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Experimental Biology and Medicine 228:836-842 (2003)
© 2003 Society for Experimental Biology and Medicine

ORIGINAL RESEARCH ARTICLE

Hydrogen Peroxide Inhibits Insulin Signaling in Vascular Smooth Muscle Cells

Carla D. Gardner*, Satoru Eguchi{dagger}, Cherilynn M. Reynolds*, Kunie Eguchi*{ddagger}, Gerald D. Frank{dagger} and Evangeline D. Motley*,1

* Department of Anatomy and Physiology, Meharry Medical College, Nashville, Tennessee 37208 and
{dagger} Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Both insulin resistance and reactive oxygen species (ROS) have been reported to play essential pathophysiological roles in cardiovascular diseases, such as hypertension and atherosclerosis. However, the mechanistic link between ROS, such as H2O2 and insulin resistance in the vasculature, remains undetermined. Akt, a Ser/Thr kinase, mediates various biological responses induced by insulin. In this study, we examined the effects of H2O2 on Akt activation in the insulin-signaling pathway in vascular smooth muscle cells (VSMCs). In VSMCs, insulin stimulates Akt phosphorylation at Ser473. Pretreatment with H2O2 concentration- and time-dependently inhibited insulin-induced Akt phosphorylation with significant inhibition observed at 50 µM for 10 min. A ROS inducer, diamide, also inhibited insulin-induced Akt phosphorylation. In addition, H2O2 inhibited insulin receptor binding partially and inhibited insulin receptor autophosphorylation almost completely. However, pretreatment with a protein kinase C inhibitor, GF109203X (2 µM), for 30 min did not block the inhibitory effects of H2O2 on insulin-induced Akt phosphorylation, suggesting that protein kinase C is not involved in the inhibition by H2O2. We conclude that ROS inhibit a critical insulin signal transduction component required for Akt activation in VSMCs, suggesting potential cellular mechanisms of insulin resistance, which would require verification in vivo.

Key Words: hydrogen peroxide • Akt • protein kinase B • insulin resistance • vascular smooth muscle cells


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The vasculature is an insulin-responsive tissue and alterations in insulin action in the vasculature, mediated via the insulin receptors, have been proposed to contribute to atherosclerosis and the regulation of vascular tone (1, 2). Both increased insulin concentration and decreased insulin sensitivity (insulin resistance) have been identified as independent risk factors for cardiovascular disease (3, 4). However, little is known regarding the pathways of insulin signaling, and their regulation in vascular smooth muscle cells (VSMCs).

Reactive oxygen species (ROS) also play pathogenic roles in vascular remodeling associated with hypertension and/or atherosclerosis (5). It has been demonstrated that lipid peroxidation is increased in individuals with noninsulin-dependent diabetes mellitus (NIDDM) (6). Furthermore, hyperglycemia leads to the production of H2O2 within the cell (7). However, the relationship between insulin resistance and oxidative stress in the vasculature remains unclear. Angiotensin II, a potent vasoconstrictor and hypertrophic inducer of VSMCs, increases the production of ROS in VSMCs (8). Angiotensin II and a protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), impair coupling of the insulin receptor pathway to phosphatidylinositol 3 (PI3)-kinase in cultured rat VSMCs (9). These data indicate that cultured VSMCs are an interesting model to study the mechanism of insulin resistance, possibly induced by ROS.

A Ser/Thr kinase, Akt/protein kinase B, is believed to be the major downstream mediator of PI3 kinase and becomes activated by the insulin receptor through insulin receptor substrate (IRS)-dependent PI3 kinase activation (10, 11). Because the activation of Akt mimics the ability of insulin to stimulate various biological responses, such as glucose transport, glycogen synthase, protein synthesis, antilipolysis, and suppression of hepatic gluconeogenesis (12), its negative regulation by ROS in VSMCs could contribute to insulin resistant states and play a role in the pathogenesis of vascular diseases.

