Insulin Receptor Substrate-1 and Phosphoinositide-Dependent Kinase-1 Are Required for Insulin-Stimulated Production of Nitric Oxide in Endothelial Cells

Monica Montagnani, Lingamanaidu V. Ravichandran, Hui Chen, Diana L. Esposito and Michael J. Quon

Cardiology Branch (M.M., L.V.R., H.C., M.J.Q.), National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892; and Department of Oncology and Neurosciences (D.L.E.), University G. D’Annunzio, Chieti 66013, Italy

Address all correspondence and requests for reprints to: Michael J. Quon, M.D., Ph.D., Cardiology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Building 10, Room 8C-218, 10 Center Drive MSC 1755, Bethesda, Maryland 20892-1755. E-mail: quonm{at}nih.gov.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Vasodilator actions of insulin are mediated by signaling pathways involving phosphatidylinositol 3-kinase (PI 3-kinase) and Akt that lead to activation of endothelial nitric oxide synthase (eNOS) in endothelium. Signaling molecules immediately upstream and downstream from PI 3-kinase involved with production of NO in response to insulin have not been previously identified. In this study, we evaluated roles of insulin receptor substrate 1 (IRS-1) and phosphoinositide-dependent kinase 1 (PDK-1) in production of NO. The fluorescent dye 4,5-diamine fluorescein diacetate was used to directly measure NO in NIH-3T3IR cells transiently cotransfected with eNOS and various IRS-1 or PDK-1 constructs. In control cells, transfected with only eNOS, insulin stimulated a rapid dose-dependent increase in NO. Overexpression of wild-type IRS-1 increased the maximal insulin response 3-fold. Overexpression of IRS1-F6 (mutant that does not bind PI 3-kinase) or an antisense ribozyme against IRS-1 substantially inhibited insulin-stimulated production of NO. Likewise, overexpression of wild-type PDK-1 enhanced insulin-stimulated production of NO, whereas a kinase-inactive mutant PDK-1 inhibited this action of insulin. Qualitatively similar results were observed in vascular endothelial cells. Production of NO by a calcium-dependent mechanism in response to lysophosphatidic acid was unaffected by either wild-type or mutant IRS-1 and PDK-1. We conclude that IRS-1 and PDK-1 play necessary roles in insulin-signaling pathways leading to activation of eNOS. Furthermore, classical Ca2+-mediated pathways for activation of eNOS are separable from IRS-1- and PDK-1-dependent insulin-signaling pathways.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
INSULIN HAS PHYSIOLOGICAL actions in the vasculature to promote vasodilation and blood flow that may help couple regulation of metabolic and hemodynamic homeostasis (1, 2). Previously, we identified the insulin receptor tyrosine kinase, phosphatidylinositol 3-kinase (PI 3-kinase), and Akt as essential components of insulin-signaling pathways related to production of NO in vascular endothelium (3, 4). We also demonstrated that phosphorylation of endothelial nitric oxide synthase (eNOS) by Akt is necessary for its activation by insulin (5). Moreover, this phosphorylation-dependent mechanism for insulin-stimulated activation of eNOS is independent and separable from classical calcium-dependent pathways (5). However, a complete biochemical pathway linking the insulin receptor to phosphorylation and activation of eNOS has not been directly demonstrated.

Many receptor tyrosine kinases bind and activate src homology 2 (SH2) domain-containing proteins such as PI 3-kinase (6). Although the autophosphorylated insulin receptor is also capable of directly binding to SH2 domains of PI 3-kinase (7, 8), it generally propagates signals by phosphorylating intracellular substrates. Specific tyrosine-phosphorylated motifs in these insulin receptor substrates then bind to SH2 domains of downstream signaling molecules (9). Substrates of the insulin receptor that function as docking proteins for molecules containing SH2 domains include insulin receptor substrate (IRS)-1, -2, -3, and -4, Grb-2 associated binder-1, p62dok, and Shc (10, 11). When tandem SH2 domains on the p85 regulatory subunit of PI 3-kinase bind to phosphorylated YXXM motifs on these insulin receptor substrates, the preassociated p110 catalytic subunit of PI 3-kinase is activated. Although IRS family members share many similarities, the various isoforms maintain specificity with respect to particular biological actions of insulin (10). Therefore, it is important to identify the main receptor substrate involved with insulin-stimulated production of NO in endothelium. Lipid products of PI 3-kinase such as phosphatidylinositol 3,4,5-triphosphate generated in response to insulin stimulation help localize and activate the ser/thr kinase phosphoinositide- dependent kinase 1 (PDK-1) in signaling complexes (12, 13). PDK-1 can then phosphorylate and activate downstream kinases including Akt, protein kinase C (PKC)-{zeta}/{lambda}, serum- and glucocorticoid-dependent kinase, and p70 S6 kinase that mediate diverse biological actions of insulin (14). However, in some cases, Akt can be activated independently of PI 3-kinase and PDK-1 (15, 16, 17, 18, 19). Thus, it is also important to explicitly test for the participation of PDK-1 in the production of NO in response to insulin. In the present study, we used wild-type and inhibitory mutants of IRS-1 and PDK-1 as well as an antisense ribozyme against IRS-1 to demonstrate necessary roles for IRS-1and PDK-1 in insulin-stimulated production of NO in vascular endothelium. With inclusion of these components, we have identified a complete biochemical pathway leading from the insulin receptor to phosphorylation and activation of eNOS.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Role of IRS-1 in Insulin-Stimulated Production of NO
To investigate insulin-signaling pathways related to production of NO, we used a previously validated transfection system employing the NO-specific fluorescent dye 4,5-diaminofluorescein diacetate (DAF-2 DA) (5). NIH-3T3IR fibroblasts that do not express endogenous eNOS were cotransfected with expression vectors for red fluorescent protein (RFP), eNOS, and either the empty expression vector pCIS2 or IRS1-wild type (WT). Transfected cells were identified by expression of RFP (Fig. 1AGo, first column). As previously demonstrated (4), cotransfection efficiency was nearly 100% so that cells expressing RFP also expressed eNOS. Importantly, transfected cells were the only cells that produced detectable NO in response to insulin. Thus, we could directly evaluate effects of various transgenes on insulin-stimulated production of NO. We first evaluated the consequences of overexpressing wild-type IRS-1 (Fig. 1Go). Consistent with previous studies (5), in control cells expressing eNOS alone, insulin stimulated production of NO in a dose-dependent manner with an ED50 of 50 nM (Fig. 1AGo, top panels; Fig. 1BGo, solid circles). By contrast, overexpression of IRS1-WT resulted in a 3-fold increase in production of NO in response to maximal insulin stimulation without causing a significant change in insulin sensitivity (Fig. 1AGo, bottom panels; Fig. 1BGo, solid triangles). These data suggest that IRS-1 is capable of mediating production of NO in response to insulin.



