2008.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Pi-Sunyer FX. Obesity: determinants and therapeutic initiatives. Nutrition. 1991; 7: 292–294.[Medline] [Order article via Infotrieve]
2. Taniguchi CM, Emanuelli B, Kahn CR. Critical nodes in signalling pathways: insights into insulin action. Nat Rev Mol Cell Biol. 2006; 7: 85–96.[CrossRef][Medline] [Order article via Infotrieve]
3. Krieger-Brauer HI, Kather H. 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 Invest. 1992; 89: 1006–1013.[Medline] [Order article via Infotrieve]
4. Bedard K, Krause KH. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev. 2007; 87: 245–313.
5. Ambasta RK, Kumar P, Griendling KK, Schmidt HH, Busse R, Brandes RP. Direct interaction of the novel Nox proteins with p22phox is required for the formation of a functionally active NADPH oxidase. J Biol Chem. 2004; 279: 45935–45941.
6. Sturrock A, Huecksteadt TP, Norman K, Sanders K, Murphy TM, Chitano P, Wilson K, Hoidal JR, Kennedy TP. Nox4 mediates TGF-beta1-induced retinoblastoma protein phosphorylation, proliferation, and hypertrophy in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol. 2007; 292: L1543–L1555.
7. Dardick I, Poznanski WJ, Waheed I, Setterfield G. Ultrastructural observations on differentiating human preadipocytes cultured in vitro. Tissue Cell. 1976; 8: 561–571.[CrossRef][Medline] [Order article via Infotrieve]
8. Anilkumar N, Weber R, Zhang M, Brewer A, Shah AM. Nox4 and nox2 NADPH oxidases mediate distinct cellular redox signaling responses to agonist stimulation. Arterioscler Thromb Vasc Biol. 2008; 28: 1347–1354.
9. Yang VW, Christy RJ, Cook JS, Kelly TJ, Lane MD. Mechanism of regulation of the 422(aP2) gene by cAMP during preadipocyte differentiation. Proc Natl Acad Sci U S A. 1989; 86: 3629–3633.
10. Sul HS, Smas C, Mei B, Zhou L Function of pref-1 as an inhibitor of adipocyte differentiation. Int J Obes Relat Metab Disord. 2000; 24 Suppl 4: S15–S19.[CrossRef]
11. Hwang J, Kleinhenz DJ, Lassegue B, Griendling KK, Dikalov S, Hart CM. Peroxisome proliferator-activated receptor-gamma ligands regulate endothelial membrane superoxide production. Am J Physiol Cell Physiol. 2005; 288: C899–C905.
12. Von KA, Brune B. Activation of peroxisome proliferator-activated receptor gamma by nitric oxide in monocytes/macrophages down-regulates p47phox and attenuates the respiratory burst. J Immunol. 2002; 169: 2619–2626.
13. Cao Z, Umek RM, McKnight SL. Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells. Genes Dev. 1991; 5: 1538–1552.
14. Marumo T, Schini-Kerth VB, Brandes RP, Busse R. Glucocorticoids inhibit superoxide anion production and p22phox mRNA expression in human aortic smooth muscle cells. Hypertension. 1998; 32: 1083–1088.
15. Iuchi T, Akaike M, Mitsui T, Ohshima Y, Shintani Y, Azuma H, Matsumoto T. Glucocorticoid excess induces superoxide production in vascular endothelial cells and elicits vascular endothelial dysfunction. Circ Res. 2003; 92: 81–87.
16. Benian GM, Gruener N. Protein kinase A downregulates the phosphorylation of p47phox in human neutrophils: a possible pathway for inhibition of the respiratory burst. Cell Signal. 1996; 8: 291–296.[CrossRef][Medline] [Order article via Infotrieve]
17. Clempus RE, Sorescu D, Dikalova AE, Pounkova L, Jo P, Sorescu GP, Schmidt HH, Lassegue B, Griendling KK. Nox4 is required for maintenance of the differentiated vascular smooth muscle cell phenotype. Arterioscler Thromb Vasc Biol. 2007; 27: 42–48.
18. Cucoranu I, Clempus R, Dikalova A, Phelan PJ, Ariyan S, Dikalov S, Sorescu D. NAD(P)H oxidase 4 mediates transforming growth factor-{beta}1-induced differentiation of cardiac fibroblasts into myofibroblasts. Circ Res. 2005; 97: 900–907.
19. Krieger-Brauer HI, Medda PK, Kather H. Insulin-induced activation of NADPH-dependent H2O2 generation in human adipocyte plasma membranes is mediated by Gai2. J Biol Chem. 1997; 272: 10135–10143.
20. Mahadev K, Motoshima H, Wu X, Ruddy JM, Arnold RS, Cheng G, Lambeth JD, Goldstein BJ. The NAD(P)H oxidase homolog Nox4 modulates insulin-stimulated generation of H2O2 and plays an integral role in insulin signal transduction. Mol Cell Biol. 2004; 24: 1844–1854.
21. Nakano Y, Banfi B, Jesaitis AJ, Dinauer MC, Allen LA, Nauseef WM. Critical roles for p22phox in the structural maturation and subcellular targeting of Nox3. Biochem J. 2007; 403: 97–108.[CrossRef][Medline] [Order article via Infotrieve]
22. Yu L, Zhen L, Dinauer MC. Biosynthesis of the phagocyte NADPH oxidase cytochrome b558. Role of heme incorporation and heterodimer formation in maturation and stability of gp91phox and p22phox subunits. J Biol Chem. 1997; 272: 27288–27294.
