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Am J Physiol Endocrinol Metab 289: E95-E104, 2005. First published February 8, 2005; doi:10.1152/ajpendo.00397.2004
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Acute treatment with TNF-{alpha} attenuates insulin-stimulated protein synthesis in cultures of C2C12 myotubes through a MEK1-sensitive mechanism

David L. Williamson, Scot R. Kimball, and Leonard S. Jefferson

Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania

Submitted 25 August 2004 ; accepted in final form 3 February 2005


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Insulin and TNF-{alpha} exert opposing effects on skeletal muscle protein synthesis that are mediated in part by the rapamycin-sensitive mammalian target of rapamycin (mTOR) pathway and the PD-98059-sensitive, extracellular signal-regulated kinase (ERK)1/2 pathway. The present study examined the separate and combined effects of insulin (INS), TNF, PD-98059, or dnMEK1 adenovirus on the translational control of protein synthesis in C2C12 myotubes. Cultures were treated with INS, TNF, PD-98059, dnMEK1, or a combination of INS + TNF with PD-98059 or dnMEK1. INS stimulated protein synthesis, enhanced eIF4E·eIF4G association, and eIF4G phosphorylation and repressed eIF4E·4E-BP1 association vs. control. INS also promoted phosphorylation of ERK1/2, S6K1, and 4E-BP1 and dephosphorylation of eIF4E. TNF alone did not have an effect on protein synthesis (vs. control), eIF4E·eIF4G association, or the phosphorylation of eIF4G, S6K1, or 4E-BP1, although it transiently increased ERK1/2 and eIF4E phosphorylation. When myotubes were treated with TNF + INS, the cytokine blocked the insulin-induced stimulation of protein synthesis. This appeared to be due to an attenuation of insulin-stimulated eIF4E·eIF4G association, because other stimulatory effects of INS, e.g., phosphorylation of ERK1/2, 4E-BP1, S6K1, eIF4G, and eIF4E and eIF4E·4E-BP1 association, were unaffected. Finally, treatment of myotubes with PD-98059 or dnMEK1 adenovirus before TNF + INS addition resulted in a derepression of protein synthesis and the association of eIF4G with eIF4E. These findings suggest that TNF abrogates insulin-induced stimulation of protein synthesis in myotubes through a decrease in eIF4F complex assembly independently of S6K1 and 4E-BP1 signaling and dependently on a MEK1-sensitive signaling pathway.

tumor necrosis factor-{alpha}; extracellular signal-regulated kinase; translation initiation; eukaryotic initiation factor 4F assembly; skeletal muscle; eukaryotic initiation factor 4G; PD-98059

TUMOR NECROSIS FACTOR-{alpha} (TNF-{alpha}) is a cytokine that is elevated during several cachectic conditions, such as cancer, AIDS, and sepsis (5, 6, 23, 32, 46), all of which cause a reduction in skeletal muscle protein synthesis and a loss of muscle mass. One common characteristic of these conditions is the insulin-resistant state of skeletal muscle for both glucose and protein metabolism. As a specific example, the increased rate of skeletal muscle loss induced by sepsis is partially due to a reduction in protein synthesis that is associated with an inhibition of the initiation of mRNA translation (26, 46). Under these conditions, insulin treatment does not attenuate the sepsis-induced reduction in protein synthesis (45), regardless of preserved insulin signaling through the mammalian target of rapamycin (mTOR) pathway. The failure of insulin to attenuate the sepsis-induced decrease in skeletal muscle protein synthesis is associated with a reduction in translation initiation, although the mechanism is not fully understood.

TNF-{alpha} administration is typically associated with a loss of skeletal muscle mass and an inhibition of protein synthesis, although its effects have not been clearly demonstrated to be due to alterations in translation initiation. The effects of TNF-{alpha} are primarily associated with activation of stress-activated kinases, such as the mitogen-activated protein kinases (MAPK), p38 MAPK, stress-activated protein kinase/Jun-NH2-terminal kinase (SAPK/JNK), and extracellular signal-regulated kinase (ERK)1/2, subsequently increasing the activation of stress-related proteins [e.g., nuclear factor-{kappa}B (NF-{kappa}B) and suppressor of cytokine signaling (SOCS)] (42, 43). Induction of ERK1/2 and p38 MAPK signaling also stimulates the phosphorylation of the eukaryotic initiation factor (eIF)4E (47, 48) through activation of the MAPK-interacting kinase (MNK)1/2.

Elevated concentrations of TNF-{alpha} in circulating blood or in cell culture medium have been shown to inhibit insulin-stimulated pathways. TNF-{alpha} stimulates the phosphorylation of insulin receptor substrate (IRS)-1 on serine residues, thereby inhibiting the phosphatidylinositol 3-kinase pathway (17). Likewise, under conditions of elevated circulating concentrations of TNF-{alpha} and other cytokines, such as sepsis, endotoxin, or TNF-{alpha} treatment, signaling through mTOR, as assessed by the phosphorylation status of the mTOR substrates eIF4E-binding protein (4E-BP)1 and the 70-kDa ribosomal protein S6 kinase (S6K1) is inhibited, along with a reduction in eIF4G associated with eIF4E (23, 25, 26, 46). However, these studies do not partition out the acute effects of the many hormones that are simultaneously elevated or decreased during these treatments, which could confound the individual effects of each hormone.

