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Originally published In Press as doi:10.1074/jbc.M312021200 on February 4, 2004

J. Biol. Chem., Vol. 279, Issue 17, 17070-17078, April 23, 2004
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Tumor Necrosis Factor {alpha} Produces Insulin Resistance in Skeletal Muscle by Activation of Inhibitor {kappa}B Kinase in a p38 MAPK-dependent Manner*

Cristina de Alvaro{ddagger}, Teresa Teruel§, Rosario Hernandez{ddagger}, and Margarita Lorenzo

From the Departamento de Bioquimica y Biologia Molecular II, Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain

Received for publication, November 3, 2003 , and in revised form, January 28, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Insulin stimulation produced a reliable 3-fold increase in glucose uptake in primary neonatal rat myotubes, which was accompanied by a similar effect on GLUT4 translocation to plasma membrane. Tumor necrosis factor (TNF)-{alpha} caused insulin resistance on glucose uptake and GLUT4 translocation by impairing insulin stimulation of insulin receptor (IR) and IR substrate (IRS)-1 and IRS-2 tyrosine phosphorylation, IRS-associated phosphatidylinositol 3-kinase activation, and Akt phosphorylation. Because this cytokine produced sustained activation of stress and proinflammatory kinases, we have explored the hypothesis that insulin resistance by TNF-{alpha} could be mediated by these pathways. In this study we demonstrate that pretreatment with PD169316 or SB203580, inhibitors of p38 MAPK, restored insulin signaling and normalized insulin-induced glucose uptake in the presence of TNF-{alpha}. However, in the presence of PD98059 or SP600125, inhibitors of p42/p44 MAPK or JNK, respectively, insulin resistance by TNF-{alpha} was still produced. Moreover, TNF-{alpha} produced inhibitor {kappa}B kinase (IKK)-{beta} activation and inhibitor {kappa}B-{beta} and -{alpha} degradation in a p38 MAPK-dependent manner, and treatment with salicylate (an inhibitor of IKK) completely restored insulin signaling. Furthermore, TNF-{alpha} produced serine phosphorylation of IR and IRS-1 (total and on Ser307 residue), and these effects were completely precluded by pretreatment with either PD169316 or salicylate. Consequently, TNF-{alpha}, through activation of p38 MAPK and IKK, produces serine phosphorylation of IR and IRS-1, impairing its tyrosine phosphorylation by insulin and the corresponding activation of phosphatidylinositol 3-kinase and Akt, leading to insulin resistance on glucose uptake and GLUT4 translocation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Insulin increases glucose transport into cells of target tissues, primarily muscle (skeletal and cardiac) and fat (white and brown). Acute insulin treatment stimulates glucose transport in adipocytes and myocytes largely by mediating translocation of GLUT4 from an intracellular compartment to the plasma membrane, as reviewed previously (1–3). This effect is accomplished by activation of phosphatidylinositol 3-kinase (PI3K),1 Akt, and the atypical protein kinase C isoforms {zeta} and {lambda} in most cellular models (4–6), although other protein kinase C isoforms seem to play a role in skeletal muscle (7, 8). Recent discoveries have shown the presence of a second insulin signaling pathway leading to GLUT4 translocation in a PI3K-independent manner, involving the adaptor protein Cbl and the activation of a small GTP-binding protein, TC10 (3, 9). Furthermore, insulin can activate glucose uptake without producing GLUT4 translocation; this effect involves activation of p38 mitogen-activated protein kinase (MAPK), as has been specifically reported for muscle cells (10).

Insulin resistance, defined as a smaller than normal response to a given amount of insulin, is an important contributor to the pathogenesis of type 2 diabetes mellitus. Both genetic and environmental factors can contribute to the development of insulin resistance. Targeted disruption of insulin-like growth factor I and insulin receptor (IR) or of GLUT4, selectively, in skeletal muscle causes insulin resistance and insulin intolerance (11–13). Tumor necrosis factor (TNF)-{alpha} has been proposed as a link between adiposity and the development of insulin resistance because (a) the majority of type 2 diabetics are obese, (b) TNF-{alpha} is highly expressed in adipose tissues of obese animals and humans (14, 15), and (c) obese mice lacking either TNF-{alpha} or TNF-{alpha} receptors showed protection against developing insulin resistance (16, 17). Infusion of TNF-{alpha} to adult rats reduces systemic insulin sensitivity associated with major changes in adipocyte gene expression, favoring free fatty acid release without changes on muscle gene expression (18). These data suggest that impaired activity of the insulin signaling transduction pathways rather than changes in gene expression may be contributing to the development of insulin resistance in the muscle of TNF-{alpha}-treated animals.

Direct exposure of fat cells to TNF-{alpha} inhibits insulin-stimulated glucose uptake in several systems including 3T3-L1 cells, human primary adipocytes, and primary brown adipocytes (19, 20). The mechanism proposed involves serine phosphorylation of IR substrate (IRS)-1 that converts IRS-1 into an inhibitor of the insulin receptor tyrosine kinase activity (21). Furthermore, Ser307 has been identified as a site for TNF-{alpha}-induced phosphorylation of IRS-1, with activation of c-Jun NH2-terminal kinase (JNK) involved in the phosphorylation of this residue (22). Other studies suggest that p42/p44 and p38 MAPKs could inhibit insulin signaling by diverse mechanisms in 3T3-L1 adipocytes (23). In this regard, an enhanced basal activation of MAPKs in adipocytes from type 2 diabetic patients has been reported recently (24). Moreover, other works have also implicated inhibitor {kappa}B kinase (IKK) activation by TNF-{alpha} on serine phosphorylation of IRS-1 (25), whereas IKK inhibition with salicylates or targeted disruption of Ikk-{beta} produces reversal of obesity- and diet-induced insulin resistance (26).

