TNF-alpha inhibits flow and insulin signaling leading to NO production in aortic endothelial cells

Francis Kim, Byron Gallis, and Marshall A. Corson

Department of Medicine, Division of Cardiology, Harborview Medical Center, University of Washington, Seattle, Washington 98104


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Endothelial cells release nitric oxide (NO) acutely in response to increased "flow" or fluid shear stress (FSS), and the increase in NO production is correlated with enhanced phosphorylation and activation of endothelial nitric oxide synthase (eNOS). Both vascular endothelial growth factor and FSS activate endothelial protein kinase B (PKB) by way of incompletely understood pathway(s), and, in turn, PKB phosphorylates eNOS at Ser-1179, causing its activation. In this study, we found that either FSS or insulin stimulated insulin receptor substrate-1 (IRS-1) tyrosine and serine phosphorylation and increased IRS-1-associated phosphatidylinositol 3-kinase activity, phosphorylation of PKB Ser-473, phosphorylation of eNOS Ser-1179, and NO production. Brief pretreatment of bovine aortic endothelial cells with tumor necrosis factor-alpha (TNF-alpha ) inhibited the above described FSS- or insulin-stimulated protein phosphorylation events and almost totally inhibited FSS- or insulin-stimulated NO production. These data indicate that FSS and insulin regulate eNOS phosphorylation and NO production by overlapping mechanisms. This study suggests one potential mechanism for the development of endothelial dysfunction in disease states with alterations in insulin regulation and increased TNF-alpha levels.

insulin receptor substrate-1; tyrosine phosphorylation; phosphatidylinositol 3-kinase; endothelial nitric oxide synthase phosphorylation; tumor necrosis factor-alpha ; nitric oxide


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ONE OF THE MAJOR ROLES of the endothelium is to mediate the vasodilatory response by regulating the production of nitric oxide (NO). Endothelium-derived NO contributes to blood vessel homeostasis by regulating vessel tone (33), cell growth (18), platelet aggregation (26), and leukocyte binding to endothelium (37). Endothelial cells synthesize NO tonically and increase NO in response to physiological stimuli, such as vascular endothelial growth factor (VEGF) (21) and fluid shear stress (FSS) (10, 11). Endothelial nitric oxide synthase (eNOS or type III NOS) is one of three isoenzymes that converts L-arginine to L-citrulline and NO. VEGF or an increase in FSS activates phosphatidylinositol 3-kinase (PI 3-kinase) in endothelial cells, leading to activation of protein kinase B (PKB). PKB, in turn, phosphorylates eNOS at Ser-1179 and activates the enzyme, leading to release of NO (14, 17). Insulin also stimulates NO production in human umbilical vein endothelial cells (HUVEC) by a mechanism that is sensitive to wortmannin (a PI 3-kinase inhibitor) or Nomega -nitro-L-arginine methyl ester, an inhibitor of eNOS (46). Likewise, NG-monomethyl-L-arginine, an alternate eNOS inhibitor, prevents insulin-induced vasodilation in humans (6, 39). In mice lacking insulin receptor substrate-1 (IRS-1), a major substrate for the insulin receptor (IR) tyrosine kinase, there is impaired endothelium-dependent vascular relaxation (1). Together, these studies suggest that insulin, VEGF, and FSS may use elements of the same pathway(s) to regulate NO production in endothelial cells.

The molecular basis of insulin receptor signal transduction has been well characterized due to its importance in insulin-sensitive target tissues (42, 43). Insulin binding to IR activates receptor tyrosine kinases, which phosphorylates numerous substrates, including IRS-1. Many other ligands signal through IRS-1, including integrins (20), interferon (7), interleukins (7, 27), and gastrin (29). In each of these cases, IRS-1 appears to be specifically regulated according to receptor type and cell context. However, little is known about the function of IRS-1 in vascular endothelial cells. On the basis of our previous observations that FSS activates eNOS through a PI 3-kinase-dependent mechanism, we hypothesized that IRS-1 could participate in the mechanotransduction of FSS to NO production.