Insulin stimulates protein synthesis in skeletal muscle (3, 21, 22), in part by accelerating the binding of the initiator form of met-tRNA (31, 35) to the 40S ribosomal subunit to form the 43S preinitiation complex (20). Insulin also stimulates the mTOR-mediated signaling pathway, causing 4E-BP1 to become phosphorylated and dissociate from eIF4E, thus allowing for an increase in the interaction of eIF4G with eIF4E (21, 22). Similar to 4E-BP1, S6K1 (36) is phosphorylated upon insulin stimulation (21). Activation of S6K1 is associated with alterations in global rates of protein synthesis, as well as enhanced translation of mRNAs containing terminal oligopyrimidine (TOP) sequences (28), which encode for many of the ribosomal proteins and other proteins involved in mRNA translation. Finally, following insulin stimulation, the ERK1/2 pathway is also activated (8, 21), leading to a dephosphorylation of eIF4E in skeletal muscle (21, 45). The ERK1/2 pathway is inhibited by the compound PD-98059, which reduces the activation of the ERK1/2 protein kinase, MEK1, and subsequent insulin-stimulated protein synthesis in myoblasts (21).

The purpose of the study described herein was to examine the action of TNF-{alpha} on insulin-stimulated protein synthesis and translation initiation in cultures of mouse (C2C12) myotubes and to gain insight into the interaction of these two hormones when they are acting in concert devoid of extraneous hormonal effects. The results from this study demonstrate that TNF-{alpha} abrogates the stimulatory effect of insulin on protein synthesis in C2C12 myotubes. However, TNF-{alpha} does not alter the insulin-induced phosphorylation of the mTOR substrates S6K1 and 4E-BP1 or the association of eIF4E with 4E-BP1. Instead, the attenuation of insulin-stimulated protein synthesis by TNF-{alpha} appears to be due to a reduction in the association of eIF4G with eIF4E through a MEK1-sensitive pathway.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Bovine insulin was purchased from Sigma (St. Louis, MO) and recombinant murine tumor necrosis factor-{alpha} (TNF-{alpha}) from R & D Systems. Polyvinylidene difluoride (PVDF) membranes were purchased from Pall Life Sciences. [35S]Easytag express protein labeling mix was purchased from PerkinElmer. Anti-eIF4E, anti-phospho-Ser209 eIF4E, anti-phospho-Ser1108 eIF4G, and anti-phospho-Thr202/Tyr204 ERK1/2 antibodies were purchased from Cell Signaling Technology. S6K1 antibodies were purchased from Santa Cruz Biotechnology. Anti-4E-BP1 was purchased from Bethyl Laboratories. The eIF4G antibody was produced in the authors' laboratory (22). Enhanced chemiluminescence (ECL) detection kits were purchased from Amersham Biosciences, and the donkey anti-rabbit and sheep anti-mouse horseradish peroxidase-conjugated IgG were purchased from Bethyl Laboratories.

Cell culture. C2C12 myoblasts (American Type Culture Collection) were seeded in 100-mm culture dishes in DMEM, supplemented with 10% fetal calf serum (Atlas Biologicals, Fort Collins, CO) and 1% penicillin and streptomycin. Cells were grown to ~95% confluence and then induced to differentiate into myotubes by replacement of DMEM, devoid of serum and antibiotics, with ITS-media supplement (Sigma) for three days. Before the start of the experiments, the myotubes were maintained in serum and antibiotic-free DMEM for 1 h. Insulin (20 nM), TNF-{alpha} (20 ng/ml), PD-98059 (25 µM), or dominant negative (dn)MEK1 adenovirus (50 pfu), when present, were added to the medium for the time indicated in the figure legends. For the last 5 min of treatment, 25 µl of [35S]Easytag Express protein labeling mix (14 mCi/ml) were added to the cell culture medium to allow for measurement of protein synthesis. The myotubes were then harvested by scraping in a buffer containing 20 mM HEPES (pH 7.4), 2 mM EGTA, 50 mM NaF, 100 mM KCl, 0.2 mM EDTA, 50 mM {beta}-glycerophosphate, 1% Triton X-100, 1% deoxycholate, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM benzamidine, 1 mM DTT, and 0.5 mM sodium vanadate. The resulting cell homogenate was mixed on a platform rocker for 30 min at 4°C and then clarified by a 1,000-g centrifugation (4°C). Aliquots of the supernatant were used for protein synthesis measurements, Western blot analysis, and eIF4E immunoprecipitation or stored at –80°C until analyzed.

Replication-deficient dnMEK1 adenoviral infections. dnMEK1 (Ser222 to Ala, dnMEK1) and empty vector adenoviruses (a kind gift from Dr. Jeffery Molkentin, Cincinnati Children's Hospital Medical Center, Cincinnati, OH), were plaque purified, expanded, and titered by detection of visible plaques in HEK293 monolayer cells by use of the agarose gel overlay method, as previously described (34, 37). Adenoviral infections were performed on differentiated myotubes following a rinse in phosphate-buffered saline. The adenovirus (50 pfu) was layered onto the myotube cultures for 2 h in serum-free DMEM and then replaced with 2% fetal calf serum containing DMEM for 22 h. Before the start of the experiments, the myotubes were maintained in serum- and antibiotic-free DMEM for 1 h. The cultures were then treated with insulin and/or TNF-{alpha} as described above under Cell culture.