Ceramide and fatty acids have been reported to induce insulin resistance in skeletal muscle (27–29), and production of insulin resistance could be a consequence of sphingomyelinase or lipolytic activation by TNF-{alpha} (30, 31). However, a direct effect of TNF-{alpha} on insulin resistance in muscle, which is responsible for 80% of the glucose disposal of the body, is controversial. Several reports did not detect TNF-{alpha} inhibitory action on insulin-induced glucose uptake, although TNF-{alpha} per se highly increased basal glucose uptake (32–34). However, others (8, 35) observed that TNF-{alpha} had an inhibitory effect on insulin action without modifying basal glucose uptake in muscle cells. On the other hand, in most of these studies, insulin stimulation of glucose uptake was very poor because virtually all cultured skeletal muscle cell lines have been found to be deficient in GLUT4 expression. Accordingly, in this work, we have prepared primary cultures of neonatal rat myotubes that, under physiological conditions, respond to insulin by increasing glucose uptake 3-fold and increasing GLUT4 translocation to the plasma membrane. Both effects were impaired by chronic treatment with TNF-{alpha}. This cytokine, through activation of p38 MAPK and IKK, produces phosphorylation of the residue Ser307 in IRS-1, impairing the insulin signaling cascade at the level of IR and IRS-1 tyrosine phosphorylation and IRS-1-associated PI3K and Akt activities.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Insulin, bovine serum albumin (fraction V, essentially fatty acid-free), myelin basic protein (MBP), and anti-{beta}-actin antibody were from Sigma. TNF-{alpha} was purchased from Pharma Biotechnologie (Hannover, Germany). PD169316 (PD*) and PD98059 (PD) were purchased from Calbiochem-Novabiochem. SP600125 (SP) and SB203580 (SB) were from Alexis. Horse serum, phosphate-buffered saline (PBS), trypsin-EDTA, culture media, and Trizol were from Invitrogen. Nylon membranes were GeneScreenTM (PerkinElmer Life Sciences Research Products). Autoradiographic films were Kodak X-Omat AR (Eastman Kodak Co.). 2-Deoxy-D[1-3H]glucose (11.0 Ci/mmol), [{gamma}-32P]ATP, and protein G-Sepharose were purchased from Amersham Biosciences. The rabbit anti-GLUT1, anti-GLUT4, and anti-P-Ser antibodies were supplied by Chemicon (Temecula, CA). The anti-phospho- and anti-Akt, -p42/p44 MAPK, -p38 MAPK, and -JNK antibodies were from Cell Signaling (Beverly, MA). Antibodies against IRS-1, IRS-2, P-Tyr (4G10), and P-IRS-1(Ser307) were from Upstate Biotechnology (Lake Placid, NY). Antibodies against P-Tyr (PY20) (sc-508), IR {beta}-chain (sc-09), caveolin-1(N-20) (sc-894), IKK-{beta} (sc-7607), and I{kappa}B-{beta} (sc-945) were from Santa Cruz (Palo Alto, CA). The anti-IR {beta}-chain antibody was from Oncogene Science (Uniondale, NY). All other reagents used were of the purest grade available.

Cell Culture—Skeletal muscle cells were prepared from thigh muscles obtained from 3–5-day-old neonatal rats according to the method described in Ref. 7, with some modifications. Neonatal rats were killed by cervical dislocation. The muscles from the fore and hind limbs were removed (carefully dissected to discard fat and connective tissues), washed in PBS (pH 7.5) to remove excess blood cells, triturated, and homogenized with scissors. Then the minced muscle was transferred to a Ca2+-free 0.25% trypsin solution containing 1 mM EDTA for incubation, with continuous stirring at 37 °C. Cells were collected after successive 20–30-min trypsinization periods of incubation, until all tissue was dispersed, and then centrifuged for 5 min at 1000 x g. The successive supernatants were filtered and reserved in growth medium. For the last incubation, the resting tissue was digested with a solution of collagenase II (Worthington) in Ca2+- and Mg2+-free PBS (100 IU/ml), 20–30 min at 37 °C with stirring. Finally, all the supernatants were centrifuged for 5 min at 2000 x g. The last pellet was washed with PBS, resuspended in growth medium, and pre-plated for 20–30 min in 100-mm-diameter tissue culture dishes to reduce the number of fibroblasts. The supernatant was collected, and the remaining myoblasts were diluted with growth medium to a concentration of 1 x 106 cells/ml. To facilitate adhesion of muscular cells to the plastic surface and to avoid attachment of fibroblasts, cells were seeded in collagen-coated dishes. Optimal plating densities were 1 x 106 cells in 6-well dishes and 5 x 106 cells in 100-mm-diameter dishes. Cells were cultured in growth medium (Dulbecco's minimal essential medium plus 10% horse serum) in a water-saturated atmosphere of 95% air, 5% CO2 at 37 °C during the first 3–4 days, when cells proliferated until reaching confluence, as observed under inverse light microscopy. Next, cells were cultured for an additional 4–5 days in differentiation medium (2% horse serum-Dulbecco's minimal essential medium), when cells fused and differentiated into multinucleated myotubes. Finally, myotubes were cultured overnight in serum-free, low-glucose (1000 mg/liter glucose) Dulbecco's minimal essential medium supplemented with 1% (w/v) bovine serum albumin before starting different treatments.