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Figure 1. Overexpression of IRS-1 Increases Insulin-Stimulated Production of NO

NIH-3T3IR cells transiently cotransfected with RFP, eNOS, and either pCIS2 ({bullet}) or IRS1-WT ({blacktriangleup}) were loaded with DAF-2 DA and treated with insulin as described in Materials and Methods. Transfected cells expressing RFP were identified by emission of red light upon excitation at 558 nm. Increasing concentrations of insulin were added to the cells at 3-min intervals. Production of NO in the transfected cells visualized by emission of green light upon excitation at 489 nm was quantified using a digital camera. A, Transfected cells expressing RFP are shown in the left column. Progressive increases in green fluorescence (indicative of increased NO production) are observed in response to increasing concentrations of insulin (last three columns on the right). B, Relative changes in green fluorescence 2 min after the addition of each insulin dose are shown. Results are the mean ± SEM of five independent experiments.

 
We next inquired whether overexpression of IRS-1 would also enhance production of NO in response to lysophosphatidic acid (LPA, a phospholipid growth factor that activates eNOS by mobilization of intracellular Ca2+). Cells cotransfected with RFP, eNOS, and either pCIS2 or various IRS-1 constructs were sequentially stimulated with LPA and then insulin. Comparable expression of eNOS and IRS-1 constructs in each experimental group was demonstrated by immunoblotting (Fig. 2CGo). The time courses of LPA- and insulin-stimulated production of NO in control cells expressing eNOS alone were similar to those reported previously (5). That is, LPA treatment (5 µM) resulted in a rapid increase in NO production that peaked at 30 sec and came back to basal after 3 min (Fig. 2AGo, upper panels; Fig. 2BGo, open circles). Subsequent insulin treatment (500 nM) resulted in a slower rise in NO that reached a maximum after 2 min and rapidly returned to basal within the next 1 min (Fig. 2AGo, upper panels; Fig. 2BGo, solid circles). Interestingly, overexpression of wild-type IRS-1 had no significant effect on the magnitude or time course of LPA-mediated production of NO (Fig. 2AGo, middle panels; Fig. 2BGo, open triangles). Although the time course of insulin-stimulated production of NO was unaffected, the magnitude of the NO response to insulin was greatly enhanced by overexpression of IRS1-WT consistent with results shown in Fig. 1Go (Fig. 2AGo, middle panels; Fig. 2BGo, solid triangles). Thus, in contrast with insulin signaling pathways, IRS-1 does not appear to interact with pathways used by LPA to activate eNOS.



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Figure 2. IRS-1 Plays an Essential Role in Mediating Actions of Insulin, But Not of LPA, to Stimulate Production of NO

NIH-3T3IR cells transiently transfected with RFP, eNOS, and either pCIS2 (empty vector) or various IRS-1 constructs were loaded with DAF-2 DA and then sequentially stimulated with LPA and insulin. A, Expression of RFP in transfected cells was detected with excitation at 558 nm (left column). NO production in transfected cells was detected by green fluorescence in response to LPA treatment (5 µM, 30 sec) and insulin treatment (500 nM, 2 min). B, The time course for NO production in response to LPA and insulin treatment is shown for control cells transfected with pCIS2 (circles), cells overexpressing IRS1-WT (triangles), and cells overexpressing IRS1-F6 (squares). Results are the mean ± SEM of at least six independent experiments. C, Anti-HA and anti-eNOS immunblots of lysates derived from transfected cells demonstrating comparable expression of transfected eNOS and HA-tagged IRS-1 constructs in each group.

 
To determine whether IRS-1 is playing a necessary role to couple signaling from the insulin receptor to PI 3-kinase and subsequent activation of eNOS, we evaluated the consequences of overexpressing IRS1-F6 [a mutant unable to bind and activate PI 3-kinase in response to insulin stimulation (20)]. Expression of IRS1-F6 did not significantly modulate LPA-stimulated production of NO (Fig. 2AGo, lower panels; Fig. 2BGo, open squares). However, production of NO in response to insulin was completely inhibited in cells expressing IRS1-F6 (Fig. 2AGo, lower panels, Fig. 2BGo, solid squares). These results suggest that IRS1-F6 is behaving in a dominant inhibitory manner and that IRS molecules are essential substrates linking signaling from the insulin receptor to PI 3-kinase-dependent pathways involved with activation of eNOS. Moreover, unlike insulin-mediated mechanisms, LPA-mediated production of NO may involve distinct mechanisms to activate eNOS that do not interact with IRS-1.

