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Endocrinology Vol. 148, No. 7 3356-3363
Copyright © 2007 by The Endocrine Society

Tumor Necrosis Factor-{alpha} Induces Insulin Resistance in Endothelial Cells via a p38 Mitogen-Activated Protein Kinase-Dependent Pathway

Guolian Li, Eugene J. Barrett, Matthew O. Barrett, Wenhong Cao and Zhenqi Liu

Division of Endocrinology and Metabolism (G.L., E.J.B., M.O.B., Z.L.), Department of Internal Medicine, University of Virginia Health System, Charlottesville, Virginia 22908; and Endocrine Biology Program (W.C.), The Hamner Institutes for Health Sciences, Research Triangle Park, North Carolina 27709

Address all correspondence and requests for reprints to: Zhenqi Liu, M.D., Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Virginia Health System, P.O. Box 801410, Charlottesville, Virginia 22908-1410. E-mail: zl3e{at}virginia.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Chronic inflammation contributes to vascular insulin resistance and endothelial dysfunction. Systemic infusion of TNF-{alpha} abrogates insulin’s action to enhance skeletal muscle microvascular perfusion. In skeletal muscle TNF-{alpha} induces insulin resistance via the p38 MAPK pathway. To examine whether p38 MAPK also regulates TNF-{alpha}-induced vascular insulin resistance, bovine aortic endothelial cells (bAECs) were incubated ± TNF-{alpha} (5 ng/ml) for 6 h in the presence or absence of SB203580 (p38 MAPK specific inhibitor, 10 µM) after serum starvation for 10 h. For the last 30 min, cells were treated ± 1 nM insulin, and insulin receptor substrate (IRS)-1, Akt, endothelial nitric oxide synthase (eNOS), p38 MAPK, ERK1/2, c-Jun N-terminal kinase, and AMP-activated protein kinase (AMPK) phosphorylation, and eNOS activity were measured. TNF-{alpha} increased p38 MAPK phosphorylation, potently stimulated IRS-1 serine phosphorylation, and blunted insulin-stimulated IRS-1 tyrosine and Akt phosphorylation and eNOS activity. TNF-{alpha} also potently stimulated the phosphorylation of ERK1/2 and AMPK. Treatment with SB203580 decreased p38 MAPK phosphorylation back to the baseline and restored insulin sensitivity of IRS-1 tyrosine and Akt phosphorylation and eNOS activity in TNF-{alpha}-treated bAECs without affecting TNF-{alpha}-induced ERK1/2 and AMPK phosphorylation. We conclude that in cultured bAECs, TNF-{alpha} induces insulin resistance in the phosphatidylinositol 3-kinase/Akt/eNOS pathway via a p38 MAPK-dependent mechanism and enhances ERK1/2 and AMPK phosphorylation independent of the p38 MAPK pathway. This differential modulation of TNF-{alpha}’s actions by p38 MAPK suggests that p38 MAPK plays a key role in TNF-{alpha}-mediated vascular insulin resistance and may contribute to the generalized endothelial dysfunction seen in type 2 diabetes mellitus and the cardiometabolic syndrome.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TYPE 2 DIABETES ACCELERATES atherosclerosis, leading to increased morbidity and mortality in these patients. Although the underlying mechanisms remain to be fully defined, strong evidence indicates that insulin resistance and endothelial dysfunction are key players in the pathogenesis of atherosclerosis (1, 2, 3, 4). Systemic insulin resistance and endothelial dysfunction are closely related and may be mutually sustaining. Many factors are able to cause insulin resistance and endothelial dysfunction. Among them, inflammatory cytokines are of particular importance as type 2 diabetes is associated with a low-grade inflammation with increased levels of inflammatory cytokines such as TNF-{alpha}, IL-6, resistin, IL-1, monocyte chemoattractant protein 1, and C-reactive protein (5).

Under normal physiology, the vascular endothelium preserves vascular integrity and regulates vascular tone and permeability, immune surveillance, and coagulation. In response to insulin, endothelial cells produce nitric oxide (NO) via the phosphatidylinositol 3-kinase (PI3-kinase)/protein kinase B (or Akt)/endothelial nitric oxide synthase (eNOS) pathway (6, 7, 8, 9, 10, 11), resulting in increased total muscle blood flow. Insulin also enhances muscle microvascular perfusion (12, 13, 14, 15, 16). This action is also NO dependent because inhibition of eNOS with N{omega}-nitro-L-arginine-methyl ester completely abolishes insulin-mediated microvascular perfusion (14, 15).