Glucose Transport Determination—Glucose uptake was measured during the last 10 min of culture by incorporation of labeled 2-deoxyglucose. Parallel dishes were used for protein determination, and individual values were expressed as disintegrations/min/µg protein, as described previously for brown adipocytes (36). Results were expressed as percentage of stimulation over basal (control = 100).

Subcellular Fractionation—Cells were washed with ice-cold PBS and scraped into homogenization buffer containing 20 mM Tris-HCl, 2 mM EGTA, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mM {beta}-mercaptoethanol, 10 µg/ml aprotinin, and 10 µg/ml leupeptin (pH 7.4). After a 10-min incubation, cells were homogenized with 30 strokes of a Dounce homogenizer using a tight-fitting pestle. Nuclei were pelleted by centrifugation at 500 x g for 5 min, and the low-speed supernatant was centrifuged at 100,000 x g for 30 min. The high-speed supernatant constituted the internal membrane fraction. The pellet was washed three times and extracted in ice-cold homogenization buffer containing 1% Triton X-100 for 60 min. The Triton-soluble component (plasma membrane fraction) was separated from the Triton-insoluble material (cytoskeletal fraction) by centrifugation at 100,000 x g for 15 min. Internal and plasma membrane fractions were kept at –70 °C before protein quantification and Western blotting with GLUT4 and caveolin-1 antibodies (20).

Immunoprecipitations—Cells were extracted with lysis buffer I containing 10 mM Tris, 50 mM NaCl, 1% Triton X-100, 5 mM EDTA, 20 mM sodium pyrophosphate, 50 mM NaF, 100 µM Na3VO4, and 1 µM phenylmethylsulfonyl fluoride (pH 7.5) and immunoprecipitated with different antibodies against IRSs, IR, p38 MAPK, or IKK-{beta}. PI3K activity was measured in anti-IRS immunoprecipitates by in vitro phosphorylation of phosphatidylinositol as described previously (20).

p38 and IKK in Vitro Kinase Assays—These assays were performed in anti-p38 MAPK {alpha} and {beta} and IKK-{beta} immunoprecipitates as described previously (37). Immune complexes were washed five times with ice-cold lysis buffer containing 0.5 M NaCl and two times with kinase buffer (35 mM Tris, pH 7.5, 10 mM MgCl2, 0.5 mM EGTA, and 1 µM Na3VO4). The kinase reaction was performed in a kinase buffer containing 1 µCi of [{gamma}-32P]ATP, 60 µM ATP, and 1 µg of MBP as a substrate for 30 min at 30 °C and terminated by the addition of 2x SDS-PAGE sample buffer followed by boiling for 5 min at 95 °C. Samples were resolved in 12% SDS-PAGE, and gels were dried out and subjected to autoradiography.

Western Blotting—Cells were lysed in lysis buffer I, and cellular proteins (30 µg) were subjected to SDS-PAGE, transferred to Immobilon membranes, blocked using 5% nonfat dried milk in 10 mM Tris-HCl and 150 mM NaCl (pH 7.5), and incubated overnight with several antibodies as indicated in each case in 0.05% Tween 20 and 1% nonfat dried milk in 10 mM Tris-HCl and 150 mM NaCl (pH 7.5). Immunoreactive bands were visualized using the enhanced chemiluminescence (ECL-Plus) Western blotting protocol (Amersham Biosciences).

Data Analysis—Results are means ± S.E. (n = 4 or 10) for duplicate dishes from 4 to 10 independent experiments. Statistical significance was tested with a one-way analysis of variance followed by the protected least-significant different test. p values of <0.05 were considered significant. In experiments using x-ray films (Hyperfim), different exposure times were used to ensure that bands were not saturated.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
TNF-{alpha} Inhibits Insulin-induced Glucose Transport in a p38 MAPK-dependent Manner in Skeletal Muscle—Isolated neonatal muscle cells were differentiated in low serum until the formation of myotubes. Then, cells were shifted overnight to serum-free, low-glucose medium and further cultured for 24 h in the absence or presence of 1 nM TNF-{alpha} before stimulation for 30 min with 50 nM insulin. Glucose uptake was measured during the last 10 min of culture by incorporation of labeled 2-deoxy glucose, and results were expressed as the percentage of stimulation over basal level (control = 100) (Fig. 1A). Insulin stimulation for 30 min significantly increased (3-fold) basal glucose uptake. Cells pretreated with TNF-{alpha} for 24 h showed a 3-fold higher glucose uptake than untreated cells, and under this circumstance, insulin stimulation did not further stimulate glucose uptake. Because insulin stimulation of glucose transport is mediated by the translocation of GLUT4 to the plasma membrane, we decided to confirm the above-mentioned data on glucose uptake by examining GLUT4 translocation. After subcellular fractionation, GLUT4 protein in plasma membrane fractions was detected by immunoblotting (Fig. 1B). Insulin increased GLUT4 translocation to plasma membrane fraction 3-fold, but this effect was not produced when cells were pretreated with TNF-{alpha} before insulin stimulation. In parallel, the disappearance of GLUT4 localization in internal membrane produced by insulin was not observed when TNF-{alpha} was present together with insulin. Caveolin-1, an integral membrane protein from caveolae, was used as a marker protein of the plasma membrane, and its amount remained essentially unaltered by the different treatments used (Fig. 1B). Moreover, the increase in basal glucose uptake observed under TNF-{alpha} treatment was not the result of GLUT4 translocation, as shown in Fig. 1B. However, the levels of GLUT1 protein and mRNA detected under TNF-{alpha} treatment were increased compared with controls, without affecting the expression of GLUT4 protein (Fig. 1C). Moreover, because TNF-{alpha} treatment was performed in differentiated myotubes, no changes in the state of differentiation of the cells were observed (data not shown).