Numerous clinical observations demonstrate that elevated tumor necrosis factor-alpha (TNF-alpha ) levels induce insulin resistance in a variety of catabolic states, including cancer, sepsis, and trauma (4, 30). Neutralization of TNF-alpha with a soluble TNF receptor immunoglobulin fusion protein was found to improve insulin receptor signaling and insulin sensitivity in obese and insulin-resistant rats (22). Because of the inflammatory characteristics of coronary atherosclerosis (38) and studies demonstrating a negative modulatory effect of TNF-alpha on insulin signaling (12, 16, 23, 24, 28, 34), we hypothesized that TNF-alpha could impair aspects of signaling to eNOS and NO production.

This study shows that both insulin and FSS signal through IRS-1 in bovine aortic endothelial cells (BAEC) to cause activation of PI 3-kinase by association with IRS-1, and both stimuli enhance PKB and eNOS phosphorylation as well as NO production. We also demonstrate that brief pretreatment of BAEC with TNF-alpha before FSS or insulin stimulation profoundly decreases NO synthesis while impairing many of the above signaling events.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
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Materials. The monoclonal antibody (H32) to eNOS was purchased from Biomol (Plymouth Meeting, MA). Anti-phospho (Ser-473) PKB and anti-PKB polyclonal antibodies were purchased from New England Biolabs (Beverly, MA). Anti-IRS-1 rabbit polyclonal antibody and anti-phosphotyrosine monoclonal antibody (4G10) were purchased from Upstate Biotechnology (Lake Placid, NY). Rabbit polyclonal antiserum to IRS-1 was a kind gift from Dr. Hagit Eldar-Finkelman (Harvard Medical School). Recombinant human TNF-alpha was obtained from R&D Systems (Minneapolis, MN). Recombinant human insulin was obtained from Calbiochem (La Jolla, CA). [32P]orthophosphoric acid (185 MBq) and [gamma -32P]ATP (37 MBq) were purchased from NEN Life Science (Boston, MA). Sequencing-grade modified trypsin was obtained from Promega (Madison, WI). Phosphoserine, phosphothreonine, and phosphotyrosine were purchased from Sigma (St. Louis, MO). Thin-layer cellulose chromatography plates were purchased from EM Science (Gibbstown, NJ). Bradford protein assay reagents were from Bio-Rad Laboratories (Hercules, CA). Phosphatidylinositol was purchased from Avanti Polar Lipid (Alabaster, AL).

BAEC culture and exposure to FSS. BAEC cultures were established and maintained in medium 199 (GIBCO BRL) supplemented with 10% fetal bovine serum as described (10). Cells from passages 3-8 were seeded in 100-mm culture dishes and used at 60-80% confluency for labeling and shear stress experiments. Cells were exposed to laminar FSS in a cone and plate viscometer. Before shear stress or incubation with insulin, BAEC were starved for 3-5 h in serum-free endothelial cell basal medium (Clonetics, San Diego, CA). FSS stimulation of BAEC was for 2 min at 12 dyn/cm2 unless otherwise indicated.

Cell labeling, lysis, and immunoprecipitation of eNOS and IRS-1. These procedures were performed as described (17). All immunoprecipitations and Western blots were carried out using equal amounts of total protein for each condition for each experiment. Protein concentrations were determined by the Bradford assay.

SDS gel electrophoresis, Western blotting, and transfer of proteins to membranes. These procedures were performed as described (17), except that IRS-1 was transferred to nitrocellulose for 1 h at 100 V in transfer buffer (24 mM Tris base and 181 mM glycine) containing 0.1% SDS and 20% methanol.

Phosphopeptide mapping and phosphoamino acid analysis. These procedures were performed as described (17).

PI 3-kinase assay. After immunoprecipitation with anti-IRS-1 antibody, the immune complexes were washed, resuspended in 50 µl of 10 mM Tris · HCl (pH 7.4), 150 mM NaCl, and 5 mM EDTA, and then incubated with 20 µg of phosphatidylinositol and 5 µl of a stock solution [30 µCi of [gamma -32P]ATP (3,000 Ci/mmol), 0.88 mM ATP, and 20 mM MgCl2] for 12 min at 30°C. The reaction was stopped with 20 µl of 6N HCl, and 160 µl of CHCl3:methanol (1:1) was added. The mixture was centrifuged, and the lower organic phase was removed and applied to a silica gel thin-layer chromatography (TLC) plate (Merck) that was precoated with 1% potassium oxalate. The plates were developed by ascending chromatography in CHCl3:MeOH:H2O:NH4OH (60:47:11.3:2) and dried, and the radiolabeled lipids were visualized and quantified by PhosphorImager analysis.