To confirm that results from our model system reflect mechanisms operative in a physiologically relevant cell type, we repeated key experiments using bovine aortic endothelial cells (BAEC) in primary culture (Fig. 3Go). As in our model system, in untransfected BAEC, insulin stimulated the production of NO in a dose-dependent manner (Fig. 3AGo, solid circles). Moreover, overexpression of wild-type IRS-1 significantly enhanced the magnitude of the NO response to insulin without affecting insulin sensitivity (Fig. 3AGo, solid triangles). Because it is possible that IGF-I receptors may be activated by high doses of insulin, we also tested the effects of IGF-I to stimulate NO production in BAEC. Importantly, comparable doses of IGF-I did not elicit as large a response as insulin in either untransfected BAEC (Fig. 3AGo, open circles) or in cells overexpressing IRS-1 WT (Fig. 3AGo, open triangles). Because there are 10 times as many IGF-I receptors as insulin receptors on endothelial cells (3), we conclude that the effects of IRS-1 constructs on production of NO in response to insulin, which we observed in endothelial cells, is specifically linked to insulin receptor signaling. Although the absolute magnitude of the response was approximately 8 times lower in untransfected BAEC than in NIH-3T3IR control cells expressing eNOS alone, the time course and relative dynamics for LPA- and insulin-stimulated production of NO were similar (Fig. 3BGo, open and solid circles). Overexpression of either IRS1-WT or IRS1-F6 in BAEC did not significantly alter the production of NO in response to LPA (Fig. 3BGo, open triangles and squares). As with NIH-3T3IR cells, overexpression of IRS1-WT in BAEC significantly enhanced production of NO in response to insulin (Fig. 3BGo, solid triangles), whereas insulin-stimulated production of NO was substantially inhibited in BAEC expressing IRS1-F6 (Fig. 3BGo, solid squares). Thus, our results in endothelial cells agree qualitatively with results from our minimal system and strongly support a necessary role for IRS proteins in mediating actions of insulin, but not of LPA, to stimulate production of NO in vascular endothelium.



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Figure 3. IRS-1 Plays an Essential Role in Mediating Actions of Insulin, But Not of LPA, to Stimulate Production of NO in BAEC

BAEC were transfected with various IRS-1 constructs and treated as described in the legend to Fig. 2Go. A, Increasing concentrations of insulin (closed symbols) or IGF-I (open symbols) were added to the cells at 3-min intervals. Production of NO is shown 2 min after each dose was added in untransfected control BAEC (circles) or BAEC overexpressing IRS1-WT (triangles). Results are the mean ± SEM of at least three independent experiments. B, The time course for NO production in response to LPA (5 µM) and insulin treatment (500 nM) is shown for untransfected control cells (circles), cells overexpressing IRS1-WT (triangles), and cells overexpressing IRS1-F6 (squares). Production of NO was quantified as described in Materials and Methods. Results shown are the mean ± SEM of five independent experiments.

 
To explicitly link the effects of wild-type IRS-1 and IRS1-F6 on production of NO to PI 3-kinase activity, we directly assessed the ability of the p85 regulatory subunit of PI 3-kinase and PI 3-kinase activity to coimmunoprecipitate with the IRS-1 constructs in response to insulin in our system (Fig. 4Go). As expected, the amount of p85 and PI 3-kinase activity coimmunoprecipitated with wild-type IRS-1 significantly increased in response to insulin stimulation in transfected NIH-3T3IR cells (Fig. 4Go, lanes 3 and 4), whereas there was no detectable p85 or PI 3-kinase activity associated with IRS1-F6 either before or after insulin stimulation (Fig. 4Go, lanes 5 and 6). Similar results were observed in endothelial cells (data not shown). Thus, the effects of wild-type IRS-1 and IRS1-F6 on production of NO in response to insulin parallel their ability to engage p85 and subsequently activate PI 3-kinase.



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Figure 4. PI 3-Kinase Activity and p85 Associated with IRS-1 Constructs

NIH-3T3IR cells transfected with pCIS2 or HA-tagged IRS-1 constructs were serum starved overnight and treated with insulin (100 nM, 3 min) as indicated. Recombinant IRS-1 was recovered from cell lysates by immunoprecipitation with an anti-HA antibody. PI 3-kinase activity associated with anti-HA immunoprecipitates (upper panel) was measured by thin layer chromatography, as previously described (20 21 ). Coimmunoprecipitation of p85 with IRS-1 was assessed by immunoblotting anti-HA immunoprecipitates with an anti-p85 antibody (middle panel). Comparable recovery of IRS-1 constructs is demonstrated by immunoblotting anti-HA immunoprecipitates with anti-IRS-1 antibody (lower panel). Representative blots are shown from experiments that were repeated independently three times.

 
Because IRS1-F6 may potentially inhibit all IRS family members from interacting with insulin receptors, we used an antisense ribozyme directed specifically against rat IRS-1 to further evaluate the role of endogenous IRS-1 in insulin-stimulated production of NO. In rat fibroblasts coexpressing human insulin receptor (hIR), eNOS, and rIRS1-ribozyme, insulin-stimulated production of NO was significantly reduced when compared with cells expressing the Ribo-CTRL (Fig. 5Go, solid circles and squares). Production of NO in response to LPA was not affected by expression of either rIRS1-ribozyme or the Ribo-CTRL (Fig. 5Go, open circles and squares). Qualitatively similar results were obtained in rat endothelial cells (Fig. 5BGo). Moreover, the rat-specific IRS-1 ribozyme did not inhibit insulin-stimulated production of NO in bovine endothelial cells (data not shown). These results further confirm a necessary role for IRS-1 in mediating signaling from the insulin receptor to activation of eNOS and increased production of NO.