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  		FIG. 1.
TNF-{alpha} inhibits insulin-induced glucose transport in a p38 MAPK-dependent manner in skeletal muscle. A, primary neonatal rat myotubes were cultured for 24 h in the absence (C) or presence of 1 nM TNF-{alpha} (T) without or with 800 nM PD*, 10 µM SB, 20 µM PD, or 3 µM SP and stimulated for 30 min with 50 nM insulin (I) or not stimulated. Glucose uptake was measured during the last 10 min by incorporation of 2-deoxy-D[1-3H]glucose into the cells. Results are expressed as the percentage of stimulation over control (C = 100) and are means ± S.E. (n = 10). Statistical significance was tested, and differences between values in the presence of I versus C are represented by , differences between values in the presence of T versus C are represented by {diamondsuit}, differences between T+I versus T are represented by {triangleup}, and differences between values in the presence of T+I+PD* versus T+I are represented by {blacktriangleup}. B, cells cultured as described in A were harvested and subjected to subcellular fractionation. Plasma and internal membrane proteins were subjected to SDS-PAGE, blotted onto nylon membrane, immunodetected with anti-GLUT4 and anti-caveolin-1 (Cav-1) antibodies, and developed with enhanced chemiluminescence. Representative experiments are shown. Histograms from densitometric analysis expressed in arbitrary units are means ± S.E. (n = 4). Statistical significance was tested, and differences between values in the presence of I versus control (C) are represented by , differences between values in the presence of T+I versus I are represented by {triangleup}, and differences between T+I+PD* versus T+I are represented by {blacktriangleup}. C, cells were cultured for 24 h in the absence or presence of TNF-{alpha} and inhibitors, at the doses used in A, and after harvest, extracts were analyzed by Western blotting with the corresponding antibodies against GLUT4 and GLUT1 proteins or analyzed by Northern blotting with a GLUT1 cDNA. Representative experiments are shown.

 
The mechanism by which TNF-{alpha} produced insulin resistance on glucose uptake is far from clear and prompted us to investigate the signaling pathways elicited by chronic TNF-{alpha} treatment in skeletal muscle. Cells were deprived of serum overnight and further cultured for up to 24 h in the presence of 1nM TNF-{alpha}. At the end of the culture time, cells were collected, and protein extracts were analyzed for activation of stress kinases by Western blotting using the corresponding antibodies against phosphorylated and total p38 and p42/p44 MAPK and JNK (Fig. 2A). TNF-{alpha} produced a peak phosphorylation of p38 MAPK at 5 min, and this activation was sustained for at least 6 h of treatment. Furthermore, TNF-{alpha}-induced phosphorylation of p42/p44 MAPK was detectable for 6 h of treatment; these activations on MAPKs were not detectable after 24 h. JNK phosphorylation by TNF-{alpha} was detected after 30 min of stimulation, and this kinase remained active for 6 h. The changes observed in the state of phosphorylation of these kinases seem to reflect changes in their activities because the protein levels are similar in all the conditions. Next, we investigated which p38 MAPK isoform was activated by TNF-{alpha}, using in vitro kinase assays of MBP phosphorylation in immunoprecipitates against the p38 MAPK isoforms {alpha} and {beta} (Fig. 2B). Stimulation for 5 min with TNF-{alpha} (1 nM) activated mainly the p38 MAPK activity associated with the {beta} isoform, whereas 50 nM insulin, which has been shown to stimulate phosphorylation of p38 MAPK in C2C12 cells (38), activated mainly the p38{alpha} isoform. Both isoforms were activated to a similar extent after stimulation with 500 mM sorbitol.