Measurement of nitrogen oxide concentrations. NO released by BAEC was measured as its nitrogen oxide (NOx) metabolites, using a chemiluminescence detector as described in detail (10).

Statistics. Statistical analysis was performed using Stata version 6.0 (Stata). Group differences were determined using the standard Student's t-test. All variables are expressed as means ± SE. P < 0.05 was considered statistically significant.


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Effects of FSS and insulin on tyrosine phosphorylation of endothelial IRS-1; inhibition by TNF-alpha . One of the eNOS serine residues phosphorylated in response to increased FSS is contained within the PKB substrate consensus phosphorylation sequence RXRXXSH (in which H is a hydrophobic residue and X is any amino acid) (17). Since PI 3-kinase is an important upstream activator of PKB (3) and is itself activated in some cell contexts through interaction with tyrosine phosphorylated IRS-1, we hypothesized that IRS-1 might be phosphorylated in response to FSS. BAEC were subjected to FSS (12 dyn/cm2) for 2 min in a cone and plate viscometer. Whole cell lysates were immunoprecipitated with an anti-IRS-1 antibody, and Western blot analysis was performed with an anti-phosphotyrosine antibody (4G10). FSS increased IRS-1 tyrosine phosphorylation by 5.4 ± 1.6-fold (n = 3, Fig. 1A). In view of the known antagonism of IRS-1 signal transduction by TNF-alpha in insulin-sensitive cells, we next determined whether such a mechanism is operative in endothelial cells. After pretreatment with TNF-alpha (10 ng/ml for 10 min), FSS stimulated IRS-1 tyrosine phosphorylation by 2.6 ± 0.5-fold, which represented a 48% inhibition. IRS-1 total protein content was not altered after FSS or TNF-alpha pretreatment, suggesting that FSS and TNF-alpha modify IRS-1 tyrosine phosphorylation through dynamic, likely posttranslational mechanisms.


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Fig. 1.   Tumor necrosis factor (TNF)-alpha pretreatment inhibits fluid shear stress (FSS)- and insulin-dependent increases in tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) in bovine aortic endothelial cells (BAEC). A: TNF-alpha decreases FSS-induced tyrosine phosphorylation of IRS-1. BAEC monolayers were either maintained in static culture or exposed to FSS at 12 dyn/cm2 for 2 min without or with pretreatment with TNF-alpha (10 ng/ml) for 10 min. Cell lysates were immunoprecipitated (IP) with an anti-IRS-1 antibody, fractionated by SDS-PAGE, and transferred to nitrocellulose. Nitrocellulose was probed with the anti-phosphotyrosine (anti-pY) antibody 4G10 or with an anti-IRS-1 antibody to demonstrate equal loading. Densitometric band intensities were normalized to static controls, and fold increases were calculated. The results are displayed in this and subsequent figures as means ± SE (n = 3). *Increased (P < 0.05) with respect to control; #decreased (P < 0.05) with respect to FSS or insulin stimulation alone. B: TNF-alpha decreases insulin-induced tyrosine phosphorylation of IRS-1. BAEC monolayers were exposed to diluent or to insulin (1 µM) for 2 min without or with TNF-alpha pretreatment (10 ng/ml) for 10 min. Tyrosine phosphorylation of IRS-1 was assessed by immunoblotting (IB) and densitometry as in A.

Since insulin stimulation of IRS-1 tyrosine phosphorylation has not been previously demonstrated in endothelial cells, BAEC were stimulated with 1 µM insulin. Figure 1B demonstrates that after 2 min of insulin stimulation, IRS-1 tyrosine phosphorylation increased by 2.1 ± 0.2-fold (n = 3, Fig. 1B) and pretreatment with TNF-alpha for 10 min inhibited insulin-stimulated tyrosine phosphorylation by nearly 90% (n = 3).