With insulin resistance, insulin action through the PI3-kinase/Akt pathway is blunted (3), leading to decreased NO production. However, insulin’s mitogenic actions, which are mediated via the MAPK pathway, remain intact. Thus, by this selective PI3-kinase pathway, insulin resistance may actually enhance insulin’s mitogenic actions, leading to increased production of adhesion molecules and endothelin-1 and thereby predisposing insulin-resistant patients to hypertension and atherosclerosis.

The molecular underpinnings for the inflammation-induced insulin resistance and endothelial dysfunction duet are not clear, and recent evidence has suggested that p38 MAPK may play a central role in this process. The p38 MAPK belongs to the MAPK superfamily and is a stress-activated serine/threonine protein kinase with major functions in apoptosis, cytokine production, transcriptional regulation, and cytoskeletal reorganization (17). Various inflammatory cytokines are capable of activating p38 MAPK, which has been shown to regulate inflammation and induce endothelial dysfunction (17, 18). C-reactive protein inhibits endothelium-dependent, NO-mediated dilation in coronary arterioles by activating p38 MAPK and nicotinamide adenine dinucleotide phosphate oxidase (19). Selective inhibition of p38 MAPK dose-dependently reduces TNF-{alpha} or lipopolysaccharide-induced intercellular adhesion molecule-1 expression in cultured human umbilical vein endothelial cells and restores NO-mediated endothelium-dependent relaxation in spontaneously hypertensive, stroke-prone rats (20). These results strongly suggest that p38 MAPK plays a pivotal role in vascular inflammation and endothelial dysfunction/repair.

In skeletal muscle, it has been suggested that TNF-{alpha} induces insulin resistance via p38 MAPK pathway (21, 22). In the current study, we examined the role of p38 MAPK in TNF-{alpha}-induced selective insulin resistance in the PI3-kinase pathway in endothelial cells. We here report for the first time that TNF-{alpha} induces insulin resistance in the PI3-kinase pathway via a p38 MAPK-dependent mechanism and activates ERK, c-Jun N-terminal kinase (JNK) and AMP-activated protein kinase (AMPK) pathways independent of p38 MAPK in endothelial cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Culture of bovine aortic endothelial cells (bAECs)
Cells in primary culture were purchased from Cambrex Bio-Sciences Walkersville, Inc. (Walkersville, MD) and cultured in endothelial basic media, which was supplemented with 5% fetal bovine serum, bovine brain extract, human epidermal growth factor (10 ng/ml), gentamicin sulfate (50 µg/ml), amphotericin-B (50 ng/ml), and hydrocortisone (1 µg/ml). Cells between passages 3 and 8 were used for experiments after growing to 75–80% confluence and serum starvation for 10 h. In most experiments, cells were treated with or without human TNF-{alpha} at a final concentration of 5 ng/ml in the absence or presence of 10 µM of SB203580, a specific inhibitor of p38 MAPK, for 6 h. During the last 30 min, cells were treated with or without 1 nM insulin. Cells were then washed twice with ice-cold 1x PBS solution, lysed by sonication using a Fisher XL2020 sonicator (Fisher Scientific, Pittsburgh, PA) in ice-cold lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet-P40, 0.25% sodium deoxycholate, 1 mM EGTA, 1 mM sodium orthovanadate, 1 mM NaF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride]. Cell lysates were centrifuged for 5 min at 4 C (13,000 x g) and the supernatants were used for immunoprecipitation and/or Western blotting. Additional control experiments were done to determine the time course and dose-response for TNF-{alpha} action. We also performed studies in the presence of wortmannin (an inhibitor of PI3-kinase) at a final concentration of 100 nM or PD98059 (an inhibitor of MAPK kinase 1) at a final concentration of 25 µM. Either wortmannin or PD98059 was added 30 min before the addition of TNF-{alpha}. SB203580 was purchased from EMD Bioscience (San Diego, CA). TNF-{alpha}, wortmannin, and PD98059 were obtained from Sigma-Aldrich (St. Louis, MO).