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  		FIG. 2.
TNF-{alpha} activates JNK and p38 and p42/p44 MAPK in skeletal muscle. A, primary neonatal rat myotubes were cultured for up to 24 h in the absence or presence of 1 nM TNF-{alpha}. At the end of the culture time, cells were collected, and protein extracts were analyzed by Western blotting with the corresponding antibodies against phosphorylated and total p38 MAPK and p42/p44 MAPK and JNK. Representative experiments are shown as well as the densitometric analysis of phosphorylated proteins after standardization using the total amount of protein, expressed in arbitrary units (a.u.). B, myotubes (C) were stimulated for 5 min with 1 nM TNF-{alpha}, 50 nM insulin (Ins), or 500 mM sorbitol (Sorb) and lysed and immunoprecipitated with either anti-p38 MAPK {alpha} or {beta} antibodies. p38 MAPK activity was assayed in the resulting immune complexes for MBP phosphorylation and autoradiography. Histograms resulting from the densitometric analysis of the autoradiograms represent phosphorylated MBP levels in arbitrary units (a.u.) and are means ± S.E. (n = 4). C, cells were cultured for 6 h in either the absence (C) or presence of 1 nM TNF-{alpha} without or with 800 nM PD*, 10 µM SB, 20 µM PD, or 3 µM SP. Protein extracts were analyzed by Western blotting with the corresponding antibodies against phosphorylated JNK, p38 MAPK, and p42/p44 MAPK and total p38 MAPK. Representative experiments are shown. Inhibition of p38 MAPK {beta} activity by PD* or SB is shown in a representative experiment performed for MBP phosphorylation in anti-p38 MAPK {beta} immunoprecipitates (IP), as described in B.

 
To investigate whether the sustained activation of p38 MAPK, p42/p44 MAPK, and JNK by TNF-{alpha} could contribute to insulin resistance, we decided to impair these pathways by the use of chemical inhibitors and determine whether TNF-{alpha} was able to produce insulin resistance under those circumstances. We used SP (3 µM) to inhibit JNK, PD (20 µM) to inhibit ERK, and two different compounds (PD* (800 nM) or SB (10 µM)) as p38 MAPK inhibitors after testing the specificity and optimal inhibitory doses of these chemicals in neonatal muscle cells (Fig. 2C). Glucose uptake was determined in cells cultured for 24 h in the absence or presence of 1 nM TNF-{alpha} with or without inhibitors, before stimulation for 30 min with 50 nM insulin (Fig. 1A). When cells were cultured for 24 h in the presence of PD, SP, or PD*/SB, no significant changes in insulin or TNF-{alpha}-stimulated glucose uptake were detected. However, treatment with either PD* or SB completely restored insulin stimulation of glucose uptake in the presence of TNF-{alpha}, leading to a 5-fold stimulation over basal level and to a 2-fold increase over TNF-{alpha} (Fig. 1A). This effect was not observed in the presence of PD or SP. A similar restoration on insulin-induced translocation of GLUT4 from internal membrane to the plasma membrane was detected in the presence of TNF and PD* for 24 h, whereas this effect was not observed in the presence of PD (Fig. 1B). Furthermore, the presence of the inhibitors for 24 h did not modify the expression of GLUT4 or GLUT1 proteins (Fig. 1C). The above-mentioned data seem to indicate that although TNF-{alpha} activates several MAPKs in skeletal muscle, p38{beta} MAPK could be the primary contributor to the TNF-{alpha} inhibitory effect on insulin-stimulated glucose uptake.

TNF-{alpha} Impairs the Insulin Signaling Cascade in a p38 MAPK-dependent Manner—The next step was to identify at which level TNF-{alpha} was interfering with the insulin signaling cascade, and whether that interference could be avoided in the presence of the inhibitor of p38 or p42/p44 MAPKs. Myotubes deprived of serum overnight were cultured for 24 h in the absence or presence of 1 nM TNF-{alpha} with or without PD or PD*, before stimulation for 5 min with 50 nM insulin. Insulin induced tyrosine phosphorylation of IR observed in IR immunoprecipitates, an effect that was impaired by pretreatment with TNF-{alpha} (Fig. 3A). However, treatment with PD*, but not PD, completely restored IR tyrosine phosphorylation by insulin in the presence of TNF-{alpha}. Treatment with TNF-{alpha} for 24 h not only decreased tyrosine phosphorylation of IR by insulin but also produced serine phosphorylation of the IR, as demonstrated in immunoprecipitates with P-Ser antibodies and Western blotting with IR {beta}-chain antibody. However, serine phosphorylation of IR was decreased in myotubes pretreated with PD*, contributing to restoration on tyrosine phosphorylation (Fig. 3B).



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  		FIG. 3.
TNF-{alpha} impairs insulin-induced tyrosine phosphorylation of IR in a p38 MAPK-dependent manner. Cells were cultured for 24 h in the absence (C) or presence of 1 nM TNF-{alpha} (T) without or with 800 nM PD* or 20 µM PD and stimulated for 5 min with 50 nM insulin (I) or not stimulated. A, cell lysates were immunoprecipitated with antibodies against IR and immunoblotted with anti-P-Tyr (4G10) antibody (PY) or with the antibody against the {beta}-chain of IR. B, cell lysates were immunoprecipitated with antibodies against P-Ser and immunoblotted with the antibody against the {beta}-chain of IR. Representative experiments are shown. Histograms from densitometric analysis expressed in arbitrary units (a.u.) are means ± S.E. (n = 4). Statistical significance was tested, and differences between values in the presence of I versus control C are represented by , differences between values in the presence of T or T+I+PD versus C are represented by {diamondsuit}, differences between values in the presence of T+I versus I are represented by {triangleup}, and differences between T+I+PD* versus T+I are represented by {blacktriangleup}.