Phosphoamino acid analysis of IRS-1 from BAEC subjected to FSS without or with TNF-alpha pretreatment. Inhibition of insulin signaling by TNF-alpha has been associated with TNF-induced serine phosphorylation of IRS-1 in adipocytes and Fao hepatoma cells (23, 34). To determine whether TNF-alpha also increases serine phosphorylation of IRS-1 in endothelial cells, BAEC were incubated for 3 h with [32P]orthophosphate. The cells were maintained in static culture or exposed to 12 dyn/cm2 FSS for 2 min without or with pretreatment with TNF-alpha (10 ng/ml for 10 min). IRS-1 was immunoprecipitated, size fractionated, and transferred to polyvinylidene difluoride. Figure 2A shows by autoradiography that compared with the control static condition, exposure to FSS, TNF-alpha pretreatment followed by FSS, or TNF-alpha alone, all increased 32P incorporation into IRS-1. IRS-1 was hydrolyzed, and the labeled phosphoamino acids were separated by high-voltage electrophoresis (HVE; Fig. 2B). Compared with cells maintained in static culture, all treatments were associated with enhanced serine phosphorylation of IRS-1. Similar increases in serine phosphorylation of IRS-1 were observed when BAEC were stimulated with insulin rather than FSS (data not shown). After exposure to FSS or insulin, phosphoamino acid analysis did not demonstrate changes in the phosphotyrosine content of IRS-1, in contrast to the results obtained through anti-phosphotyrosine Western blotting (Fig. 1, A and B). In fact, by phosphoamino acid analysis, insulin enhancement of IRS-1 phosphotyrosine content in Chinese hamster ovary cells transfected with IRS-1 cDNA was very modest (41). These findings likely reflect the relative lability of tyrosine phosphate bonds to acid hydrolysis (15).


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Fig. 2.   Both FSS and TNF-alpha increase serine phoshorylation of IRS-1: phosphoamino acid analysis. A: BAEC were labeled with [32P]orthophosphate for 3 h and maintained in static culture or exposed to FSS at 12 dyn/cm2 for 2 min without or with pretreatment with TNF-alpha (5 ng/ml) for 10 min. IRS-1 was immunoprecipitated, size fractionated by SDS-PAGE, and transferred to polyvinylidene difluoride. An autoradiogram is shown. B: labeled IRS-1 was exhaustively hydrolyzed with acid, mixed with phosphoserine (p-Ser), phosphothreonine (p-Thr), and phosphotyrosine (p-Tyr) standards, and separated in one dimension by high-voltage electrophoresis (HVE). C: Western blot of IRS-1 from cell lysates is shown as a reflection of equal amounts of cell protein for each condition. These data are representative of 3 similar experiments.

Effects of FSS and insulin on IRS-1 association with, and activation of, PI 3-kinase; inhibition by TNF-alpha . Since FSS increased tyrosine phosphorylation of IRS-1, we determined directly whether FSS would cause the association of IRS-1 with PI 3-kinase and activation of the latter. We have previously obtained data consistent with FSS activation of PI 3-kinase-dependent pathway(s), since pretreatment of BAEC with the PI 3-kinase inhibitor LY-294002 attenuated NO production in response to FSS (17). After exposure to FSS, whole cell lysates were immunoprecipitated with an anti-IRS-1 antibody. PI 3-kinase assays were performed as described in METHODS. FSS increased the PI 3-kinase activity associated with IRS-1 by 5.5 ± 1.3-fold (n = 3) over the control static condition (Fig. 3). Insulin stimulation increased PI 3-kinase association and activity levels (Fig. 3) by 4.9 ± 1.3-fold (n = 3). In C2C12 muscle cells (12) or Fao hepatoma cells (16), TNF-alpha pretreatment decreased insulin-mediated PI 3-kinase association with IRS-1 and PI 3-kinase activation. TNF-alpha treatment alone did not significantly affect basal IRS-1-associated PI 3-kinase activity, but did decrease insulin-stimulated PI 3-kinase activation by 96% (P < 0.05). The inhibitory effect of TNF-alpha treatment before FSS was quantitatively less, causing only a 38% decrease in FSS-induced PI 3-kinase activation; however, this inhibitory effect was not statistically significant (P = 0.27). These coimmunoprecipitation experiments demonstrate that either FSS or insulin increases IRS-1-associated PI 3-kinase activity. TNF-alpha pretreatment inhibits IRS-1-mediated activation of PI 3-kinase after insulin stimulation.