Measurement of protein synthesis. Protein synthesis was determined by the incorporation of [35S]methionine and [35S]cysteine into protein during the last 5 min of treatment, as described previously (21).

Analysis of Western blots. Protein immunoblots were visualized via ECL, as described previously (23), and then quantified by measuring the luminescent signal with a GeneGnome Bio-Imaging System (SynGene).

Quantitation of phosphorylated and unphosphorylated ERK1/2, eIF4E, and eIF4G. An aliquot of the 1,000-g supernatant was combined with an equal volume of 5x SDS sample buffer and then resolved by electrophoresis on a 12.5% (ERK1/2), a 15% (eIF4E), or a 7.5% (eIF4G) polyacrylamide gel. The proteins were transferred to PVDF membranes, which were then incubated with anti-phosphopeptide antibodies directed against Thr202/Tyr204 for ERK1/2, Ser209 for eIF4E, or Ser1108 for eIF4G. The immunoblots were developed and analyzed as described above. The blots were then stripped and reprobed with antibodies that recognize ERK1/2, eIF4E, or eIF4G independently of phosphorylation state.

Analysis of S6K1 and 4E-BP1 phosphorylation. An aliquot of the cell homogenate was combined with an equal volume of 5x SDS sample buffer and then resolved by electrophoresis on a 7.5% (S6K1) or a 15% (4E-BP1) polyacrylamide gel. The proteins were transferred to PVDF membranes, which were then incubated with anti-S6K1 or anti-4E-BP1 polyclonal antibodies. The blots were visualized by ECL. Typically, upon phosphorylation, S6K1 and 4E-BP1 resolve into multiple forms following electrophoresis on SDS-polyacrylamide gels. It is generally accepted that the electrophoretic mobility of S6K1 and 4E-BP1 is inversely proportional to the degree of their phosphorylation, whereby the fastest migrating, hypophosphorylated forms of the proteins are designated the {alpha}-form. The relative phosphorylation of S6K1 was estimated by dividing the proportion of the protein present in hyperphosphorylated forms (i.e., any form migrating slower than the hypophosphorylated {alpha}-form) by the total amount of the protein. 4E-BP1 phosphorylation was estimated as the proportion present in the hyperphosphorylated {gamma}-form relative to the total amount of the protein.

Quantitation of 4E-BP1·eIF4E and eIF4G·eIF4E complexes. The association of 4E-BP1 or eIF4G with eIF4E was determined by the use of previously described methodology (21). Briefly, eIF4E was immunoprecipitated from the supernatant fraction by using a monoclonal anti-eIF4E antibody. The immune complexes were isolated with a goat anti-mouse BioMag IgG (PerSeptive Diagnostics) bead slurry. The beads were blocked with 0.1% nonfat dry milk in buffer A [50 mM Tris·HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 0.1% {beta}-mercaptoethanol, 0.5% Triton X-100, 50 mM NaF, 50 nM {beta}-glycerophosphate, 0.1 mM PMSF, 1 mM benzamidine, and 0.5 mM sodium vanadate] and then incubated with the sample for 1 h at 4°C. The beads were collected using a magnetic stand, washed twice with buffer A, and washed once in buffer B (containing 500 mM instead of150 mM NaCl). The precipitates were eluted in 1x SDS sample buffer and then boiled for 5 min. The beads were pelleted by centrifugation and the supernatants collected and subjected to SDS-PAGE. The proteins were transferred to PVDF membranes, which were then incubated in anti-4E-BP1 antibody, anti-eIF4G antibody, or anti-eIF4E antibody overnight at 4°C. The blots were visualized by ECL, and then the ratios of 4E-BP1 to eIF4E or eIF4G to eIF4E were calculated.

Statistics. Data are expressed as means ± SE. All comparisons were made vs. control conditions of the respective treatment condition and time for the experiments (two-tailed t-test analysis; Prism v. 3.0, GraphPad Software). Comparisons for the insulin, TNF, PD-98059, dnMEK1, or a combination of the treatments were made using a repeated-measures ANOVA design vs. control (Dunnet's post hoc analysis; Prism v. 3.0). The significance level was set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Before a comparison was undertaken of the combined actions of insulin and TNF-{alpha} in cultures of C2C12 myotubes, preliminary studies were performed to establish the time course of changes in end points relevant to translation initiation and/or growth for the effects of each agent administered alone. The preliminary studies demonstrated that insulin treatment for 30 min resulted in significant changes in eIF4G and eIF4E phosphorylation that persisted for 70 min (Fig. 1, A and C), whereas ERK1/2 phosphorylation was increased within 10 min and remained elevated throughout the 70-min time course (Fig. 1B). These changes were associated with an insulin-induced stimulation of protein synthesis observed as early as the 30-min time point (Fig. 2 and other data not shown). Unlike insulin stimulation, TNF-{alpha} treatment resulted in no change in the phosphorylation of eIF4G and only transient increases in ERK1/2 and eIF4E phosphorylation at the 10-min time point (Fig. 1, D–F). Moreover, the cytokine did not affect protein synthesis at any of the time points (Fig. 2 and data not shown). On the basis of the preliminary results and previous skeletal muscle culture experiments (21), we chose to treat the myotubes with TNF-{alpha} for 30 min prior to 40 min of insulin addition, because most of the effects of TNF-{alpha} and insulin had taken place by 30 min and between 30 and 70 min, respectively.