Immunoprecipitation of insulin receptor substrate (IRS)-1
Aliquots of supernatant containing 300–500 µg protein in 500 µl lysis buffer were incubated with 4 µg primary antibody against IRS-1 (Upstate Cell Signaling, Lake Placid, NY) overnight at 4 C. Protein G agarose was then added and the mixture was kept at 4 C for 1 h with gentle rocking. After washing six times with lysis buffer, the beads were spun down (1000 x g for 30 sec), resuspended in 25 µl 2x sample buffer [375 mM Tris-HCl (pH 6.8), 12% sodium dodecyl sulfate, 60% glycerol, 300 mM dithiothreitol, and 0.06% bromophenol blue], and boiled for 5 min.

Immunoblotting
Equal amounts of IRS-1 immunoprecipitate or aliquots of cell lysate supernatant containing approximately 20 µg protein were diluted with an equal volume of sodium dodecyl sulfate sample buffer and electrophoresed on a 5 or 10% polyacrylamide gel, transferred to nitrocellulose membranes, and blocked with 5% low-fat milk in Tris-buffered saline plus 0.1% Tween 20. Subsequently the membranes from IRS-1 immunoprecipitates were probed with antiphosphotyrosine, clone 4G10, horseradish peroxidase conjugate, or antiphosphoserine antibody (Upstate Cell Signaling), and membranes from cell lysates were probed with antibodies against phospho-Akt1 (Ser473) (Upstate Cell Signaling), phospho-eNOS (Ser1177), phospho-p38 MAPK (Thr180/Tyr182), phospho-ERK1/2 (Thr202/Tyr204), phospho-SAPK/JNK (Thr183/Tyr185), or phospho-AMPK{alpha} (Thr172) (Cell Signaling Technology, Danvers, MA) overnight at 4 C. This was followed by a secondary antibody coupled to horseradish peroxidase, and the blots were developed using ECL (Amersham Life Sciences, Piscataway, NJ). To ensure equal loading, membranes were stripped and reprobed with primary antibodies against respective proteins. Autoradiographic films were scanned densitometrically (Molecular Dynamics, Piscataway, NJ) and quantitated using Imagequant 3.3. Both the total and phospho-specific densities were quantitated and the ratios of phosphospecific to total density calculated.

eNOS activity assay
Cells were treated as stated above. After washing the 10-cm plates with ice-cold 1x PBS containing 1 mM EDTA three times, cells were scraped and sonicated in 100 µl Tris buffer [25 mM Tris (pH 7.4), 1 mM EDTA, and 1 mM EGTA]. The lysate was then centrifuged at 13,000 x g for 5 min. After determining the protein content, the eNOS activity was measured using the nitric oxide synthase assay kit (Calbiochem, San Diego, CA). In brief, an aliquot (10 µl) of supernatant containing 20–30 µg protein was added to 40 µl of reaction buffer [final concentration: 25 mM Tris-HCl (pH 7.4), 3 µM tetrahydrobiopterin, 1 µM FAD, 1 µM flavin mononucleotide, 1 mM reduced nicotinamide adenine dinucleotide phosphate, 0.6 mM CaCl2, 2.4 µM calmodulin (Calbiochem), 1 µl 14C-arginine (1 µCi/20 µl; Amersham Biosciences, Piscataway, NJ)] and the mixture was incubated at 37 C for 90 min. The reaction was stopped by adding 400 µl stop buffer [50 mM HEPES (pH 5.5), 5 mM EDTA]. After the addition of 110 µl ion-exchange resin (Dowex 50WX8–200) to a spin cup, the mixture was transferred to the spin cup and centrifuged in a microcentrifuge at full speed for 30 sec which separated the 14C-citrulline from 14C-arginine. The samples containing either 14C-citrulline or 14C-arginine were counted in a LS 6500 Beckman scintillation counter (Fullerton, CA). After subtracting counts obtained in the presence of 1 mM N{omega}-nitro-L-arginine-methyl ester, the ratio of 14C-citrulline to 14C-arginine was calculated and used as indicator of NOS activity. For each experiment the samples were run in triplicate.