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Fig. 3.   TNF-alpha inhibits FSS- or insulin-dependent increases in IRS-1-associated phosphatidylinositol (PI) 3-kinase activity. BAEC were subjected to FSS at 12 dyn/cm2 for 2 min or treated with insulin (1 µM) for 2 min without or with pretreatment with TNF-alpha (10 ng/ml) for 10 min. Cell lysates were immunoprecipitated with anti-IRS-1 antibody. The immune complexes were washed and then incubated with phosphatidylinositol and [gamma -32P]ATP. The reaction was stopped with 6N HCl and then extracted with chloroform/methanol. The organic phase was spotted onto a TLC (thin-layer chromatography) plate and separated by ascending chromatography. PIP formation, which reflects PI 3-kinase activity, was quantified using a PhosphorImager. All values were normalized to control, and fold increases were calculated (n = 3). *Increased (P < 0.05) with respect to control. #Decreased (P < 0.05) with respect to insulin treatment alone.

Effects of TNF-alpha on FSS and insulin-mediated PKB phosphorylation. FSS activates both PI 3-kinase (19) and PKB in endothelial cells, and FSS-induced PKB phosphorylation can be inhibited by wortmannin (13, 14) or LY-294002 (17). Similarly, activation of PI 3-kinase activity and PKB phosphorylation by insulin have been shown previously (3). An antibody to phosphorylated Ser-473 of PKB was employed to examine the effects of FSS, insulin, and TNF-alpha on PKB phosphorylation. After FSS or insulin treatment of BAEC, Western blot analysis using anti-phosphoserine-473 PKB or anti-PKB antibodies was performed. Figure 4A demonstrates that FSS increased phosphorylation of PKB by 2.1 ± 0.2-fold and that pretreatment with TNF-alpha for 10 min before FSS reduced FSS-induced phosphorylation by 70% (n = 3). Figure 4B shows that insulin increased PKB Ser-473 phosphorylation by 2.6 ± 0.4-fold (n = 4) and that pretreatment with 10 min of TNF-alpha before insulin stimulation decreased insulin-induced PKB phosphorylation almost entirely. These results demonstrate that brief pretreatment with TNF-alpha before FSS or insulin stimulation leads to decreased levels of phosphorylation of Ser-473 in PKB.


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Fig. 4.   A and B: TNF-alpha inhibits FSS- or insulin-dependent phosphorylation of protein kinase B (PKB) Ser-473. Cells were treated with FSS, insulin, and/or TNF-alpha as in Fig. 1. Cell lysates were size fractionated by SDS-PAGE. After transfer to nitrocellulose, the blots were probed with anti-PKB-phosphoserine-473 antibody. A Western blot using anti-PKB antibody of PKB from cell lysates is shown as a reflection of an equal amount of protein used for each condition. The fold increase in PKB phosphorylation was calculated and displayed as in Fig. 1. These data are representative of 3 similar experiments. *Increased (P < 0.05) with respect to control. #Decreased (P < 0.05) with respect to FSS or insulin treatment alone.

FSS stimulates eNOS phosphorylation, which is inhibited by TNF-alpha . eNOS is basally phosphorylated, its phosphorylation is enhanced in response to FSS (10, 14, 17), and FSS enhances phosphorylation of two peptides, designated F1 and F2 (17). PKB phosphorylates eNOS at the F1 site in the cell and in vitro, increasing eNOS enzymatic activity (17). BAEC were labeled for 3 h with [32P]orthophosphate and maintained in static condition or subjected to FSS without or with TNF-alpha pretreatment. Exposure to FSS increased eNOS phosphorylation nearly twofold, as shown previously (17) and in Fig. 5. There was no change in eNOS phosphorylation relative to the static condition following 10 min of treatment with TNF-alpha , but TNF-alpha pretreatment did inhibit FSS-dependent phosphorylation of eNOS. Since TNF-alpha inhibits flow-dependent PKB Ser-473 phosphorylation, it was determined whether TNF-alpha also inhibited flow-dependent phosphorylation of peptide F1. eNOS was transferred to nitrocellulose and subjected to tryptic cleavage, and the tryptic peptides were separated by TLC and HVE. Autoradiograms of the thin-layer plates are shown in Fig. 5 (top). The relative volumes of the phosphopeptide F1 in each condition were quantified with a PhosphorImager. Compared with static conditions, the phosphopeptide F1 was increased by 2.4 ± 0.5-fold (n = 3) by FSS. Although incubation with TNF-alpha alone caused no change in the basal level of phosphorylation of eNOS tryptic peptides, preincubation with TNF-alpha before flow inhibited F1 phosphorylation by 40% (n = 3).