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Figure 5. Antisense Ribozyme Against IRS-1 Specifically Impairs Actions of insulin, But Not of LPA, to Stimulate Production of NO

A, Rat fibroblasts transiently transfected with RFP, hIR, eNOS, and either Ribo-CTRL or rIRS1-Ribozyme were loaded with DAF-2 DA and then sequentially stimulated with LPA and insulin. The time course for NO production in response to LPA and insulin treatment is shown for control cells transfected with Ribo-CTRL (circles) and cells expressing rIRS1-Ribozyme (squares). Results are the mean ± SEM of four independent experiments. B, Rat aortic endothelial cells.

 
Interactions of PI 3-Kinase with IRS-1 Mediate Its Effects to Promote Production of NO
We used previously characterized mutants of IRS-1 that contain intact YXXM motifs at either or both positions 612 and/or 632 to evaluate the importance of interactions between PI 3-kinase and IRS-1 in mediating insulin-stimulated production of NO (four other YXXM motifs at 465, 662, 941, and 989 have substitutions of Phe for Tyr). We previously demonstrated that IRS1-Y612/Y632 binds and activates PI 3-kinase and mediates translocation of glucose transporter 4 (GLUT4) in response to insulin in a manner comparable to wild-type IRS-1 (20). IRS1-Y612 and IRS1-Y632 have an impaired ability to bind and activate PI 3- kinase when compared with wild-type IRS-1 (20). LPA and insulin treatment of NIH-3T3IR cells overexpressing IRS1-Y612/Y632 gave results that were similar to those observed in cells overexpressing IRS1-WT (Fig. 6Go, open and solid diamonds). That is, LPA-stimulated production of NO was unaffected by IRS1-Y612/Y632, whereas insulin-stimulated production of NO was greatly enhanced. By contrast, cells overexpressing either IRS1-Y612 (Fig. 6Go, solid triangles) or IRS1-Y632 (Fig. 6Go, solid squares) had a slightly enhanced insulin response that was intermediate between that of the control cells and cells overexpressing IRS1-Y612/Y632. The LPA response was unaffected by overexpression of either IRS1-Y612 (Fig. 6Go, open triangles) or IRS1-Y632 (Fig. 6Go, open squares). Thus, results with our IRS-1 mutants suggest that it is the ability of IRS-1 to bind and activate PI 3-kinase that mediates insulin-stimulated production of NO. Furthermore, tyrosines at positions 612 and 632 on IRS-1 are both required to mimic the full effect of wild-type IRS-1.



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Figure 6. Tyr612 and Tyr632 in IRS-1 Are Essential for Mediating the Effects of Insulin to Stimulate Production of NO

NIH-3T3IR cells transiently transfected with RFP, eNOS, and pCIS2 (circles), IRS1-Y612/Y632 (diamonds), IRS1-Y612 (triangles) or IRS1-Y632 (squares) were loaded with DAF-2 DA and then sequentially stimulated with LPA (5 µM) and insulin (500 nM). Production of NO was quantified as described in Materials and Methods. Results shown are the mean ± SEM of at least six independent experiments.

 
We recently demonstrated that serine phosphorylation of IRS-1 by PKC-{zeta} impairs the ability of IRS-1 to bind and activate PI 3-kinase in response to insulin stimulation (21). Therefore, to further investigate the importance of IRS-1/PI 3-kinase interactions in insulin-stimulated production of NO, we examined effects of coexpressing PKC-{zeta} with IRS-1. In NIH-3T3IR cells cotransfected with IRS1-WT and PKC-{zeta}, NO production in response to LPA treatment (Fig. 7AGo, open triangles) was similar to that observed in control cells (Fig. 7Go, open circles). By contrast, insulin-stimulated production of NO was significantly reduced in cells coexpressing PKC-{zeta} and IRS-1 (Fig. 7AGo, solid triangles) when compared with control cells overexpressing only IRS1-WT (Fig. 7AGo, solid circles). Thus, the ability of PKC-{zeta} to impair IRS-1/PI 3-kinase interactions may result in decreased activation of eNOS in response to insulin when PKC-{zeta} is overexpressed. To determine whether a similar phenomenon could also be observed in cells not overexpressing IRS-1, control cells expressing only eNOS were compared with cells cotransfected with eNOS and PKC-{zeta} (but not IRS1-WT). Interestingly, even without overexpression of IRS-1, transfection of PKC-{zeta} still resulted in a significant impairment of insulin-stimulated production of NO without affecting the LPA response (when compared with control cells) (Fig. 7BGo). To further support the idea that the negative effect of PKC-{zeta} on insulin-stimulated production of NO is due to specific interactions of PKC-{zeta} with IRS-1, we coexpressed IRS-2 with or without PKC-{zeta}. PKC-{zeta} serine-phosphorylates IRS-1 but not IRS-2 (Ravichandran, L. V., and M. J. Quon, unpublished observations). Overexpression of IRS-2 enhanced production of NO in response to insulin when compared with control cells in a manner similar to IRS-1 (Fig. 7CGo, compare solid squares and diamonds). Importantly, in contrast to results with IRS-1, coexpression of PKC-{zeta} with IRS-2 did not impair the ability of overexpressed IRS-2 to enhance NO production in response to insulin (Fig. 7CGo, solid inverted triangles). As with IRS-1, IRS-2 had no effect on LPA-stimulated production of NO (Fig. 7CGo, open symbols). Taken together, these results are consistent with a negative regulatory effect of PKC-{zeta} on IRS-1 and provide additional support for the key role of IRS-1 in linking signaling from the insulin receptor with PI 3- kinase pathways mediating insulin-stimulated production of NO.