Statistical analysis
Results are expressed as mean ± SEM. Statistical analysis was performed using one-way, repeated-measures ANOVA with Turkey test or Student’s t test as appropriate. P ≤ 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We first examined the time course and dose-response of TNF-{alpha} effect on insulin signaling using insulin-stimulated Akt1 phosphorylation in cultured bAECs. Cells were serum starved for 10 h and then incubated ± 5 ng/ml TNF-{alpha} for 0–18 h (Fig. 1AGo) or 0–20 ng/ml TNF-{alpha} for 6 h (Fig. 1BGo). As shown in Fig. 1Go, insulin at 1 nM potently stimulated Akt1 phosphorylation, and this effect was blunted by TNF-{alpha} in a time- and dose-dependent manner. Because TNF-{alpha} at 5 ng/ml clearly blunted insulin-stimulated Akt1 phosphorylation and Akt1 protein content decreased after 12 h of incubation with TNF-{alpha}, we carried out all subsequent experiments using a TNF-{alpha} concentration of 5 ng/ml and incubation time of 6 h.


Figure 1
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  		FIG. 1. Time course and dose response of TNF-{alpha} effect on insulin-stimulated Akt1 phosphorylation in cultured bAECs. Cells were serum starved for 10 h and then incubated ± TNF-{alpha} (5 ng/ml) for 0–18 h (A) or TNF-{alpha} 0–20 ng/ml for 6 h (B). For insulin-treated cells, insulin (1 nM) was added for the last 30 min. Cells were then harvested as detailed in the text. Results are representative of two separate experiments.

 
To examine whether p38 MAPK mediates TNF-{alpha}-induced insulin resistance in the vascular endothelium, we examined the effect of TNF-{alpha} on insulin-stimulated Akt1 and eNOS phosphorylation in cultured bAECs in the absence or presence of 10 µM SB203580, a specific p38 MAPK inhibitor. Consistent with our previous finding (11), insulin at 1 nM potently stimulated the phosphorylation of Akt1 (P < 0.001) and eNOS (P < 0.001) (Fig. 2Go). TNF-{alpha} treatment for 6 h did not significantly alter the basal phosphorylation of either Akt1 or eNOS; however, it completely abolished insulin-stimulated Akt1 phosphorylation (Fig. 2BGo). Interestingly, insulin-mediated eNOS Ser1177 phosphorylation persisted in the presence of TNF-{alpha} (P = 0.001) (Fig. 2CGo). SB203580 treatment restored insulin sensitivity of Akt1 phosphorylation in TNF-{alpha}-treated bAECs. As expected, wortmannin abolished insulin mediated Akt1 and eNOS phosphorylation, but PD98059 did not affect either insulin- or TNF-{alpha}-induced changes in Akt1 or eNOS phosphorylation (Fig. 2DGo).


Figure 2
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  		FIG. 2. Effects of TNF-{alpha} on Akt and eNOS phosphorylation in bAECs. A–C, Cells were serum starved for 10 h and then incubated ± TNF-{alpha} (5 ng/ml) for 6 h in the absence (left four bars) or presence (right four bars) of SB203580 (10 µM). For insulin-treated cells, insulin (1 nM) was added at the last 30 min. Cells were then harvested as detailed in the text. Results are the average of four experiments. A, Representative Western blots of Akt1 and eNOS phosphorylation. B, Akt1 phosphorylation. Compared with control: *, P <0.001. Compared with control + SB203580: #, P < 0.001. C, eNOS phosphorylation. Compared with control: *, P < 0.001; **, P = 0.001. Compared with control + SB203580: #, P = 0.007; ##, P < 0.02. D, Akt1 and eNOS phosphorylation in the presence of either wortmannin (100 nM) or PD98059 (25 µM). Representative Western blots of three separate experiments.

 
Because TNF-{alpha} treatment did not blunt insulin-stimulated eNOS phosphorylation, we next examined whether TNF-{alpha} modulates insulin-mediated increase in eNOS activity (Fig. 3Go). Consistent with changes in Akt1 phosphorylation, insulin at 1 nM significantly increased eNOS activity (P = 0.001), and this increase was abolished by TNF-{alpha}. Incubation of cells with SB203580 did not affect insulin-stimulated eNOS activity (P < 0.001, compared with SB203580-treated control cells); however, it did abrogate TNF-{alpha}’s inhibitory effect on insulin to increase eNOS activity. Thus, there exists a dissociation between insulin-stimulated eNOS Ser1177 phosphorylation and activity in the presence of TNF-{alpha}.