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Fig. 5.   TNF-alpha inhibits FSS-dependent phosphorylation of endothelial nitric oxide synthase (eNOS) Ser-1179. BAEC were labeled with [32P]orthophosphate and maintained in static culture or exposed to FSS at 12 dyn/cm2 for 2 min without or with TNF-alpha pretreatment. The eNOS was immunoprecipitated, size fractionated by SDS-PAGE, and transferred to nitrocellulose. The eNOS bands were excised from the nitrocellulose, and counts per minute (cpm) incorporated into eNOS for each condition were determined by Cerenkov counting: static, 6,831 cpm; FSS, 12,474 cpm; TNF-alpha alone, 7,468 cpm; and TNF-alpha before FSS, 5,247 cpm. eNOS from each nitrocellulose strip was trypsinized, and ~85% of the counts per minute were recovered. Tryptic digests were spotted onto thin-layer plates and separated by HVE in the first dimension and TLC in the second dimension. Autoradiograms of the phosphopeptide maps are shown (top). The eNOS phosphopetide designated F1, denoted by an arrow, has the sequence TQpSFSLQER. Western blots of eNOS immunoprecipitates are shown (IB: anti-eNOS) as a reflection of equal amounts of eNOS per peptide map. These data are representative of 3 similar experiments.

Insulin stimulates eNOS phosphorylation, which is inhibited by TNF-alpha . Insulin causes vasodilation in humans by way of an eNOS-dependent mechanism (6, 39). Insulin-dependent production of NO in endothelial cells can be inhibited by wortmannin (46). These findings suggest that insulin and FSS may activate eNOS by similar pathways. We therefore determined whether insulin could stimulate eNOS F1 site phosphorylation in BAEC. In Fig. 6, insulin stimulated the phosphorylation of peptide F1 by 2.1 ± 0.1-fold (n = 3) compared with control cells. Preincubation with TNF-alpha for 10 min before the addition of insulin inhibited F1 phosphorylation by 71% (n = 3). Thus both insulin and FSS induced phosphorylation of peptide F1, which is known to activate eNOS (14, 17), and TNF-alpha pretreatment inhibited phosphorylation of F1 in response to FSS or insulin.


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Fig. 6.   TNF-alpha inhibits insulin-dependent phosphorylation of eNOS Ser-1179. BAEC were labeled with [32P]orthophosphate and treated with vehicle or insulin (1 µM) for 2 min without or with TNF-alpha pretreatment. eNOS was prepared as in Fig. 5, and the counts per minute incorporated into eNOS for each condition were determined by Cerenkov counting: control, 1,221 cpm; insulin, 2,020 cpm; and TNF-alpha before insulin, 2,131 cpm. Autoradiograms of the phosphopeptide maps are shown, along with a Western blot of eNOS immunoprecipitates (IB: anti-eNOS) as a reflection of equal amounts of eNOS per peptide map. The phosphopeptide F1 is indicated by an arrow. These data are representative of 3 similar experiments.

Pretreatment of BAEC with TNF-alpha inhibits both FSS- and insulin-induced NO production. FSS is a well-recognized activator of NO production in endothelial cells (10, 11). Zeng and Quon (46) have previously shown that insulin stimulation can increase endothelial NO production, but the effect of TNF-alpha on NO production in response to FSS or insulin stimulation has not been previously reported. We assessed NO directly released by endothelial cells during FSS stimulation by measuring NOx metabolites using ozone chemiluminescence as previously described (10). Figure 7A demonstrates that NO production increases upon exposure of static BAEC to FSS. The FSS-dependent increase was inhibited nearly 80% following TNF-alpha pretreatment. Figure 7B demonstrates that stimulation of BAEC with insulin also resulted in an increase in NO production that was inhibited ~70% by pretreatment with TNF-alpha . Thus a brief pretreatment of BAEC with TNF-alpha inhibited both FSS- and insulin-mediated NO production.