 
Insulin-induced tyrosine phosphorylation of IRS-1 and IRS-2 was significantly impaired by pretreatment with TNF-{alpha}, without significant changes in IRS expression (Fig. 4, A and B). However, treatment with PD* in the presence of TNF-{alpha} completely restored tyrosine phosphorylation by insulin at the level of IRS-1, with a partial effect at the level of IRS-2. Treatment with TNF-{alpha} for 24 h produced serine phosphorylation of IRS-1, and this phosphorylation was precluded by pretreatment with PD*, but not PD, as demonstrated in immunoprecipitates with P-Ser antibodies and Western blotting with IRS-1 antibody (Fig. 4C). Moreover, TNF-{alpha} produced phosphorylation on the Ser307 residue of IRS-1 in a p38 MAPK-dependent manner, as demonstrated by direct Western blotting with anti-P-IRS-1(Ser307)-specific antibody.



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  		FIG. 4.
TNF-{alpha} increases Ser phosphorylation and impairs insulin-induced tyrosine phosphorylation of IRS-1 and IRS-2. Cells were cultured as indicated in Fig. 3, and lysates were immunoprecipitated with anti-IRS-1 (A) or anti-IRS-2 (B) antibodies and immunoblotted with anti-P-Tyr (4G10) antibody (PY) or with the antibodies against IRSs. C, cell lysates were immunoprecipitated with antibodies against P-Ser and immunoblotted with anti-IRS-1. A direct Western blot with the anti-P-IRS-1(Ser307)-specific antibody is also shown. Representative experiments are shown. Histograms from densitometric analysis of phosphorylated IRSs expressed in arbitrary units (a.u.) are means ± S.E. (n = 4). Statistical significance was tested as described in the Fig. 3 legend.

 
PI3K activity associated with IRSs was determined in the immune complexes after immunoprecipitation with either the anti-IRS-1 or anti-IRS-2 antibodies (Fig. 5A). TNF-{alpha} impaired insulin-induced PI3K activity in IRS-2 and, more evidently, in IRS-1 immunoprecipitates, but this inhibitory effect was not produced in the presence of PD*. In addition, cell lysates were analyzed by Western blotting with anti-phospho-Akt antibodies for both regulatory residues Ser473 and Thr308 (Fig. 5B). Upon insulin stimulation, Akt was highly serine and threonine phosphorylated, and both effects were impaired by TNF-{alpha} pretreatment in a p38 MAPK-dependent manner (Fig. 5B). All these data indicated that TNF-{alpha} impaired insulin activation of the IR/IRS-1/Akt signaling cascade in a p38 MAPK-dependent, p42/p44-independent manner, in a fashion similar to that detected for glucose uptake.



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  		FIG. 5.
PD* restores insulin stimulation of PI3K and Akt activities in the presence of TNF-{alpha}. Cells were cultured as indicated in Fig. 3, and lysates were immunoprecipitated with anti-IRS-1 or anti-IRS-2 antibodies and assayed for PI3K activity (A). Cell lysates were also analyzed by Western blotting with the corresponding antibodies against phospho-Akt(Ser473), phospho-Akt(Thr308), and Akt (B). Representative experiments are shown. Histograms from densitometric analysis of PI3K activity and P-Akt expressed in arbitrary units (a.u.) are means ± S.E. (n = 4). Statistical significance was tested as described in the Fig. 3 legend.

 
Inhibition of IKKs by Salicylate or PD* Precludes Insulin Resistance by TNF-{alpha} in Skeletal Muscle—Insulin resistance by TNF-{alpha} in skeletal muscle can be impaired by inhibition of p38 MAPK. However, TNF-{alpha}, besides activating stress kinases, also activates proinflammatory kinases such as IKKs that have recently been implicated in the development of insulin resistance. Accordingly, we decided to explore the potential implication of IKKs in insulin resistance by TNF-{alpha} and determine whether there was any connection with the activation of p38 MAPK. Myotubes deprived of serum overnight were cultured for up to 24 h in the absence or presence of 1 nM TNF-{alpha}, and IKK activation was detected indirectly by degradation of I{kappa}B-{alpha} and -{beta}, monitored by Western blotting (Fig. 6A). As expected, TNF-{alpha} induced I{kappa}B-{alpha} and -{beta} degradation after 5 min of treatment, and this effect was essentially maintained for 24 h of treatment. Insulin stimulation for 5 min had no effect on I{kappa}B-{alpha} and -{beta} degradation by TNF-{alpha}. However, this effect was avoided in the presence of PD*, but not in the presence of PD, indicating that TNF-{alpha} activation of IKKs was dependent on p38 MAPK, as determined by in vitro kinase assay in IKK-{beta} immunoprecipitates as well as by Western blotting (Fig. 6B). However, p38 MAPK activation by TNF-{alpha} was independent of IKK activation because in the presence of an IKK inhibitor (salicylate), p38 MAPK phosphorylation was still detected (Fig. 6C). Accordingly, we decided to block IKK activation by TNF-{alpha} by incubating myotubes with salicylate to determine whether it was able to restore insulin signaling (Fig. 7). Cells cultured for 24 h in the presence of 1 nM TNF-{alpha} with 5 mM salicylate showed a complete restoration of insulin signaling at the level of IR and IRS-1 tyrosine phosphorylation, with PI3K activation associated with IRS-1 and Akt phosphorylation on both Ser473 and Thr308 residues, as shown in Fig. 7. Furthermore, impairment of TNF-{alpha}-induced IKK activation by salicylate completely prohibited Ser307 phosphorylation of IRS-1 by TNF-{alpha}, indicating that the phosphorylation of this residue by TNF-{alpha} plays a major role in the development of insulin resistance in neonatal myotubes (Fig. 7, B and D).