Figure 3
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  		FIG. 3. TNF-{alpha} blunts insulin-stimulated eNOS activity via p38-MAPK-dependent mechanism in cultured bAECs. Cells were serum starved for 10 h and then incubated ± TNF-{alpha} (5 ng/ml) for 6 h in the absence (left four bars) or presence (right four bars) of SB203580 (10 µM). For insulin-treated cells, insulin (1 nM) was added at the last 30 min. Cells were then harvested as detailed in the text. The eNOS activity was assayed by assessing the conversion of 14C-arginine to 14C-citrulline. Results are averages of four experiments. Compared with control: *, P = 0.001. Compared with control + SB203580: #, P < 0.001; ##, P = 0.002.

 
We next examined whether blocking p38 MAPK affects TNF-{alpha}-induced IRS-1 serine phosphorylation and insulin-stimulated IRS-1 tyrosine phosphorylation in cultured bAECs because IRS-1 is upstream of Akt and eNOS in the insulin signal transduction pathway. Insulin acts on the insulin receptor and/or IGF-I receptor and leads to tyrosine-phosphorylation of IRS-1. Many insulin-resistant factors, including TNF-{alpha}, have been shown to decrease insulin signaling by inducing IRS-1 serine phosphorylation. As shown in Fig. 4Go, insulin at 1 nM increased IRS-1 tyrosine phosphorylation nearly 10-fold (from 0.035 ± 0.003 to 0.333 ± 0.008, P < 0.001), whereas TNF-{alpha} induced a nearly 14-fold increase in IRS-1 serine phosphorylation (from 0.017 ± 0.001 to 0.253 ± 0.01, P < 0.001). This TNF-{alpha}-induced increase in IRS-1 serine phosphorylation was associated with a 54% decrease in insulin-stimulated IRS-1 tyrosine phosphorylation (P < 0.001, Fig. 4BGo). Interestingly, adding insulin to TNF-{alpha}-treated cells also significantly ameliorated TNF-{alpha}-induced IRS-1 serine phosphorylation by nearly 70% (P < 0.001, Fig. 4CGo). Inhibition of p38 MAPK with SB203580 abolished TNF-{alpha}-induced IRS-1 serine phosphorylation and completely restored insulin-stimulated IRS-1 tyrosine phosphorylation. Neither wortmannin nor PD98059 affected insulin-induced IRS-1 tyrosine phosphorylation in the presence or absence of TNF-{alpha} (Fig. 4DGo).


Figure 4
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  		FIG. 4. TNF-{alpha} stimulates IRS-1 serine phosphorylation and blunts insulin-stimulated IRS-1 tyrosine phosphorylation in bAECs via a p38 MAPK-dependent mechanism. A–C, Cells were serum starved for 10 h and then incubated ± TNF-{alpha} (5 ng/ml) for 6 h in the absence (left four bars) or presence (right four bars) of SB203580 (10 µM). For insulin-treated cells, insulin (1 nM) was added at the last 30 min. Cells were then harvested as detailed in the text. Results are averages of three to four experiments. A, Representative Western blots showing IRS-1 tyrosine and serine phosphorylation. B, IRS-1 tyrosine phosphorylation. Compared with control: *, P < 0.001; compared with insulin: #, P < 0.001. Compared with control + SB203580: **, P < 0.001. C, IRS-1 serine phosphorylation. Compared with control: *, P < 0.001; compared with TNF-{alpha}: #, P < 0.001. D, IRS-1 tyrosine phosphorylation in the presence of either wortmannin (100 nM) or PD98059 (25 µM). Representative Western blots of three separate experiments. IP, Immunoprecipitation; IB, immunoblot.