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Figure 7. PKC-{zeta} Negatively Modulates the Ability of IRS-1 to Mediate Insulin-Stimulated Production of NO

A, NIH-3T3IR cells transiently transfected with RFP, eNOS, IRS1-WT, and either pCIS2 (circles) or PKC-{zeta} (triangles) were loaded with DAF-2 DA and then sequentially stimulated with LPA (5 µM) and insulin (500 nM). Production of NO was quantified as described in Materials and Methods. Results shown are the mean ± SEM of four independent experiments. B, NIH-3T3IR cells transiently transfected with RFP, eNOS, and either pCIS2 (circles) or PKC-{zeta} (triangles) were loaded with DAF-2 DA and then sequentially stimulated with LPA and insulin. Production of NO was quantified as described in Materials and Methods. Results shown are the mean ± SEM of five independent experiments. C, NIH-3T3IR cells transiently transfected with RFP, eNOS, IRS2-WT, and either pCIS2 (squares) or PKC-{zeta} (inverted triangles) as well as control cells transfected with RFP, eNOS, and pCIS2 (diamonds) were loaded with DAF-2 DA and then sequentially stimulated with LPA and insulin. Production of NO was quantified as described in Materials and Methods. Results shown are the mean ± SEM of four independent experiments.

 
Recent studies have suggested that the calcium binding protein calmodulin can interact with IRS-1 (22, 23, 24) and that calmodulin may play a role in mediating metabolic actions of insulin (25, 26, 27, 28). Therefore, we tested the effects of two structurally distinct calmodulin inhibitors on insulin- and LPA-stimulated production of NO. NIH-3T3IR cells transfected with eNOS were pretreated with trifluoperazine (TFP; 40 µM, 20 min) or ophiobolin A (50 µM, 30 min). As expected, TFP completely inhibited LPA-mediated production of NO (Fig. 8Go, compare open circles and open triangles). Interestingly, the effects of insulin to stimulate production of NO were partially inhibited by TFP consistent with a role for calmodulin in modulating this process (Fig. 8Go, compare solid circles with solid triangles). Similar results were observed after pretreatment of cells with ophiobolin A (data not shown).



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Figure 8. Effects of TFP on LPA- and Insulin-Stimulated Production of NO

NIH-3T3IR cells transiently cotransfected with RFP and eNOS were loaded with DAF-2 DA, pretreated without (circles) or with (triangles) TFP (40 µM, 20 min), and then sequentially stimulated with LPA (5 µM) and insulin (500 nM). Results shown are the mean ± SEM of at least five independent experiments.

 
Role of PDK-1 in Insulin-Stimulated Production of NO
After identifying IRS-1 as an upstream link to PI 3-kinase that mediates insulin-stimulated production of NO, we next investigated the role of PDK-1 as a downstream effector of PI 3-kinase that couples to Akt and subsequent activation of eNOS. NIH-3T3IR cells cotransfected with eNOS and either wild-type PDK-1 or PDK1-K114A (kinase-inactive mutant) were sequentially stimulated with LPA and insulin (Fig. 9Go). As with IRS-1, overexpression of either PDK1-WT or PDK1-K114A did not significantly affect the ability of LPA to stimulate production of NO (Fig. 9AGo, open symbols). By contrast, when compared with control cells (Fig. 9AGo, solid circles), overexpression of PDK1-WT significantly enhanced insulin-stimulated production of NO (Fig. 9AGo, solid triangles). More importantly, overexpression of PDK1-K114A substantially inhibited NO production in response to insulin (Fig. 9AGo, closed squares). Qualitatively similar results were obtained when these experiments were repeated in primary endothelial cells. That is, the PDK-1 constructs did not significantly alter LPA-mediated production of NO in BAEC, whereas overexpression of PDK1-WT enhanced insulin-stimulated production of NO, and PDK1-K114A inhibited this action of insulin in BAEC (Fig. 9BGo). Thus, PDK-1 appears to be an essential component of the insulin-signaling pathway (but not the LPA-signaling pathway) leading to activation of eNOS and enhanced production of NO in endothelium.



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Figure 9. PDK-1 Is Required for Insulin-Stimulated Production of NO

A, NIH-3T3IR cells transiently transfected with RFP, eNOS, and pCIS2 (circles), PDK1-WT (triangles), or PDK1-K114A (squares) were loaded with DAF-2 DA and then sequentially stimulated with LPA (5 µM) and insulin (500 nM). Production of NO was quantified as described in Materials and Methods. Results shown are the mean ± SEM of five independent experiments. B, Untransfected BAEC (circles) and BAEC transiently transfected with RFP and either PDK1-WT (triangles) or PDK1-K114A (squares) were loaded with DAF-2 DA and then sequentially stimulated with LPA and insulin. Results shown are the mean ± SEM of five independent experiments. C, Anti-HA immunblot of lysates derived from BAEC demonstrating comparable expression of the transfected HA-tagged PDK-1 constructs in each group.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Metabolic and hemodynamic homeostasis may be coupled by physiological actions of insulin in the vascular endothelium to promote production of NO and enhance vasodilation and blood flow (1). PI 3-kinase is a central and essential signaling molecule mediating both metabolic (9, 29) and vasodilator effects of insulin (1, 3, 4, 5). Moreover, insulin-stimulated production of NO in vascular endothelium requires the PI 3-kinase effector Akt to phosphorylate eNOS at Ser1179 by a calcium-independent mechanism (4, 5). However, signaling molecules immediately upstream and downstream from PI 3-kinase that participate in insulin-stimulated production of NO have not been previously identified. With the demonstration in the present study that IRS-1 and PDK-1 play necessary roles in the insulin signaling pathways leading to increased production of NO in endothelium, there is now direct evidence for a complete biochemical pathway leading from the insulin receptor to phosphorylation and activation of eNOS. Although the concentrations of insulin used in the present study are somewhat higher than physiological insulin concentrations, this probably reflects a technical limitation of the sensitivity of the DAF-2 DA fluorescent dye to detect low NO concentrations. In fact, the ED50 for insulin-stimulated production of NO in NIH-3T3IR cells and endothelial cells evaluated using DAF-2 DA was approximately 10 times less than that reported for endothelial cells using an NO-specific electrode (3, 4). This most likely reflects improved sensitivity of DAF-2 DA over the electrode-based method as well as an ability to carry out experiments at 37 C rather than at room temperature. It seems unlikely that the effects of insulin to stimulate production of NO in our system are mediated by IGF-I receptors since comparable stimulation with IGF-I gave much smaller NO responses.