23. Serrander L, Cartier L, Bedard K, Banfi B, Lardy B, Plastre O, Sienkiewicz A, Forro L, Schlegel W, Krause KH. NOX4 activity is determined by mRNA levels and reveals a unique pattern of ROS generation. Biochem J. 2007; 406: 105–114.[CrossRef][Medline] [Order article via Infotrieve]
24. Ago T, Kitazono T, Ooboshi H, Iyama T, Han YH, Takada J, Wakisaka M, Ibayashi S, Utsumi H, Iida M. Nox4 as the major catalytic component of an endothelial NAD(P)H oxidase. Circulation. 2004; 109: 227–233.
25. Ellmark SH, Dusting GJ, Fui MN, Guzzo-Pernell N, Drummond GR. The contribution of Nox4 to NADPH oxidase activity in mouse vascular smooth muscle. Cardiovasc Res. 2005; 65: 495–504.
26. Pleskova M, Beck KF, Behrens MH, Huwiler A, Fichtlscherer B, Wingerter O, Brandes RP, Mulsch A, Pfeilschifter J. Nitric oxide down-regulates the expression of the catalytic NADPH oxidase subunit Nox1 in rat renal mesangial cells. FASEB J. 2006; 20: 139–141.
27. Sorescu D, Weiss D, Lassegue B, Clempus RE, Szocs K, Sorescu GP, Valppu L, Quinn MT, Lambeth JD, Vega JD, Taylor WR, Griendling KK. Superoxide production and expression of nox family proteins in human atherosclerosis. Circulation. 2002; 105: 1429–1435.
28. Mouche S, Mkaddem SB, Wang W, Katic M, Tseng YH, Carnesecchi S, Steger K, Foti M, Meier CA, Muzzin P, Kahn CR, Ogier-Denis E, Szanto I. Reduced expression of the NADPH oxidase NOX4 is a hallmark of adipocyte differentiation. Biochim Biophys Acta. 2007; 1773: 1015–1027.[Medline] [Order article via Infotrieve]
29. Smas CM, Sul HS. Pref-1, a protein containing EGF-like repeats, inhibits adipocyte differentiation. Cell. 1993; 73: 725–734.[CrossRef][Medline] [Order article via Infotrieve]
30. Kim KA, Kim JH, Wang Y, Sul HS. Pref-1 (preadipocyte factor 1) activates the MEK/extracellular signal-regulated kinase pathway to inhibit adipocyte differentiation. Mol Cell Biol. 2007; 27: 2294–2308.
31. McCubrey JA, May WS, Duronio V, Mufson A. Serine/threonine phosphorylation in cytokine signal transduction. Leukemia. 2000; 14: 9–21.[CrossRef][Medline] [Order article via Infotrieve]
32. Geiszt M, Kopp JB, Varnai P, Leto TL. Identification of renox, an NAD(P)H oxidase in kidney. Proc Natl Acad Sci U S A. 2000; 97: 8010–8014.
33. Kasuga M, Karlsson FA, Kahn CR. Insulin stimulates the phosphorylation of the 95,000-dalton subunit of its own receptor. Science. 1982; 215: 185–187.
34. Sun XJ, Crimmins DL, Myers MG Jr, Miralpeix M, White MF. Pleiotropic insulin signals are engaged by multisite phosphorylation of IRS-1. Mol Cell Biol. 1993; 13: 7418–7428.
35. Valverde AM, Kahn CR, Benito M. Insulin signaling in insulin receptor substrate (IRS)-1-deficient brown adipocytes: requirement of IRS-1 for lipid synthesis. Diabetes. 1999; 48: 2122–2131.[Abstract]
36. Crespo P, Font de MJ, Aaronson DS, Santos E, Gutkind JS. Transforming G protein-coupled receptors block insulin and ras-induced adipocytic differentiation in 3T3-L1 cells: evidence for a PKC and MAP kinase independent pathway. Biochem Biophys Res Commun. 1998; 245: 554–561.[CrossRef][Medline] [Order article via Infotrieve]
37. Izawa Y, Yoshizumi M, Fujita Y, Ali N, Kanematsu Y, Ishizawa K, Tsuchiya K, Obata T, Ebina Y, Tomita S, Tamaki T. ERK1/2 activation by angiotensin II inhibits insulin-induced glucose uptake in vascular smooth muscle cells. Exp Cell Res. 2005; 308: 291–299.[CrossRef][Medline] [Order article via Infotrieve]
38. Wu JJ, Roth RJ, Anderson EJ, Hong EG, Lee MK, Choi CS, Neufer PD, Shulman GI, Kim JK, Bennett AM. Mice lacking MAP kinase phosphatase-1 have enhanced MAP kinase activity and resistance to diet-induced obesity. Cell Metab. 2006; 4: 61–73.[CrossRef][Medline] [Order article via Infotrieve]
39. Keyse SM, Emslie EA. Oxidative stress and heat shock induce a human gene encoding a protein-tyrosine phosphatase. Nature. 1992; 359: 644–647.[CrossRef][Medline] [Order article via Infotrieve]
40. Chen K, Kirber MT, Xiao H, Yang Y, Keaney JF Jr. Regulation of ROS signal transduction by NADPH oxidase 4 localization. J Cell Biol. 2008; 181: 1129–1139.
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