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  		Fig. 1. Effects of insulin (INS) and tumor necrosis factor (TNF)-{alpha} on eukaryotic initiation factor (eIF)4G, extracellular signal-regulated kinase (ERK)1/2, and eIF4E phosphorylation in C2C12 myotubes. Myotubes were incubated in serum-free medium for 1 h before experiments, followed by 10-, 30-, or 70-min treatment with insulin (20 nM; A–C) or TNF-{alpha} (20 ng/ml; D–F). Cells were harvested in lysis buffer containing protease and phosphatase inhibitors. Control cells were harvested at each time point. Homogenate was assessed for phosphorylation status of eIF4G, ERK1/2, or eIF4E with an antibody specific for the Ser1108, Thr202/Tyr204, or Ser209 site, respectively, by immunoblot analysis and then reprobed for the total amount of the respective protein. Representative Western blots are shown (C, control). Ratio of phosphorylated to total form of the protein was calculated, and results were expressed as %control (addition of PBS) for the respective time point and represent means ± SE for 3 experiments. In each experiment, 3 cultures were individually analyzed. *P < 0.05 vs. control conditions.

 


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  		Fig. 2. Effects of INS and TNF-{alpha} on [35S]methionine incorporation into C2C12 myotubes. Myotubes were incubated in serum-free medium for 1 h before experiments, followed by 40-min treatment with INS (20 nM), 70-min treatment with TNF-{alpha} (20 ng/ml), or a combination of both hormones (30-min pretreatment with TNF-{alpha} followed by 40-min treatment with INS + TNF-{alpha}). Myotubes received 25 µl of [35S]Easytag Express 5 min before the end of the treatment period and then were harvested in lysis buffer containing protease and phosphatase inhibitors. Homogenate was analyzed for incorporation of radiolabel into cellular protein, as described in EXPERIMENTAL PROCEDURES. Results are expressed as %control (addition of PBS) and represent means ± SE for 3 experiments. In each experiment, 3 cultures were individually analyzed. *P < 0.05 vs. control conditions.

 
In support of the results presented above, after an acute 40-min exposure to insulin, [35S]methionine/cysteine incorporation into protein was increased to 146% of the control value (Fig. 2), indicating a significant stimulation of protein synthesis. In contrast, addition of TNF-{alpha} to the medium for 70 min did not have an effect on the incorporation of radiolabel into protein (Fig. 2), signifying no change in protein synthesis. However, in contrast to insulin treatment alone, myotubes pretreated with TNF-{alpha} for 30 min, followed by 40 min of insulin treatment, did not manifest a stimulation of protein synthesis (Fig. 2). Thus an acute exposure to TNF-{alpha} abrogated the stimulatory effect that insulin had on protein synthesis in myotubes.

Upregulation of the mTOR signaling pathway is generally associated with conditions of increased growth and/or protein synthesis (21, 22). Thus, if TNF-{alpha} repressed the stimulatory effects of insulin on mTOR signaling, this might explain the aforementioned protein synthesis results. As an index of changes in signaling through mTOR, phosphorylation of the mTOR substrates S6K1 and 4E-BP1 was assessed by gel shift analysis following insulin stimulation. It was found that the electrophoretic mobility of S6K1 and 4E-BP1 was increased 18 and 54% compared with controls (Fig. 3, A and B), respectively. In contrast, TNF-{alpha} alone did not alter the phosphorylation status of either protein. Surprisingly, when administered in combination with insulin, TNF-{alpha} did not suppress the insulin-stimulated phosphorylation of S6K1 or 4E-BP1, suggesting that the repressive effect of TNF-{alpha} on insulin-stimulated protein synthesis was independent of mTOR-signaling targets. Similarly, the rapamycin-sensitive phosphorylation site on eIF4G, Ser1108, showed the same pattern (Fig. 3C) with insulin and/or TNF-{alpha} treatment.



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  		Fig. 3. Effects of INS and TNF-{alpha} on hyperphosphorylation of ribosomal protein S6 kinase (S6K1) and eIF4E-binding protein (4E-BP1) and phosphorylation of eIF4G in C2C12 myotubes. Myotubes were incubated in serum-free medium for 1 h and then treated with INS and/or TNF-{alpha} and harvested as described in the legend to Fig. 2. Homogenate was assessed for phosphorylation status of S6K1 (A) and 4E-BP1 (B) by immunoblot analysis. Hyperphosphorylation of S6K1 was calculated as amount of protein present in the {beta}-, {gamma}-, and {delta}-forms vs. total. Similarly, hyperphosphorylation of 4E-BP1 was calculated as amount of protein present in the {gamma}-form vs. total. Representative Western blots are shown (C, control; I, INS; T, TNF; I+T, INS + TNF). Results are expressed as %control (addition of PBS), and represent means ± SE for 3 experiments. In each experiment, 3 cultures were individually analyzed. *P < 0.05 vs. control conditions.