Role of IRS-1 in Insulin-Stimulated Production of NO
IRS family members have been implicated as important substrates coupling the insulin receptor to PI 3- kinase for metabolic actions of insulin such as enhanced translocation of the insulin-responsive glucose transporter GLUT4 (20, 30, 31, 32). Other insulin receptor substrates such as Shc appear to be more important for mediating mitogenic actions of insulin (11). Thus, when investigating specific biological actions of insulin, it is of interest to identify the predominant IRS that mediates each effect. In both our model system of NIH-3T3IR cells transfected with eNOS as well as in primary endothelial cells, overexpression of wild-type IRS-1 greatly enhanced insulin-stimulated production of NO without affecting insulin sensitivity. We previously observed a similar increase in production of NO (also without a shift in sensitivity) when the insulin receptor was overexpressed in human vascular endothelial cells (4). Thus, IRS-1 is capable of participating in insulin-signaling pathways leading to activation of eNOS. More importantly, overexpression of the IRS1-F6 mutant substantially inhibited the ability of insulin to stimulate production of NO. In this study and in previous studies, we showed that IRS1-F6 (Tyr replaced by Phe in six YXXM motifs) is unable to bind and activate PI 3-kinase in response to insulin stimulation (20). It is possible that the IRS1-F6 construct is binding to the insulin receptor and preventing not only endogenous IRS-1, but also other IRS family members, from binding to the insulin receptor and activating PI 3-kinase. Therefore, we used an antisense ribozyme to specifically target rat IRS-1. We have used this construct previously to demonstrate a necessary role for IRS-1 in insulin-stimulated translocation of GLUT4 in rat adipose cells (30). In both rat fibroblasts and rat aortic endothelial cells (but not in bovine endothelial cells), expression of the antisense ribozyme significantly inhibited insulin-stimulated production of NO, whereas the ribo-CTRL construct had no effect. Thus, our data strongly suggest that IRS-1 plays a necessary role in coupling the insulin receptor to PI 3-kinase for vasodilator actions of insulin. The fact that LPA-mediated production of NO was not affected by overexpression of either wild-type or mutant IRS-1 suggests that IRS proteins do not interact with LPA-signaling pathways related to production of NO. This is consistent with previous results demonstrating that calcium-dependent mechanisms employed by LPA to activate eNOS are completely independent and separable from the phosphorylation-dependent mechanism used by insulin (5). Our finding that IRS-1 plays a necessary role in insulin-stimulated production of NO is also consistent with the phenotype of IRS-1 knockout mice who develop impaired endothelium-dependent relaxation of the aorta and hypertension (33).

We used mutants of IRS-1 where various YXXM motifs were disrupted to demonstrate that the ability of IRS-1 to bind and activate PI 3-kinase is essential for its participation in insulin-signaling pathways related to production of NO. The tandem SH2 domains of the p85 regulatory subunit of PI 3-kinase bind specifically to phosphorylated YXXM motifs on IRS proteins (10). Simultaneous occupancy of these SH2 domains is necessary for full activation of PI 3-kinase (34). The Y612/Y632 mutant of IRS-1 that has intact YXXM motifs at positions 612 and 632 and disrupted YXXM motifs at positions 465, 662, 941, and 989 can mimic the ability of wild-type IRS-1 to bind and activate PI 3-kinase and mediate translocation of GLUT4, whereas the presence of Y612 or Y632 alone results in partially impaired activation of PI 3-kinase (20). With respect to insulin-stimulated production of NO, the Y612/Y632 mutant was able to mimic the effects of wild-type IRS-1, whereas the Y612 and Y632 mutants each had a partially impaired ability to enhance production of NO in response to insulin. These results suggest that it is the binding of PI 3-kinase to IRS-1 that enables IRS-1 to mediate NO production. This conclusion is also supported by experiments in which overexpression of PKC-{zeta} partially inhibited the ability of IRS-1 to enhance NO production. We previously demonstrated that a negative feedback pathway involving ser/thr phosphorylation of IRS-1 by PKC-{zeta} impairs the ability of IRS-1 to bind and activate PI 3- kinase (21). The fact that overexpression of PKC-{zeta} did not affect LPA-mediated production of NO is consistent with the idea that the negative modulation of NO production in response to insulin by PKC-{zeta} is specific to insulin-signaling pathways. To help rule out the possibility that PKC-{zeta} may be inhibiting insulin-stimulated production of NO by affecting pathways unrelated to IRS-1 we also showed that the ability of IRS-2 to enhance NO production in response to insulin was unaffected by coexpression of PKC-{zeta}. These results are consistent with the fact that PKC-{zeta} can serine-phosphorylate IRS-1 but not IRS-2 (Ravichandran, L.V., and M. J. Quon, unpublished observations). Thus, it seems likely that PKC-{zeta} negatively modulates insulin-stimulated production of NO by phosphorylating IRS-1 on serine residues, resulting in impaired PI 3-kinase activity.