In the present study, we examined whether ROS, such as H2O2, inhibit Akt activation induced by insulin in cultured rat VSMCs. Here, we demonstrated that H2O2 inhibits insulin-induced Akt activation in VSMCs. We further showed that the mechanism by which H2O2 inhibits Akt may involve both inhibition of insulin receptor binding and inhibition of insulin receptor autophosphorylation but does not involve PKC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents.
Insulin, phorbol 12-myristate 13-acetate, diamide, and H2O2 were purchased from Sigma Chemical Company (St. Louis, MO). Antibodies directed to Akt, Ser473-phosphorylated Akt, and Thr308-phosphorylated Akt were purchased from Cell Signaling (Beverly, MA). GF109203X was purchased from Calbiochem (La Jolla, CA). Antibodies directed to IRS-1, insulin receptor-ß, and protein A/G agarose were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). An antibody-directed Tyr1158-phosphorylated insulin receptor-ß was purchased from BioSource International. An anti-phosphotyrosine antibody, 4G10, was purchased from Upstate Biotechnology Inc. (Lake Placid, NY). Dulbecco’s modified eagle’s medium (DMEM), fetal calf serum, penicillin, and streptomycin were purchased from Life Technologies, Inc. (Grand Island, NY). Peroxidase linked anti-rabbit and anti-mouse IgG antibodies were obtained from Amersham Life Science (Arlington Heights, IL).

Cell Culture.
VSMCs were prepared from 12-week-old Sprague-Dawley rats (Charles River Breeding Laboratories) by the explant method and cultured in DMEM containing 10% fetal calf serum, penicillin and streptomycin (13). Subcultured VSMCs used in the experiments showed more than 99% positive immunostaining of smooth muscle {alpha}-actin antibody (Sigma) and were negative for infection of mycoplasma (13). For the experiments, cells from passage 3–12 at about 80–90% confluence in culture were used after 3 days of serum depletion.

Immunoprecipitation.
VSMCs were stimulated with agonists at 37°C. The cells were lysed with ice-cold immunoprecipitation buffer (150 mM NaCl, 1% Triton X-100, 10% [v/v] glycerol, 1 mM EDTA, 50 mM HEPES pH 7.5, 10 mM sodium pyrophosphate, 10 mM NaF, 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml of leupeptin, and 10 µg/ml of aprotinin). The cell lysates were centrifuged at 15,000g for 5 min at 4°C and the supernatant was immunoprecipitated with the antibody and protein A/G-agarose at 4°C for 24 hr as previously described (13).

Immunoblot Analysis.
Cell lysates or immunocomplex lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted using an ECL detection kit (Amersham) as described previously (14).

Binding Assay.
Cells grown on six-well plates were serum-deprived and pretreated with or without 50 µM H2O2 for 10 min. Then, cells were incubated in 1 ml of binding buffer containing DMEM, 25 mM HEPES-HCl (pH 7.8), and 0.5% BSA with or without 8.3 pM [125I] insulin (2000 Ci/mM, Amersham) at room temperature for 4 hr. After incubation, cells were washed five times with ice-cold PBS and then dissolved in 1N NaOH for measuring radioactivity (15).