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  		FIG. 6.
TNF-{alpha} activates I{kappa}B-{alpha} and {beta} degradation and IKK-{beta} in a p38 MAPK-dependent manner in skeletal muscle. A, primary neonatal rat myotubes were cultured for up to 24 h in the absence or presence of 1 nM TNF-{alpha}. At the end of the culture time, cells were collected, and protein extracts were analyzed by Western blotting with the corresponding antibodies against I{kappa}B-{alpha}, I{kappa}B-{beta}, and {beta}-actin. Representative experiments are shown. B, cells were cultured as indicated in Fig. 3, and protein extracts were analyzed by Western blotting as described in A or by in vitro kinase assay in immunoprecipitates with anti-IKK-{beta} antibody. Representative experiments are shown. Histograms from densitometric analysis expressed in arbitrary units (a.u.) are means ± S.E. (n = 4). Statistical significance was tested, and differences between values in the presence of T or T+IorT+I+PD versus C are represented by {diamondsuit}, and differences between T+I+PD* versus T+I are represented by {triangleup}. C, cells were treated for 6 h in the absence or presence of TNF-{alpha} without or with 5 mM salicylate (Sal), and proteins were analyzed by Western blotting with the corresponding antibodies against phosphorylated and total p38 MAPK. Representative experiments are shown.

 



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  		FIG. 7.
Salicylate precludes insulin resistance by TNF-{alpha} in skeletal muscle. Cells were cultured for 24 h in the absence (C) or presence of 1 nM TNF-{alpha} (T) without or with 5 mM salicylate (Sal) and stimulated for 5 min with 50 nM insulin (I) or not stimulated. A, cell lysates were immunoprecipitated with antibodies against IR and immunoblotted with anti-P-Tyr (4G10) antibody (PY) or with the antibody against the {beta}-chain of IR. B, lysates were immunoprecipitated with anti-IRS-1 or anti-P-Ser antibodies and immunoblotted with anti-P-Tyr (4G10) antibody or with the antibody against IRS-1. A direct Western blot with the anti-P-IRS-1(Ser307)-specific antibody is also shown. C, PI3K activity was assayed in anti-IRS-1 immunoprecipitates. D, cell lysates were also analyzed by Western blotting with the corresponding antibodies against phospho-Akt(Ser473), phospho-Akt(Thr308), Akt, I{kappa}B-{alpha}, and I{kappa}B-{beta}. Results representative of four experiments are shown. Histograms from densitometric analysis from B and C are expressed in arbitrary units (a.u.) and are means ± S.E. (n = 4). Statistical significance was tested, and differences between values in the presence of I versus control (C) are represented by , differences between values in the presence of T+I versus I are represented by {triangleup}, and differences between T+I+Sal versus T+I are represented by {blacktriangleup}.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
TNF-{alpha} has been reported to produce insulin resistance in several cellular systems, such as white and brown adipocytes, and in some skeletal muscle cell lines (8, 19, 20, 35). However, insulin stimulation of glucose uptake in these cells lines, including L6 and C2C12 myotubes, was very poor. Accordingly, we have developed primary cultures of neonatal skeletal muscle that represent a suitable system for investigating the molecular basis of TNF-{alpha}-induced insulin resistance. Isolated muscle cells were differentiated in low serum until the formation of myotubes and then maintained in low-glucose medium mimicking the physiological environment. Under these experimental conditions, acute insulin stimulation produced a reliable 3-fold increase in glucose uptake, which was accompanied by a similar effect on GLUT4 disappearance from internal membrane and translocation to plasma membrane. Thus, primary neonatal rat myotubes respond to insulin to a similar extent as primary fetal rat brown adipocytes (20, 36). Chronic exposure to TNF-{alpha} impaired both insulin-stimulated glucose uptake and GLUT4 translocation, without affecting the expression of GLUT4 or the state of differentiation of the myotubes. Our results in primary neonatal myotubes are in agreement with those obtained in muscle in vivo (18) and indicated that insulin resistance by TNF-{alpha} seems to be the consequence of an antagonism in the activation of the complete insulin signaling cascade from IR to Akt rather than changes on muscle gene expression. Furthermore, the significant increase in basal glucose uptake produced by TNF-{alpha} treatment in primary skeletal muscle cells was not due to GLUT4 translocation to the plasma membrane and seemed to be associated with increased GLUT1 expression, as has been described previously in several muscle cells (29, 39).