 
Inasmuch as TNF-{alpha} has been shown to activate both p38 MAPK and JNK and as JNK has been implicated in other insulin sensitive tissues to play an important role in causing IRS-1 serine phosphorylation and insulin resistance, we carried additional experiments to confirm that our observed findings of TNF-{alpha} on Akt1, eNOS and IRS-1 are indeed due to p38 MAPK activation, not from that of JNK. As shown in Fig. 5AGo, both insulin (P < 0.001) and TNF-{alpha} (P = 0.004) potently stimulated p38 MAPK phosphorylation in cultured bAECs, and inhibition of p38 MAPK with SB203580 completely abolished the increase in p38 MAPK phosphorylation induced by insulin, TNF-{alpha}, or insulin and TNF-{alpha} combination. TNF-{alpha}, but not insulin, also potently stimulated JNK phosphorylation (P = 0.005). However, SB203580 failed to abolish this TNF-{alpha}-induced increase in JNK phosphorylation (P = 0.002) (Fig. 5BGo). This suggests that SB203580 at 10 µM selectively inhibits p38 MAPK phosphorylation and TNF-{alpha}-induced JNK phosphorylation is p38 MAPK independent.


Figure 5
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  		FIG. 5. Effects of insulin and TNF-{alpha} on p38 MAPK and JNK phosphorylation in cultured bAECs. Cells were serum starved for 10 h and then incubated ± TNF-{alpha} (5 ng/ml) for 6 h in the absence (left four bars) or presence (right four bars) of SB203580 (10 µM). For insulin-treated cells, insulin (1 nM) was added at the last 30 min. Cells were then harvested as detailed in the text. Results are the average of three to four experiments. A, p38 MAPK phosphorylation. Compared with control: *, P < 0.001; **, P = 0.004. B, JNK phosphorylation. Compared with control: *, P = 0.005; **, P < 0.001. Compared with control + SB203580: #, P = 0.002; ##, P < 0.001.

 
We further examined the effect of TNF-{alpha} on insulin-induced ERK1/2 phosphorylation in cultured bAECs and whether this effect is p38 MAPK dependent. As shown in Fig. 6AGo, both insulin (P = 0.002) and TNF-{alpha} (P = 0.01) significantly increased the phosphorylation of ERK1/2. SB203580 treatment had no effect on basal, TNF-{alpha}-stimulated, or insulin-mediated ERK1/2 phosphorylation. Interestingly, insulin and TNF-{alpha} combination induced less ERK1/2 phosphorylation than insulin alone (P < 0.02 in the absence and P = 0.008 in the presence of SB203580). This insulin- or TNF-{alpha}-induced ERK1/2 phosphorylation was not affected by wortmannin but was blocked by PD98059 (Fig. 6BGo).


Figure 6
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  		FIG. 6. Effects of insulin and TNF-{alpha} on ERK1/2 phosphorylation in cultured bAECs. A, Cells were serum starved for 10 h and then incubated ± TNF-{alpha} (5 ng/ml) for 6 h in the absence (left four bars) or presence (right four bars) of SB203580 (10 µM). For insulin-treated cells, insulin (1 nM) was added at the last 30 min. Cells were then harvested as detailed in the text. Results are the average of three experiments. Compared with control: *, P = 0.002; **, P = 0.01; ***, P < 0.01. Compared with insulin: @, P < 0.02. Compared with control + SB203580: #, P < 0.001; ##, P = 0.01; ###, P < 0.05. Compared with insulin + SB203580: @@, P = 0.008. B, ERK1/2 phosphorylation in the presence of either wortmannin (100 nM) or PD98059 (25 µM). Representative Western blots of three separate experiments.

 
Finally, we examined the role of p38 MAPK in TNF-{alpha}-induced AMPK phosphorylation because previous studies demonstrated that AMPK plays important metabolic functions including the phosphorylation of eNOS in endothelial cells and TNF-{alpha} enhances AMPK phosphorylation. As shown in Fig. 7Go, TNF-{alpha}, both in the absence and the presence of insulin, significantly increased the phosphorylation of AMPK{alpha} (P < 0.001). Inhibition of p38 MAPK did not change TNF-{alpha}-induced AMPK{alpha} phosphorylation (P = 0.002), suggesting this is also a p38 MAPK-independent process. Insulin did not alter either basal or TNF-{alpha}-stimulated AMPK phosphorylation.