Statistical Analysis.
Unless stated otherwise, results are the mean ± SEM of determinations of at least three separate experiments giving similar results. The data were analyzed using one-way analysis of variance (* or {dagger}, P < 0.05) followed by Newman–Keuls post-hoc analysis. Data are expressed as fold basal with basal defined as 1.0 in unstimulated cells.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
H2O2 Inhibits Akt Activation Induced by Insulin in VSMCs.
To examine whether H2O2 inhibits Akt activation, VSMC were pretreated with 50 µM H2O2 for various time periods and Akt phosphorylation at Ser473 induced by insulin was determined. H2O2 pretreatment from 2.5 to 20 min significantly inhibited insulin-induced phosphorylation of Akt (Fig. 1AGo). Also, 12.5–50 µM H2O2 attenuated insulin-induced Akt phosphorylation in a concentration-dependent manner. Although it was not statistically significant, 6.25 µM H2O2 slightly enhanced Akt phosphorylation induced by insulin (Fig. 1BGo). Control experiments of 50 µM H2O2 treatment in the absence of insulin showed a weak and transient increase in Akt phosphorylation in VSMC (Fig. 1CGo), whereas no enhancement of phosphorylation was observed with 6.25 µM H2O2 stimulation (data not shown). Treatment with insulin also caused partial reduction of the amount of Akt detected by anti-Akt antibody (Fig. 1DGo). We also measured Thr308 phosphorylation of Akt, the major input required for the activation of Akt (16). Pretreatment of VSMC with H2O2 markedly inhibited insulin-induced Thr308 phosphorylation of Akt (Fig. 1EGo, left), whereas direct treatment of Akt immunoprecipitates with H2O2 had no significant effect on Ser473 (Fig. 1EGo, right) or Thr308 phosphorylation (data not shown) of Akt induced by insulin. To determine the effects of intracellular ROS production on Akt activation, VSMC were pretreated with 1 mM diamide, a ROS inducer (17, 18), and stimulated with or without insulin for 5 min. Like H2O2, diamide markedly inhibited insulin-induced Akt activation (Fig. 2Go), suggesting that the inhibition of Akt phosphorylation in VSMC occurs via a mechanism involving intracellular ROS production.







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  		Figure 1. H2O2 inhibits Akt activation induced by insulin in VSMCs. (A) VSMCs were pretreated with 50 µM H2O2 for the indicated time periods and stimulated with insulin (1 µM) for 5 min without washout of H2O2. The top panel is a representative experiment of cell lysates immunoblotted by phospho-Akt and Akt antibodies. The bottom panel represents data quantified by densitometric analysis. *P < 0.05 vs basal, {dagger}P < 0.05 vs insulin. (B) VSMCs were pretreated with H2O2 for 10 min at the indicated concentrations and stimulated with insulin (1 µM) for 5 min. The top panel is a representative experiment of cell lysates immunoblotted by phospho-Akt and Akt antibodies. The bottom panel represents data quantified by densitometric analysis. *P < 0.05 vs basal, {dagger}P < 0.05 vs insulin. (C) VSMCs were stimulated with H2O2 (50 µM) for indicated time periods. (D) VSMCs were stimulated with insulin (1 µM) for 5 min. The top panel is a representative experiment of cell lysates immunoblotted by phospho-Akt and Akt antibodies. The bottom panel represents data quantified by densitometric analysis. *P < 0.05 vs. basal. (E) On the left panel, VSMCs were not pretreated or were pretreated with 50 µM H2O2 and stimulated with insulin (1 µM) for 5 min. On the right panel, VSMCs were stimulated with insulin for 5 min. Then cell lysates were immunoprecipitated with anti-Akt antibody. The immunoprecipitates were treated with 50 µM H2O2 at room temperature for 10 min.

 


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  		Figure 2. Diamide inhibits insulin-induced Akt phosphorylation in VSMCs. VSMCs were pretreated with 1 mM diamide for the indicated time periods and stimulated with insulin (1 µM) for 5 min. The top panel is a representative experiment of cell lysates immunoblotted by phospho-Akt and Akt antibodies. The bottom panel represents data quantified by densitometric analysis. *P < 0.05 vs basal, {dagger}P < 0.05 vs insulin.

 
H2O2 Inhibits Insulin Receptor Binding and Autophosphorylation in VSMCs.
Because IRS mediates insulin-induced PI3 kinase activation and subsequent Akt activation, the effect of H2O2 on insulin-induced IRS-1 tyrosine phosphorylation was determined. VSMCs were pretreated with 50 µM H2O2 for 10 min and stimulated with or without insulin for 2 min. Insulin-induced tyrosine phosphorylation of immunoprecipitated IRS-1 was markedly inhibited by H2O2 (Fig. 3AGo). Figure 3BGo shows that H2O2 decreased the amount of immunoprecipitated IRS-1 from VSMC lysate, suggesting the possible degradation of IRS-1 in response to H2O2. Although approximately 20% inhibition was observed in the ratio between the pTyr and the immunoprecipitated IRS-1 (Fig. 3CGo), IRS-1 content in total cell lysate was unchanged by the H2O2 treatment (Fig. 3DGo). This indicates that modification of IRS-1 by H2O2 may affect efficiency of IRS-1 immunoprecipitation but not its immunoblotting. Thus, the ratio may not represent the actual status of total IRS-1 function in VSMCs. Therefore, we further determined insulin receptor ß autophosphorylation by phospho-specific antibody and insulin binding to the receptor by binding assay. H2O2 treatment partially inhibited the binding (Fig. 4AGo), and almost completely inhibited the autophosphorylation (Fig. 4BGo). Therefore, inhibition of Akt by H2O2 treatment involves both inhibition of receptor binding and inhibition of receptor autophosphorylation.