 
Further support for an S6K1- and 4E-BP1-independent effect of TNF-{alpha} on insulin-stimulated protein synthesis was provided by an analysis of the association of 4E-BP1 with eIF4E. As stated above, hyperphosphorylation of 4E-BP1 augments its release from eIF4E, increasing the availability of eIF4E to bind to eIF4G for eIF4F complex assembly. Results presented in Fig. 3B show an insulin-induced increase of 4E-BP1 in the {gamma}-form, suggesting that the amount of the protein bound to eIF4E should be reduced. Immunoprecipitation of eIF4E confirmed this assumption, revealing a decreased association of 4E-BP1 with eIF4E to 75% of the control value following insulin stimulation (Fig. 4A). TNF-{alpha} alone did not have any effect on the association of 4E-BP1 with eIF4E (vs. control; Fig. 4A). Moreover, in combination with insulin, TNF-{alpha} did not prevent a decline in the insulin-induced dissociation of 4E-BP1 from eIF4E, again confirming that the repressive effect of TNF-{alpha} on insulin-stimulated protein synthesis was independent of mTOR signaling.



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  		Fig. 4. Effects of INS and TNF-{alpha} on 4E-BP1 and eIF4G association with eIF4E in C2C12 myotubes. Myotubes were incubated in serum-free medium for 1 h and then treated with INS and/or TNF-{alpha} and harvested as described in the legend to Fig. 2. Homogenate was immunoprecipitated with monoclonal eIF4E antibody and immunoprecipitates were assessed for total amount of 4E-BP1 (A), eIF4G (B), and eIF4E in the immunoprecipitate by immunoblot analysis, and a ratio of 4E-BP1 or eIF4G to eIF4E was calculated. Representative Western blots are shown. Results are expressed as %control (addition of PBS) and represent means ± SE for 3 experiments. In each experiment, 3 cultures were individually analyzed. *P < 0.05 vs. control conditions.

 
When eIF4E availability increases, there is typically a concomitant enhancement of eIF4F assembly (27, 41). In the present experiments, insulin treatment resulted in an increased amount of eIF4G associated with eIF4E to 153% of the control value (Fig. 4B). Although treatment with TNF-{alpha} alone did not alter the association of the two initiation factors, pretreatment of the myotubes with TNF-{alpha} attenuated the increased association of eIF4G with eIF4E caused by insulin. This finding signifies that TNF-{alpha}'s inhibitory effects on insulin-stimulated protein synthesis may be due to inhibition of translation initiation, mediated through reductions in eIF4F assembly, independent of S6K1 or 4E-BP1.

Another signaling pathway sensitive to the stimulatory effects of insulin is the ERK1/2 pathway, which leads to activation of transcription factors and the translational machinery and has potent effects on skeletal muscle growth (10, 19). In the present experiments, insulin treatment enhanced ERK1/2 phosphorylation to 142% of the control values (Fig. 5), whereas treatment with TNF-{alpha} alone had no significant effect at this time point, in agreement with the data shown in Fig. 1E. TNF-{alpha} also did not attenuate the insulin-stimulated phosphorylation of ERK1/2. Downstream of ERK1/2, phosphorylation of eIF4E may be another point of control in translation initiation. Previous studies have reported that TNF-{alpha} and insulin conversely affect eIF4E phosphorylation (21, 29); thus an inverse change in eIF4E phosphorylation could be hypothesized when the two hormones are administered in combination. Rather, insulin alone or in combination with TNF-{alpha} elicited a reduction in eIF4E phosphorylation to ~50% of the control value (Fig. 6). Thus it appears that alterations in ERK1/2 or eIF4E phosphorylation do not explain the attenuating effect of TNF-{alpha} on insulin-stimulated protein synthesis.



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  		Fig. 5. Effects of INS and TNF-{alpha} on ERK1/2 Thr202/Tyr204 phosphorylation in C2C12 myotubes. Myotubes were incubated in serum-free medium for 1 h and then treated with INS and/or TNF-{alpha} and harvested as described in the legend to Fig. 2. Homogenate was assessed for phosphorylation status of ERK1/2 with antibody specific for Thr202/Tyr204 sites by immunoblot analysis and reprobed for total ERK1/2. Ratio of Thr202/Tyr204 phosphorylation to total ERK1/2 was calculated. Representative Western blots are shown. Results are expressed as %control (addition of PBS) and represent means ± SE for 3 experiments. In each experiment, 3 cultures were individually analyzed. *P < 0.05 vs. control conditions.