Figure 7
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  		FIG. 7. TNF-{alpha} stimulates AMPK phosphorylation independent of p38 MAPK in bAECs. Cells were serum starved for 10 h and then incubated ± TNF-{alpha} (5 ng/ml) for 6 h in the absence (left four bars) or presence (right four bars) of SB203580 (10 µM). For insulin-treated cells, insulin (1 nM) was added at the last 30 min. Cells were then harvested as detailed in the text. Results are averages of four experiments. Compared with control: *, P < 0.001. Compared with control + SB203580: #, P = 0.002; ##, P < 0.001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Chronic vascular inflammation causes insulin resistance, endothelial dysfunction, and atherosclerosis. However, the underlying signaling pathways remain unclear. Our current results demonstrate for the first time that p38 MAPK directly mediates TNF-{alpha}-induced insulin resistance in the IRS-1/PI3-kinase/Akt signaling pathway, without affecting insulin’s or TNF-{alpha}’s actions via the MAPK pathways in endothelial cells. These suggest that p38 MAPK may be the key node in the pathophysiology of inflammation-induced endothelial insulin resistance and dysfunction.

Our findings of TNF-{alpha} increasing IRS-1 serine phosphorylation, decreasing insulin-stimulated IRS-1 tyrosine phosphorylation, Akt phosphorylation, and eNOS activity in endothelial cells confirm that inflammatory cytokines cause endothelial insulin resistance and dysfunction. That TNF-{alpha} blunts insulin-mediated IRS-1 tyrosine phosphorylation and insulin partially reverts TNF-{alpha}-induced IRS-1 serine phosphorylation suggests a reciprocal interplay/crosstalk between the insulin and TNF-{alpha} signaling pathways in the vascular endothelium. TNF-{alpha} also significantly increased the phosphorylation of p38 MAPK, which correlates with increased p38 MAPK kinase activity (23). Because p38 MAPK regulates stress responses, inflammation and apoptosis and has been implicated in the pathogenesis of endothelial dysfunction and atherosclerosis (18), we speculated that this enhanced p38 MAPK activity might have played a crucial role in the insulin resistance induced by inflammatory cytokines. Indeed, inhibition of p38 MAPK activity with its specific inhibitor SB203580 completely abolished TNF-{alpha}-induced IRS-1 serine phosphorylation and restored insulin sensitivity of IRS-1 tyrosine phosphorylation, Akt phosphorylation, and eNOS activity in the presence of TNF-{alpha}. This strongly suggests that TNF-{alpha} induces endothelial cell insulin resistance via a p38 MAPK-dependent pathway.

Inasmuch as both p38 MAPK and JNK regulate apoptosis, stress responses, and inflammation (5, 18, 24), JNK has been shown to increase IRS-1 serine phosphorylation and cause insulin resistance (5, 25) and we observed an increased JNK phosphorylation in the presence of TNF-{alpha}, it is possible that JNK may have also played some role in mediating TNF-{alpha}-induced insulin resistance in vascular endothelium. However, our results suggest that JNK most likely does not play a major role in our experimental setting as treatment of endothelial cells with SB203580 abolished TNF-{alpha}-induced insulin resistance despite persistent elevation in JNK phosphorylation.

The observations of TNF-{alpha} blunting insulin-stimulated IRS-1 tyrosine phosphorylation, Akt phosphorylation, and eNOS activity and stimulating ERK1/2 phosphorylation suggest that TNF-{alpha} induces insulin resistance selectively in the PI3-kinase pathway and enhances MAPK pathway signaling. Because insulin stimulates endothelial production of NO, a major vasodilator, via the PI3-kinase pathway (6, 7, 8, 9, 10, 11) and endothelin-1, a major vasoconstrictor, via the MAPK pathway (26, 27, 28, 29), this selective insulin resistance tilts the ET-1/NO balance toward increased vascular tone and decreased blood flow to target organs (3, 28). Indeed, physiological concentrations of insulin induce endothelin-dependent vasoconstriction in skeletal muscle arterioles in the presence of TNF-{alpha} (30) or PI3-kinase blockade (31), and infusion of TNF-{alpha} blocks insulin-stimulated capillary recruitment in rats (32). Thus, it appears that p38 MAPK and ERK1/2 may coordinately mediate TNF-{alpha}-induced insulin resistance and dysfunction in the vascular endothelium.