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  		Figure 3. Effects of H2O2 on insulin-induced IRS-1 tyrosine phosphorylation in VSMCs. VSMCs were pretreated with 50 µM H2O2 for 10 min and stimulated with insulin (1 µM) for 2 min. Cell lysates were immunoprecipitated with IRS-1 antibody and immunoblotted by (A) anti-phosphotyrosine (pTyr) and (B) IRS-1 antibodies (top panel). The bottom panel represents data quantified by densitometric analysis. *P < 0.05 vs basal, {dagger}P < 0.05 vs insulin. The ratio (pTyr/IRS-1) of the insulin-stimulated samples is shown in (C). (D) VSMCs were stimulated with 50 µM H2O2 for the indicated time periods. Cell lysates were immunoblotted with anti-IRS-1 antibody.

 



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  		Figure 4. H2O2 inhibits insulin binding and IRß phosphorylation in VSMCs. (A) VSMCs were pretreated with or without 50 µM H2O2 for 10 min. Then, radiolabeled insulin binding in VSMCs were determined. (B) VSMCs were pretreated with or without 50 µM H2O2 for 10 min. The cells were then stimulated with insulin for 2 min, and cell lysates were immunoprecipitated with anti-IRß antibody and immunoblotted with pIRß and IRß antibodies, respectively (top panel). The bottom panel represents data quantified by densitometric analysis. *P < 0.05 vs basal, {dagger}P < 0.05 vs insulin.

 
Role of PKC in the H2O2 Inhibition of Insulin-Induced Akt Phosphorylation.
It has been shown that prior activation of PKC can inhibit the subsequent ability of insulin to stimulate the enzymatic activity of Akt in 3T3-L1 adipocytes (19). To test whether PKC is responsible for the inhibition of Akt activation by H2O2, we examined the effect of a PKC inhibitor, GF109203X, on the H2O2 and PMA-induced inhibition of Akt activation. GF109203X inhibits PKC-{alpha}, -ß, -{gamma}, -{delta}, and -{varepsilon} isoforms (20), and VSMCs were shown to express PKC-{alpha}, -ß1, -{delta}, -{varepsilon}, and -{zeta} isoforms, but not -ß2, -{gamma}-{theta}, -{eta}, or -{lambda} (21). As shown in Figure 5Go, a PKC activator, PMA, inhibited insulin-induced Akt activation, and GF109203X was able to reverse the effects of PMA. In contrast, GF109203X did not reverse the effects of H2O2, suggesting that PKC is not required for the H2O2–induced inhibition of insulin-induced Akt activation. However, possible involvement of atypical PKCs, such as PKC-{zeta}, remains to be studied.



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  		Figure 5. Role of PKC in the H2O2 inhibition of insulin-induced Akt phosphorylation. VSMCs were pretreated with 2 µM GF109203X for 30 min and then pretreated with H2O2 or PMA for 10 min. The cells were then stimulated with insulin for 5 min. The top panel is a representative experiment of cell lysates immunoblotted by phospho-Akt and Akt antibodies. The bottom panel represents data quantified by densitometric analysis. *P < 0.05 vs basal, {dagger}P < 0.05 vs insulin.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The major findings of the present study are (i) that H2O2 and diamide inhibit Akt phosphorylation in VSMCs, (ii) H2O2 also inhibits insulin receptor binding and receptor autophosphorylation, and (iii) the activation of PKC is most likely not involved in the H2O2–induced inhibition of insulin-induced Akt activation. These data will add new insight to the potential molecular mechanism by which ROS cause cellular insulin resistance.