 


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  		Fig. 6. Effects of INS and TNF-{alpha} on eIF4E Ser209 phosphorylation in C2C12 myotubes. Myotubes were incubated in serum-free medium for 1 h and then treated with INS and/or TNF-{alpha} and harvested as described in the legend to Fig. 2. Homogenate was assessed for phosphorylation status of eIF4E with antibody specific for Ser209 site by immunoblot analysis and reprobed for total eIF4E. Ratio of Ser209 phosphorylation to total eIF4E was calculated. Representative Western blots are shown. Results are expressed as %control (addition of PBS) and represent means ± SE for 3 experiments. In each experiment, 3 cultures were individually analyzed. *P < 0.05 vs. control conditions.

 
Given the changes that were observed in ERK1/2 and eIF4E phosphorylation during the TNF-{alpha} time course (Fig. 1, E and F), the possibility existed that the transient nature of these events contributed to the attenuation of insulin-stimulated protein synthesis. Therefore, the compound PD-98059, which inhibits MEK1, was employed to effectively reduce the phosphorylation of ERK1/2 as well as its downstream substrate eIF4E. First, to confirm the effectiveness of the compound on ERK1/2 phosphorylation, Western blot analysis was performed for specific phosphorylation sites on ERK1/2 (Fig. 7). The analysis showed a reduction in Thr202/Tyr204 phosphorylation on ERK1/2 when PD-98059 was present, thus establishing the efficacy of the compound. Furthermore, when the myotubes were pretreated with the MEK1 inhibitor prior to TNF-{alpha} and insulin addition (Fig. 8), the insulin-induced stimulation of [35S]methionine/cysteine incorporation was not observed. Interestingly, pretreatment with PD-98059 attenuated the repressive effect of TNF-{alpha} on insulin-stimulated protein synthesis. These data were further supported by the infection of the myotube cultures with an adenovirus expressing a dominant negative variant of MEK1 (dnMEK1). Expression of dnMEK1 in C2C12 myotubes attenuated the repressive effects of TNF-{alpha} on insulin-stimulated [35S]methionine/cysteine incorporation (Fig. 9). When the myotubes were treated with a control adenovirus, there was no effect on [35S]methionine/cysteine incorporation control, insulin, or insulin- plus TNF-treated cultures (unpublished findings). Similarly, PD-98059 prevented the attenuation of insulin-induced eIF4G binding to eIF4E caused by TNF-{alpha} (Fig. 10), although PD-98059 did not prevent insulin-induced eIF4G·eIF4E association in the absence of the cytokine. However, PD-98059 did not prevent the insulin-induced phosphorylation of eIF4G regardless of the presence of TNF-{alpha} (Fig. 11).



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  		Fig. 7. Effects of INS, TNF-{alpha}, or PD-98059 (PD) on ERK1/2 Thr202/Tyr204 phosphorylation in C2C12 myotubes. Myotubes were placed in serum-free medium for 1 h before experiments, followed by treatment with INS (20 nM) for 40 min, TNF (20 ng/ml) for 70 min, PD (25 µM) for 85 min, or a combination of the 3 (where PD was present throughout, TNF was present for the last 70 min, and INS during the final 40 min). Cells were harvested in lysis buffer containing protease and phosphatase inhibitors. Homogenate was assessed for phosphorylation status of ERK1/2 with antibody specific for Thr202/Tyr204 site by immunoblot analysis and reprobed for total ERK1/2. Ratio of Thr202/Tyr204 phosphorylation to total ERK1/2 was calculated. Representative Western blots are shown. Results are expressed as %control (addition of PBS or DMSO) and represent means ± SE for 3 experiments. In each experiment, 3 cultures were individually analyzed. *P < 0.05 vs. control conditions.

 


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  		Fig. 8. Effects of INS, TNF-{alpha}, or PD on [35S]methionine incorporation into C2C12 myotubes. Myotubes were incubated in serum-free medium for 1 h and then treated with INS, TNF, and/or PD, as described in the legend to Fig. 7. Myotubes received 25 µl of [35S]Easytag Express 5 min before end of treatment and then were harvested in lysis buffer containing protease and phosphatase inhibitors. Homogenate was assessed for incorporation of radiolabel into cellular protein, as described in EXPERIMENTAL PROCEDURES. Results are expressed as %control (addition of PBS or DMSO) and represent means ± SE for 3 experiments. In each experiment, 3 cultures were individually analyzed. *P < 0.05 vs. control conditions.

 


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  		Fig. 9. Effects of INS, TNF-{alpha}, or dominant negative (dn)MEK1 adenovirus on [35S]methionine incorporation into C2C12 myotubes. Myotubes were incubated in serum-free medium for 1 h and then treated with INS (20 nM), TNF (20 ng/ml), and/or dnMEK1 adenovirus (50 pfu), as described in EXPERIMENTAL PROCEDURES. Myotubes received 25 µl of [35S]Easytag Express 5 min before end of the treatment and then were harvested in lysis buffer containing protease and phosphatase inhibitors. Homogenate was assessed for incorporation of radiolabel into cellular protein, as described in EXPERIMENTAL PROCEDURES. Results are expressed as %control (addition of PBS) and represent means ± SE for 3 experiments. In each experiment, 3 cultures were individually analyzed. *P < 0.05 vs. control conditions.