The molecular mechanism underlying TNF-{alpha}-mediated insulin resistance could involve activation of different serine/threonine kinases by TNF-{alpha}, and in this regard, MAPKs (JNK, ERK, and p38) as well as IKK were activated by TNF-{alpha} in fat cells (40). In this work, we show that TNF-{alpha} produced a sustained phosphorylation of JNK, p38 MAPK, and p42/p44 MAPK during the first 6 h of treatment. Acute insulin stimulation (5 min) produces a transient phosphorylation of MAPKs, as we reported previously (38, 41), but insulin activates p38{alpha} MAPK, whereas TNF-{alpha} activates the {beta} isoform, as we demonstrate in this work. To evaluate the contribution of sustained activation of these kinases to insulin resistance, we used the chemical inhibitors SP, PD*/SB, and PD, which specifically prohibited activation of these pathways by TNF-{alpha} in primary muscle cells. Inhibition of p38 MAPK with either PD* or SB completely restored insulin-stimulated glucose uptake and insulin signaling, whereas inhibition of p42/p44 MAPK or JNK did not abrogate TNF-{alpha}-induced insulin resistance. Although activation of both isoforms of p38 MAPK by insulin has been proposed to activate glucose uptake without producing GLUT4 translocation (10, 42), other reports indicate that p38 MAPK could play a role in oxidant-induced inhibition of insulin-regulated glucose transport in L6 muscle cells (43). Furthermore, adenovirus-mediated transfections of constitutively active MAPK kinase 6/3 mutants in L6 myotubes has been reported to diminish glucose transport induced by insulin via down-regulation of GLUT4 gene expression (44).

Once we identified p38 MAPK as the potential kinase by which TNF-{alpha} was producing insulin resistance, we explored in depth how it interfered with the insulin signaling cascade. Chronic treatment with TNF-{alpha} was producing serine phosphorylation of IRS-1 and IR in a p38 MAPK-dependent manner; this serine phosphorylation was weakening the tyrosine phosphorylation induced by insulin and impairing the normal response to insulin on glucose uptake. Both p42/p44 MAPK and JNK have been proposed to mediate TNF-{alpha} serine/threonine phosphorylation of IRS-1 in white fat cells (22). Very recent works have indicated that p42/p44 MAPK, to a greater extent than p38 MAPK and JNK, could inhibit insulin signaling at the level of IRS-1 and IRS-2 in 3T3-L1 adipocytes (40), whereas JNK could mediate the feedback inhibitory effect of insulin (45). Our data indicate that p38 MAPK, but not p42/p44 MAPK or JNK, could be the major player in TNF-{alpha}-induced insulin resistance in neonatal skeletal muscle. Furthermore, Ser307 of IRS-1 seems to be one of the residues phosphorylated by TNF-{alpha} via p38 MAPK, although we cannot discard the possibility that other residues in either IRS-1 or IRS-2 or IR could also be a target for TNF-{alpha}-induced insulin resistance in skeletal muscle.

Several reports have also implicated IKK activation by TNF-{alpha} on serine phosphorylation of IRS-1 (25, 26), and a recent work has indicated that aspirin rescued insulin-induced glucose uptake in 3T3-L1 adipocytes treated with TNF-{alpha} (46). In this regard, activation of IKK detected indirectly by I{kappa}B-{alpha} or -{beta} degradation or directly by in vitro kinase assay in IKK-{beta} immunoprecipitates was observed during the chronic treatment with TNF-{alpha}; more importantly, this activation was dependent on the functionality of p38 MAPK. Inhibition of IKK activation with salicylate completely restores insulin signaling to normal levels, despite the presence of TNF-{alpha}. Furthermore, salicylate did not affect p38 MAPK activation by TNF-{alpha}. These data seem to indicate that IKK could act downstream of p38 MAPK and mediate TNF-{alpha}-induced insulin resistance on skeletal muscle. Our results are consistent with the requirement for p38 MAPK in the activation of nuclear factor-{kappa}B transcriptional factor in response to interleukin-1{beta} (47).

Accordingly, chronic TNF-{alpha} treatment impaired insulin stimulation of glucose uptake and GLUT4 translocation to the plasma membrane. This cytokine, through activation of IKK in a p38 MAPK-dependent manner, produces phosphorylation of the residue Ser307 in IRS-1, impairing the insulin signaling cascade at the level of IR and IRS-1 tyrosine phosphorylation and IRS-1-associated PI3K and Akt activity.


   FOOTNOTES
 
* This work was supported in part by Grant BMC2002-01322 from Direccion General de Investigacion Cientifica, Ministerio de Ciencia y Tecnologia and Grant 08.6/0002.1/2003 from Comunidad de Madrid, Spain. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Recipients of postgraduate fellowships from the Ministerio de Educacion y Cultura. Back

§ Recipient of a postdoctoral fellowship from the Comunidad Autonoma de Madrid. Back

To whom correspondence should be addressed: Departamento de Bioquimica y Biologia Molecular II, Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain. Tel.: 34-91-3941858; Fax: 34-91-3941779; E-mail: mlorenzo{at}farm.ucm.es.

1 The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; TNF, tumor necrosis factor; IR, insulin receptor; I{kappa}B, inhibitor {kappa}B; IKK, inhibitor {kappa}B kinase; IRS, insulin receptor substrate; MAPK, mitogen-activated protein kinase; JNK, c-Jun NH2-terminal kinase; PBS, phosphate-buffered saline; MBP, myelin basic protein; PD, PD98059; PD*, PD169316; SP, SP600125; SB, SB203580; P, phospho. Back


   ACKNOWLEDGMENTS
 
We thank the Cooperation in the Field of Scientific and Technical Research (COST) B17 Action on "Insulin Resistance, Obesity and Diabetes Mellitus in the Elderly" from the European Commission, and the Red de Grupos de Diabetes Mellitus G03/212 from Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo for support.



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 EXPERIMENTAL PROCEDURES
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 DISCUSSION
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