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Fig. 7.   TNF-alpha inhibits FSS- and insulin-dependent nitric oxide production in BAEC. BAEC were exposed to FSS at 12 dyn/cm2 for 5 min (A) or treated with insulin (1 µM) for 5 min (B) without or with pretreatment with TNF-alpha (10 ng/ml) for 10 min. Samples of buffer were removed, and nitrogen oxide (NOx) concentrations at 0, 1, 3, and 5 min were measured using ozone chemilumenscence. The percent increase over baseline at time = 1, 3, and 5 min was calculated, and the results are displayed as means ± SE (n = 3).


    DISCUSSION
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INTRODUCTION
METHODS
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DISCUSSION
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The major findings of this study are that either insulin or FSS stimulates IRS-1 tyrosine and serine phosphorylation with a subsequent increase in IRS-1-associated PI 3-kinase activity, phosphorylation of PKB Ser-473, phosphorylation of eNOS at Ser-1179, and NO production in endothelial cells. Brief pretreatment with TNF-alpha inhibits the above described FSS- or insulin-stimulated protein phosphorylation events and FSS- or insulin-stimulated NO production. IRS-1 is tyrosine phosphorylated and activated upon insulin binding to the IR and in response to activation by FSS by an unknown tyrosine kinase. As in other cell types, exposure to TNF-alpha is associated with increased IRS-1 serine phosphorylation and concomitant inhibition of its tyrosine phosphorylation and activation, implying a form of cross-talk between serine- and tyrosine-specific protein kinases. IRS-1 appears to be a focal point for the agonists we have utilized, from the perspectives of both activation and inhibition in being independently regulated by insulin, FSS, and TNF-alpha . A logical extension is that disease states characterized by alterations in insulin function and increased TNF-alpha may manifest endothelial dysfunction and insulin resistance due to alteration of the normal balance between these modulators. The model system utilized for these studies should be helpful for identifying the TNF-alpha -dependent protein kinase(s) that phosphorylate IRS-1. It can be used as a basis for determining whether the form of "insulin resistance" suggested by these studies is physiologically relevant in vivo.

Recently, Zeng and colleagues (45) have shown that insulin requires insulin receptor tyrosine kinase, PI 3-kinase, and PKB for insulin-induced NO production in HUVEC. Similarly, we report signaling events induced by FSS and insulin in endothelial cells that have commonly been described for insulin in "insulin-responsive cells," namely, increased tyrosine and serine phosphorylation of IRS-1 (41), increased association of IRS-1 with PI 3-kinase (5, 32, 41), and increased PKB phosphorylation (3). The similarities between signaling events induced by either insulin or FSS leading to eNOS phosphorylation and NO production are striking and suggest that insulin and FSS use elements of overlapping pathways to generate NO in BAEC. However, this study has not proven that insulin and FSS use the same pathway, or only one pathway, to stimulate eNOS phosphorylation and NO production.

A key question in vascular biology remains: the identity of the mechanotransducers of FSS to biochemical signals that regulate vessel physiology. FSS could produce a deformation of the endothelial cytoskeleton, changes in membrane physical chemistry, and/or activation of receptors or sensors that trigger intracellular signaling pathways. Growth factor receptors are appropriate candidate mediators, because they may be activated by an array of biological signals independent of hormone/receptor binding (8). Recently, FSS has been shown in BAEC to increase the tyrosine phosphorylation and clustering of the fetal liver kinase 1 (FLK-1) VEGF receptor and to induce its association with the adapter protein shc (9). Although insulin causes tyrosine phosphorylation of the IR in BAEC, FSS does not (Kim, unpublished observations). Thus FLK-1 remains a potential candidate to be the FSS-dependent tyrosine kinase "upstream" of IRS-1. The "downstream" coupling of IRS-1 to PI 3-kinase also appears to be a stimulus-specific response, since NO production was not increased after exposure of endothelial cells to platelet-derived growth factor, a known stimulus of PI 3-kinase in endothelial cells (46).