 


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  		Fig. 10. Effects of INS, TNF-{alpha}, or PD on eIF4G association with eIF4E in C2C12 myotubes. Myotubes were incubated in serum-free medium for 1 h and then treated with INS, TNF-{alpha}, and/or PD and harvested as described in the legend to Fig. 7. Homogenate was immunoprecipitated with monoclonal eIF4E antibody and immunoprecipitates were assessed for total amount of eIF4G and eIF4E in the immunoprecipitate by immunoblot analysis, and a ratio of eIF4G to eIF4E was calculated. Representative Western blots are shown. Results are expressed as %control (addition of PBS and DMSO) and represent means ± SE for 3 experiments. In each experiment, 3 cultures were individually analyzed. *P < 0.05 vs. control conditions.

 


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  		Fig. 11. Effects of INS, TNF-{alpha}, or PD on eIF4G Ser1108 phosphorylation in C2C12 myotubes. Myotubes were incubated in serum-free medium for 1 h and then treated with INS, TNF-{alpha}, and/or PD and harvested as described in the legend to Fig. 7. Homogenate was assessed for phosphorylation status of eIF4G with antibody specific for Ser1108 site by immunoblot analysis and reprobed for total eIF4G. Ratio of Ser1108 phosphorylation to total eIF4G was calculated. Representative Western blots are shown. Results are expressed as %control (addition of PBS or DMSO) and represent means ± SE for 3 experiments. In each experiment, 3 cultures were individually analyzed. *P < 0.05 vs. control conditions.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the experiments presented herein, we have established a direct effect of TNF-{alpha} to reduce insulin-stimulated protein synthesis and activation of biomarkers of translation initiation in cultures of C2C12 myotubes. More specifically, we have found that the insulin-induced stimulation of eIF4G association with eIF4E was prevented by pretreatment with TNF-{alpha} via a MEK1-sensitive mechanism. This suggests that TNF-{alpha} abrogates insulin-stimulated protein synthesis through an ERK1/2-dependent signaling pathway that regulates the binding of capped mRNA to the 40S preinitiation complex.

Insulin stimulates (21, 22), whereas sepsis, TNF-{alpha}, or lipopolysaccharide treatment inhibits (5, 23, 25, 26, 46), translation initiation and/or protein synthesis in skeletal muscle. Even when septic rats are administered insulin in an attempt to attenuate the inhibitory affects of the bacteria, skeletal muscle protein synthesis is still repressed (45). Likewise, several studies in cultures of myotubes (2, 8, 14, 15) demonstrate inhibitory effects of TNF-{alpha} on insulin- or IGF-stimulated glucose uptake or protein synthesis. In the present study, acute TNF-{alpha} treatment did not affect protein synthesis (Fig. 2), although, when administered in combination with insulin, the cytokine attenuated the insulin response.

Several signaling pathways are activated by insulin and TNF-{alpha}, although the most relevant to translation initiation and protein synthesis are the mTOR and the MEK/ERK pathways. Two rapamycin-sensitive substrates of mTOR, S6K1 and 4E-BP1 (4), are hyperphosphorylated under conditions of enhanced growth. Thus, by inhibiting S6K1 and/or 4E-BP1 phosphorylation with rapamycin, growth effects are negated (21). The present data suggest that the inhibitory effect of TNF-{alpha} on insulin-stimulated protein synthesis is independent of changes in S6K1 and 4E-BP1 phosphorylation (Fig. 3, A and B). These results are in agreement with the findings of Vary et al. (45) in insulin-treated, septic rat skeletal muscle. Thus, under conditions of acute elevations in TNF-{alpha}, decreases in insulin-stimulated protein synthesis appear to be independent of S6K1 and 4E-BP1 phosphorylation.

Another possible mechanism through which TNF-{alpha} might suppress insulin-stimulated protein synthesis by TNF-{alpha} could involve ERK1/2 and/or eIF4E. Previous studies (26, 29, 44) have established that TNF-{alpha} increases the phosphorylation of both ERK1/2 and eIF4E, whereas insulin increases ERK1/2 but reduces eIF4E phosphorylation in skeletal muscle (21). Our studies showed that the hormones have the same effects in C2C12 myotubes as in skeletal muscle; i.e., TNF-{alpha} transiently increased ERK1/2, consistent with others (11), and eIF4E phosphorylation, whereas insulin increased ERK1/2 but decreased eIF4E phosphorylation. Surprisingly, we did not observe any changes in phosphorylation of p38 MAPK or SAPK/JNK with insulin or TNF-{alpha} treatment (unpublished findings). Phosphorylation of eIF4E on Ser209 (18) has been associated with increased and decreased cap binding, as well as both increased and decreased protein synthesis and growth (7, 24, 30, 39, 40). Thus the exact function of eIF4E phosphorylation remains unclear. Here, we show a stimulation of ERK1/2 phosphorylation, whereas the downstream protein eIF4E was dephosphorylated after 40 min of insulin stimulation, regardless of TNF-{alpha} addition (Fig. 6), paralleling results of insulin-treated skeletal muscle from septic rats (for eIF4E only) (45). The lack of increase or preservation of eIF4E phosphorylation by TNF-{alpha}/insulin was surprising given the activation of the upstream kinase ERK1/2 under the same conditions, which suggests that alternative processes are responsible for the TNF-{alpha}-induced attenuation of insulin-stimulated protein synthesis.