Activation of eNOS by classical calcium-dependent pathways involves interaction of the calcium-binding protein calmodulin with eNOS (35, 36). Interestingly, calmodulin can be phosphorylated by the insulin receptor, and calmodulin can directly interact with IRS-1 and PI 3-kinase in intact cells (22, 23, 24). Moreover, some experiments using specific calcium chelators and calmodulin inhibitors have suggested that calcium and calmodulin may play important roles as positive mediators of metabolic actions of insulin in adipose and skeletal muscle cells (25, 26, 27, 28). However, other studies have proposed that interactions of calmodulin with IRS-1 cause insulin resistance because inhibitors of calmodulin enhance insulin signaling through IRS-1 and PI 3-kinase (37). Thus, the exact roles for calmodulin in insulin action are unclear. Previously, we demonstrated that the calcium chelator 1,2-bis(2-aminophenoxy ethane-N,N,N',N'-tetraacetic acid completely inhibited the ability of LPA to stimulate production of NO but was unable to block insulin’s ability to activate eNOS (5). In the present study, the calmodulin inhibitors, TFP and ophiobolin A, completely blocked LPA-mediated production of NO but only partially inhibited the effects of insulin. Although interactions between calmodulin and IRS-1 or PI 3-kinase may be responsible for the partial effects of calmodulin inhibitors on insulin-stimulated production of eNOS, it seems clear that the calcium-dependent mechanisms used by LPA to activate eNOS are at least partially separable from the mechanisms involved with insulin-stimulated production of NO.

Role of PDK-1 in Insulin-Stimulated Production of NO
Immediately downstream from PI 3-kinase, PDK-1 phosphorylates and activates a number of ser/thr kinases that play important roles in insulin action (14). In particular, PDK-1 activates both PKC-{zeta} and Akt to promote translocation of GLUT4 (13, 38, 39, 40). Although it is clear that PDK-1 plays an important role in activation of Akt by phosphorylating Thr308 in the regulatory loop of Akt, it is important to directly demonstrate a role for PDK-1 in insulin-stimulated activation of eNOS because there may be alternative mechanisms for activating Akt that do not require PI 3-kinase or PDK-1. For example, in Caenorhabditis elegans, the PDK-1 isoform PIAK is able to phosphorylate Thr308 in Akt and activate Akt in the presence of PI 3-kinase inhibitors (41). Similarly, activation of Akt by leptin to stimulate NO production in endothelium appears to be independent of PI 3-kinase (15). In addition, Akt activation by protein kinase A pathways (16), cAMP (17), CaM-KK (18), and heat-shock (19), also appears to be independent of PI 3-kinase. Finally, kinases such as ILK that phosphorylate Akt at the additional regulatory site Ser473 may be able to activate Akt in a PI 3-kinase-independent manner (42).

Similar to IRS-1, in both NIH-3T3IR cells and primary endothelial cells, overexpression of PDK-1 significantly enhanced insulin-stimulated production of NO, whereas overexpression of the kinase-inactive mutant PDK-1 resulted in substantial inhibition of NO production in response to insulin. Taken together, these data strongly suggest that PDK-1 plays a necessary role in insulin-signaling pathways related to activation of eNOS. Unlike our previous studies in which inhibitory Akt mutants were overexpressed (4, 5), we did not observe complete inhibition of insulin-stimulated production of NO with the kinase inactive PDK-1 mutant. It is possible that this mutant may not be very efficient in completely inhibiting endogenous PDK-1 activity. This PDK-1 mutant appears to be inhibitory in some contexts (43, 44, 45) but not in others (13, 46, 47). Alternatively, there may be a small contribution by a PDK-1-independent pathway. As with IRS-1, PDK-1 does not appear to influence LPA-mediated production of NO, providing additional evidence that insulin and LPA use different mechanisms to activate eNOS.

In summary, IRS-1 and PDK-1 are signaling molecules immediately upstream and downstream from PI 3-kinase, respectively, that play important roles in insulin signaling pathways related to activation of eNOS and production of NO in vascular endothelium. Taken together with previous studies (3, 4, 5), we now provide direct evidence for a complete biochemical pathway involving the insulin receptor, IRS-1, PI 3-kinase, PDK-1, Akt, and eNOS that can account for important physiological actions of insulin to stimulate production of NO in the vasculature.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmid Constructs
The pCIS2 mammalian expression vector (48) was the parent vector for some constructs and was used as the empty vector control in some experiments.

pCIS2-RFP: XhoI/NotI fragment containing cDNA for RFP obtained from pDsRed1–1 (CLONTECH Laboratories, Inc., Palo Alto, CA) was subcloned into pCIS2 as described previously (5).

eNOS-WT: cDNA for bovine eNOS (gift from Dr. Thomas Michel) was subcloned into pCIS2 (5).

hIR: cDNA for wild-type human insulin receptor was subcloned into pCIS2 (49).

IRS1-WT: cDNA for human IRS-1 containing an hemagglutinin (HA)-epitope tag fused to the C terminus was constructed in the pCIS2 expression vector as described previously (20).

IRS1-F6: Expression vector for mutant human IRS-1 was derived from IRS1-WT with substitution of Phe for Tyr in six YXXM motifs at positions 465, 612, 632, 662, 941, and 989 (20).

IRS1-Y612: Expression vector for mutant IRS-1 equivalent to IRS1-F6 with Tyr612 added back (20).

IRS1-Y632: Expression vector for mutant IRS-1 equivalent to IRS1-F6 with Tyr632 added back (20).