This study demonstrates that, similar to TNF-alpha 's effects in Fao hepatoma cells (16), C2C12 myotubes (12) and adipocytes (23, 24), this cytokine suppresses insulin-induced tyrosine phosphorylation of IRS-1, increases the serine phosphorylation of IRS-1 (24, 34), and decreases insulin-stimulated association of IRS-1 with PI 3-kinase (12). Studies in endothelial (31) and other cell types show that TNF-alpha activates PI 3-kinase in the cell globally, in contrast to our and other investigations (2, 12, 28) that focus on the TNF-alpha inhibition of insulin-activated PI 3-kinase associated with IRS-1. In these studies, TNF-alpha induced serine phosphorylation of IRS-1 correlated with decreased IR tyrosine kinase activity and tyrosine phosphorylation of IRS-1 (23, 24, 28). TNF-alpha -induced serine phosphorylation of IRS-1 has been proposed to alter IRS-1 conformation and thereby impair its ability to bind PI 3-kinase (2). Increased adipocyte TNF-alpha mRNA levels observed in rodent models of obesity and diabetes have led to the suggestion that insulin resistance is mediated by the changes observed in IRS-1 phosphorylation and protein kinase activity observed in cell cultures in the presence of TNF-alpha (23-25). Interestingly, eNOS knockout mice exhibit insulin resistance in the liver and peripheral tissues (40); it is speculated that reduced blood flow may impair delivery of insulin and/or glucose to target tissues.

TNF-alpha pretreatment significantly inhibits FSS-dependent IRS-1 tyrosine phosphorylation, phosphorylation of PKB Ser-473, eNOS phosphorylation at Ser-1179, and, finally, FSS-dependent NO production. TNF-alpha pretreatment, however, did not significantly alter IRS-1-associated PI 3-kinase activity following FSS. The reasons for this modest TNF-alpha effect is unclear; however, differences in insulin- and FSS-dependent serine and tyrosine phosphorylation of IRS-1 as well as differences in FSS- and insulin-dependent phosphatases may play a role in the observed differences. Nevertheless, the effects of short-term pretreatment with TNF-alpha on FSS-dependent NO production has been clearly demonstrated in our cell culture model. This investigation examines the acute effects of TNF-alpha on FSS and insulin signaling following a brief (10-min) pretreatment of endothelial cells. Long-term elevated levels of TNF-alpha may also contribute to decreased NO production, since chronic incubation of endothelial cells with TNF-alpha reduces eNOS mRNA half-life by >90% (44).

Important questions that remain include the vascular origins of TNF-alpha , the identity of the TNF-alpha receptor(s) stimulated, and the precise pathway(s) through which TNF-alpha receptor stimulation is linked to serine phosphorylation of IRS-1. TNF-alpha is likely produced in an autocrine or paracrine manner and may be closely cell associated before stimulation of TNF receptors. The known p55 and p75 TNF-alpha receptors are devoid of intrinsic or associated catalytic activity, but in some cell types couple to sphingomyelinases, liberating fatty acid autocoids, and they can activate the protein kinase C and mitogen-activated protein kinase superfamilies (35, 36). A set of IRS-1 serine residues (612, 632, 662, and 731) has been identified as being dually regulated by these kinases, but the relevance of these sites to the events described in the current report is unclear. Subsequent work will address these questions, with a particular focus on determining the identity of the TNF-alpha -dependent IRS-1 phosphorylation sites. Identifying these sites should provide the molecular tools to dissect these pathways in vivo and should suggest targets for therapeutic agents to treat insulin resistance syndromes.


    ACKNOWLEDGEMENTS

We thank Dr. Edwin G. Krebs for careful review of this manuscript.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute National Research Service Awards 1F32-HL-09878-01 (to F. Kim) and 1R21-HL-62885 and 1RO1-HL-64228 (both to B. Gallis and M. A. Corson).

Address for reprint requests and other correspondence: F. Kim, Dept. of Medicine, Div. of Cardiology, Box 359748, Harborview Medical Center, Univ. of Washington, 325 Ninth Ave., Seattle, WA 98104 (E-mail: fkim{at}u.washington.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 10 August 2000; accepted in final form 15 December 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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