Oxidative stress is increased in vivo in the diabetic state, and NIDDM is associated with accelerated production of ROS as well as a decreased scavenging of ROS (6, 7). Moreover, increased cardiovascular oxidative stress is a risk factor that clusters with syndrome X (3), suggesting that ROS may mediate insulin resistance in cardiovascular disease states. Here, we have shown that H2O2 inhibits Akt activation in VSMCs. This notion is supported by recent works in other cells demonstrating that H2O2 and other oxidative stress impairs insulin-induced stimulated GLUT4 translocation, PI3-kinase activation and Akt activation (22–24). Activation of PI3-kinases by insulin is controlled by IRS protein tyrosine phosphorylation coupled with the insulin receptor (25). Similar to the report by Hansen et al. (26), we have also shown that H2O2 decreased the autophosphorylation of insulin receptor ß. However, this inhibition may partially involve inhibition of receptor binding by H2O2. Thus, inhibition of insulin function by H2O2 may be operated at multiple levels proximal to Akt.

H2O2 increases PKC activation in several cell lines. Studies have shown that activation of PKC decreases insulin-induced tyrosine phosphorylation of IRS-1 and its ability to activate PI3-kinase (27–29). In this regard, Barthel et al. (19) recently demonstrated that PKC inhibits the activation of Akt induced by insulin in 3T3-L1 adipocytes. We, and others, have recently shown the inhibitory effects of PKC on insulin signaling in VSMC (9, 30–32). However, our results clearly demonstrate that H2O2 elicits its inhibitory effects through a PKC independent pathway, indicating cell-type specificity mechanisms regarding ROS-dependent insulin resistance.

In our experiments, we recognized that insulin partially reduced the amount of Akt in VSMCs. Caspase-dependent degradation of Akt was demonstrated in many cell lines undergoing apoptosis (33, 34). However, a different mechanism could mediate the degradation in VSMC because this mechanism is much quicker than the caspase-dependent degradation and associated with kinase phosphorylation. In addition, the present study showed an enhanced phosphorylation of Akt induced by 50 µM H2O2. This may involve the insulinomimetic action of H2O2 as previously shown in VSMCs (35). Although it is not significant, Akt phosphorylation tended upward as exposure to diamide was prolonged. This could also involve the insulinomimetic action of ROS and/or induction of endogenous antioxidant activity.

Currently, there is no established pathophysiological meaning of insulin’s action in VSMC relevant to cardiovascular diseases. Earlier studies suggest that insulin promotes these diseases by inducing growth and synthesis of extracellular matrix proteins in VSMC (2). By contrast, recent papers showed that insulin action in VSMC might protect VSMC from remodeling by inhibiting apoptosis (36) and migration (37). Thus, our findings that ROS inhibit insulin signal transduction in VSMC could be a potential cellular mechanisms of insulin resistance leading to cardiovascular diseases, which would require future verification in vivo.


   Footnotes
 
This work was supported in part by the National Institutes of Health-National Heart, Lung and Blood Institute Grants HL-03320, HL-58205, and HL-07864; by the National Institutes of Health-National Center for Research Resources Grant 2G12 RR-03032; and by the American Heart Association Grants 0150829B and 0130053N. Dr. Frank was supported by a United Negro College Fund/Merck Postdoctoral Science Research Fellowship. Dr. Eguchi was supported by the Diabetes Center Pilot and Feasibility Program of Vanderbilt University.

1 To whom requests for reprints should be addressed at the Department of Anatomy and Physiology, Meharry Medical College, Nashville, TN 37208. E-mail: emotley{at}mmc.edu Back


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 Introduction
 Materials and Methods
 Results
 Discussion
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Received for publication March 14, 2002. Accepted for publication February 12, 2003.





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