Changes in assembly of the eIF4F complex through alterations in 4E-BP1 and/or eIF4G association with eIF4E, are mechanisms of regulating translation initiation. Insulin stimulates the phosphorylation of 4E-BP1 and promotes its dissociation from eIF4E (16), allowing eIF4G to associate with eIF4E and form the eIF4F complex (27). Sepsis, TNF-{alpha}, and endotoxin (23, 26, 45) are associated with increased amounts of 4E-BP1 complexed with eIF4E and decreased eIF4F complex formation. However, when septic rodents are treated with insulin (45), the association of 4E-BP1 with eIF4E is reduced to levels similar to that of control animals treated with insulin. Similarly, in the present study, when the myotubes were treated with insulin, regardless of TNF-{alpha} addition, the relative amount of 4E-BP1 found in the eIF4E immunoprecipitates was less compared with control myotube cultures (Fig. 4A), making more eIF4E available for eIF4F complex assembly. However, rather than enhancing eIF4G binding to eIF4E, TNF-{alpha} pretreatment inhibited the insulin-stimulated assembly of the eIF4F complex by reducing the association of eIF4G with eIF4E (Fig. 4B), independent of 4E-BP1 association with eIF4E phosphorylation of eIF4E or eIF4G. The lack of effect of TNF-{alpha} alone on either association of 4E-BP1 or eIF4G with eIF4E provides evidence of a complex interaction between the insulin and TNF-{alpha} signaling pathways.

Studies reporting alterations in eIF4F complex assembly or protein synthesis, independent of S6K1 and 4E-BP1 phosphorylation and the 4E-BP1 association with eIF4E, are limited. The work of Vary et al. (45) showed that the inhibitory effects of sepsis on insulin-stimulated protein synthesis were related to reductions in eIF4F assembly, independent of S6K1 and 4E-BP1 phosphorylation and 4E-BP1 association with eIF4E. Similarly, diabetic rats displayed an increased protein synthetic response when treated with leucine, independent of changes in S6K1 and 4E-BP1 phosphorylation or eIF4F assembly (1). A few studies (12, 13) have shown a decrease in eIF4G association with eIF4E, independent of a change in the 4E-BP1-associated eIF4E, in Xenopus kidney cells treated with serum or anisomycin in combination with rapamycin or SB-203580 (a p38 MAPK inhibitor). Likewise, recent data from Naegele and Morley (33) propose that an inhibition of MEK1 vs. MEK1/2 results in differing actions on translation initiation and protein synthesis. Collectively, the current and aforementioned studies suggest multiple means of regulating eIF4F assembly and protein synthesis by rapamycin-sensitive and -insensitive processes.

Consistent with the above-mentioned studies, we show that the TNF-{alpha}-induced inhibition of insulin-stimulated eIF4G-associated eIF4E, and subsequent protein synthesis, act through a MEK1-sensitive mechanism (Figs. 8–10). Although not fully understood, similar results for L6 myoblasts have been reported following insulin/PD-98059 treatment (21). Despite the uncertainty, by inhibiting the transient stimulatory effects of TNF-{alpha} on ERK1/2 and eIF4E phosphorylation with the MEK1 inhibitor and dnMEK1, we have demonstrated a MEK1-sensitive mechanism for eIF4F complex assembly. This effect was independent of a change in eIF4G phosphorylation (Fig. 11), supporting the findings that Ser1108 phosphorylation on eIF4G is rapamycin sensitive and PD-98059 insensitive (38). MEK1 inhibition by PD-98059 has also been shown to attenuate TNF-{alpha} effects on adipocyte lipolysis (49) and IRS-1 tyrosine and serine phosphorylation (9), lending support to the current study.

In summary, the results of the present investigation indicate that TNF-{alpha} abrogates the stimulatory effect of insulin on protein synthesis in cultures of myotubes and that the changes in protein synthesis are associated with alterations in translation initiation. A likely mechanism through which TNF-{alpha} acts involves the attenuation of the stimulatory effect of insulin on eIF4F complex assembly, specifically the association of eIF4G with eIF4E. The reduction of the insulin-stimulated eIF4G association with eIF4E by TNF-{alpha} was attenuated by inhibiting the ERK1/2 pathway, suggesting that a MEK1-sensitive mechanism controls eIF4F assembly. Understanding the function of how TNF-{alpha} affects the association of eIF4G with eIF4E could contribute to further understanding of diseases and disorders involving skeletal muscle wasting associated with high circulating TNF-{alpha} levels.


   GRANTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
These experiments were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-15658 to L. S. Jefferson.


   ACKNOWLEDGMENTS
 
We thank Lynne Hugendubler for technical assistance. We also thank Dr. Douglas Bolster for input during the experiments, as well as technical assistance. Finally, we thank Dr. Jan McAllister for help with the dnMEK1 plaque purification, expansion, and titering.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. S. Jefferson, Dept. of Cellular and Molecular Physiology, H166, The Pennsylvania State Univ. College of Medicine, 500 Univ. Dr., Hershey, PA 17033 (e-mail: jjefferson{at}psu.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.


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