IRS1-Y612/Y632: Expression vector for mutant IRS-1 derived from IRS1-Y612 by adding back Tyr632 (20).

rIRS1-Ribozyme: Expression vector for antisense ribozyme directed specifically against rat IRS-1 (30).

Ribo-CTRL: Control construct for rIRS1-ribozyme with sequence of the antisense ribozyme ligated in the reverse orientation (30).

IRS2-WT: cDNA for wild-type murine IRS-2 ligated into pCIS2 as described previously (31).

PDK1-WT: cDNA for murine PDK1 containing a hemagglutinin (HA)-epitope tag ligated into pBEX expression vector (13).

PDK1-K114A: cDNA for kinase-inactive PDK1 derived from PDK1-WT with alanine substituted for lysine in the ATP-binding site (13).

PKC{zeta}-WT: cDNA for the rat wild-type PKC-{zeta} with an N-terminal HA-epitope tag subcloned into pCIS2 (21).

Cell Culture and Transfection
NIH-3T3 fibroblasts stably expressing human insulin receptors (NIH-3T3IR) (50) were seeded into 35-mm tissue culture dishes (Delta TC3, Bioptechs, Inc., Butler, PA) and grown in DMEM supplemented with 10% fetal bovine serum, penicillin G (100 U/ml), and streptomycin (100 µg/ml) at 37 C in a humid atmosphere with 5% CO2. Cells at 60% confluency were transiently cotransfected with 0.1 µg pCIS2-RFP, 0.45 µg of eNOS-WT, and 0.45 µg of either pCIS2 or various IRS, PDK-1, or PKC-{zeta} constructs using Lipofectamine Plus (Life Technologies, Inc., Gaithersburg, MD). For some experiments, cells were cotransfected with 0.1 µg pCIS2-RFP, 0.45 µg of eNOS-WT, 0.45 µg of IRS1-WT, and 0.45 µg of either pCIS2 or PKC{zeta}-WT. For experiments with rat fibroblasts, cells were cotransfected with 0.1 µg pCIS2-RFP, 0.45 µg of eNOS-WT, 0.45 µg of hIR, and 0.45 µg of either rIRS1-Ribozyme or Ribo-CTRL. For experiments with endothelial cells, BAEC (Clonetics Corp., San Diego, CA) or rat aortic endothelial cells (VEC Tech., Rensselaer, NY) in primary culture were grown in EGM-2 MV as described previously (3, 4) and used at passages 3 to 4.

Measurement of NO Production in Cells
Production of NO in transfected cells was assessed using the NO-specific fluorescent dye DAF-2 DA as previously described (5). Briefly, 1 d after transfection, NIH-3T3IR cells or BAEC were serum starved for 2 h after washing with DMEM-A [DMEM without red phenol, 15 mM HEPES (pH 7.4), 0.1% BSA]. L-Arginine (100 µM) was added 1 h before each study. Serum-starved cells were loaded with DAF-2 DA (Calbiochem, La Jolla, CA) for 20 min at 37 C at a final concentration of 3 µM. Cells were then rinsed and maintained at 37 C with a warming stage (Bioptechs, Inc.) on an epifluorescent microscope. Transfected cells were identified by expression of RFP (emission of red light upon excitation at 558 nm). Cells were treated sequentially with LPA, and insulin and NO production was visualized by emission of green light (515 nm) upon excitation at 489 nm over a period of up to 9 min. In some experiments, cells were pretreated with calmodulin inhibitors TFP (40 µM, 20 min) or ophiobolin A (50 µM, 30 min). Green fluorescence intensity indicative of NO production was quantified using IP Labs Software (Scanalytics, Inc., Fairfax, VA). Data for each experiment were normalized to a reference image of the basal state.

Coimmunoprecipitation Experiments
NIH-3T3IR cells or BAEC were transfected with HA-tagged IRS-1 constructs, serum starved overnight, and then stimulated without or with insulin (100 nM, 3 min). Cell lysates were immunoprecipitated with an anti-HA antibody and then immunoblotted with antibody against p85 (Upstate Biotechnology, Inc., Lake Placid, NY) or subjected to a PI 3-kinase assay as described previously (20, 21).

Immunoblotting
For immunoblotting experiments, cells were serum starved overnight before initiation of experiments. Cell lysates were prepared using 500 µl lysis buffer [100 mM NaCl, 20 mM HEPES (pH 7.4), 1% Triton X-100, 1 mM Na3VO4, 4 mM Na pyrophosphate, 10 mM EDTA, 1 mM PMSF, 10 mM NaF, and the complete protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN)]. Samples (45 µg total protein) were separated by 8% SDS-PAGE and immunoblotted with antibodies against eNOS (Transduction Laboratories, Inc., Lexington, KY), and HA (Covance Laboratories, Inc., Princeton, NJ) according to standard methods.


    FOOTNOTES
 
This work was supported, in part, by a Research Award grant from the American Diabetes Association to M.J.Q.

Abbreviations: BAEC, Bovine aortic endothelial cells; eNOS, endothelial nitric oxide synthase; DAF-2 DA, 4,5- diaminofluorescein diacetate; GLUT4, glucose transporter 4; HA, hemagglutinin; hIR, human insulin receptor; IRS-1, insulin receptor substrate 1; LPA, lysophosphatidic acid; PDK-1, phosphoinositide-dependent kinase; PI 3-kinase, phosphatidylinositol 3-kinase; PKC, protein kinase C; RFP, red fluorescent protein; SH2, src homology 2; TFP, trifluoperazine; WT, wild-type.

Received for publication February 18, 2002. Accepted for publication May 7, 2002.


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