The finding of insulin stimulating eNOS phosphorylation without increasing its activity in the presence of TNF-{alpha} suggests dissociation between eNOS phosphorylation and activity in the presence of TNF-{alpha}. Insulin activates eNOS via Akt phosphorylating eNOS at serine 1179. However, a functional eNOS requires the presence of sufficient substrate arginine, cofactor (6R-)-5,6,7,8-tetrahydrobiopterin, and an intact zinc-thiolate moiety (33, 34). It is well known that inflammatory cytokines produce an uncoupling state within the enzyme via several mechanisms including activating arginase with resultant decrease in intracellular arginine level, increasing nicotinamide adenine dinucleotide phosphate oxidase activity with increased production of reactive oxygen species and peroxynitrite (33, 34, 35). Peroxynitrite increases basal and agonist-stimulated Ser1179 phosphorylation of eNOS, decreases arginine to citrulline conversion and NO production, and increases superoxide production by endothelial cells (36). In addition, changes in the phosphorylation of other eNOS phosphorylation sites, such as Ser (633) (stimulatory) and/or Thr (495) (inhibitory) (37, 38), may have contributed to this dissociation. Thus, in the presence of inflammatory cytokines, increased phosphorylation of eNOS does not equate to increased arginine to citrulline conversion and NO production. Whether p38 MAPK is involved in the eNOS uncoupling process in the state of chronic inflammation is unclear and warrants further study.

Because TNF-{alpha} activates AMPK and stimulates reactive oxygen species production in cultured endothelial cells and previous studies have demonstrated that peroxynitrite increases AMPK-dependent Ser1179 phosphorylation of eNOS, uncouples eNOS, and increases enzymatic production of superoxide anions (36, 39), we further examined whether p38 MAPK is also involved in these AMPK-dependent processes. Our results indeed confirm that TNF-{alpha}, in the presence or absence of insulin, potently stimulated AMPK phosphorylation. However, this increase in AMPK phosphorylation persisted even in the presence of SB203580, thus suggesting that the TNF-{alpha}-mediated AMPK phosphorylation is not regulated by the p38 MAPK.

We used SB203580 in the current study to inhibit p38 MAPK. SB203580, like SB202190, is a potent, cell-permeable, and specific inhibitor of p38 MAPK and does not suppress other MAPKs including ERK1/2 and JNK, even at 100 µM (40, 41). We recently confirmed that SB203580 potently inhibits p38 MAPK activity and reverses free fatty acids-induced apoptosis in cultured human coronary artery endothelial cells (42). Similar to the findings in the current study, SB203580 and SB202190 both potently inhibit p38 MAPK and reverse free fatty acid-induced insulin resistance in Akt phosphorylation and IRS tyrosine phosphorylation in cultured hepatocytes (43).

In conclusion, TNF-{alpha} induces insulin resistance in the PI3-kinase/Akt/eNOS pathway via a p38 MAPK-dependent pathway and enhances ERK1/2 and AMPK phosphorylation in a p38 MAPK-independent fashion in cultured bAECs. This differential modulation of TNF-{alpha} action by p38 MAPK suggests that p38 MAPK plays a key role in TNF-{alpha}-mediated insulin resistance and endothelial dysfunction frequently observed in the insulin-resistant states such as type 2 diabetes mellitus and the cardiometabolic syndrome.


   Footnotes
 
This work was supported by a research grant from the American Diabetes Association (to Z.L.) and National Institutes of Health Grants DK-DK057878 and DK-073759 (to E.J.B.) and P30-DK063609 (to the University of Virginia Diabetes Endocrinology Research Center).

Disclosure Statement: G.L., M.O.B., W.C., and Z.L. have nothing to declare. E.J.B. received lecture fees from Novo Nordisk and Pfizer Inc.

First Published Online April 19, 2007

Abbreviations: AMPK, AMP-activated protein kinase; bAEC, bovine aortic endothelial cell; eNOS, endothelial nitric oxide synthase; IRS, insulin receptor substrate; JNK, c-Jun N-terminal kinase; NO, nitric oxide; PI3-kinase, phosphatidylinositol 3-kinase.

Received October 27, 2006.

Accepted for publication April 11, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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