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Endocrinology, doi:10.1210/en.2007-1045
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Endocrinology Vol. 149, No. 4 1880-1889
Copyright © 2008 by The Endocrine Society

The Proinflammatory Cytokine Tumor Necrosis Factor-{alpha} Increases the Amount of Glucose Transporter-4 at the Surface of Muscle Cells Independently of Changes in Interleukin-6

Nerea Roher, Victor Samokhvalov, Mònica Díaz, Simon MacKenzie, Amira Klip and Josep V. Planas

Department de Fisiologia (N.R., M.D., J.V.P.), Facultat de Biologia, Universitat de Barcelona and Institut de Biomedicina de la Universitat de Barcelona (IBUB), 08028 Barcelona, Spain; Program in Cell Biology (V.S., A.K.), The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8; and Unitat de Fisiologia Animal (S.M.), Departament de Biologia Cellular, Fisiologia i d’Immunologia, Facultat de Ciencies, Universitat Autònoma de Barcelona, Bellaterra, 08035 Barcelona, Spain

Address all correspondence and requests for reprints to: Dr. Josep V. Planas, Departament de Fisiologia, Facultat de Biologia, Universitat de Barcelona and Institut de Biomedicina de la Universitat de Barcelona, 08028 Barcelona, Spain. E-mail: jplanas{at}ub.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
TNF{alpha} is a proinflammatory cytokine secreted by macrophages in response to bacterial infection. Recently new evidence has emerged suggesting that stressed or injured myocytes produce TNF{alpha} that then acts as an autocrine and/or paracrine mediator. TNF{alpha} receptors types 1 and 2 are present in skeletal muscle cells, and muscle cells can secrete, in addition to TNF{alpha}, other cytokines such as IL-1β or IL-6. Furthermore, the plasma concentration of TNF{alpha} is elevated in insulin-resistant states associated with obesity and type 2 diabetes. Here we show that TNF{alpha} increased the amount of glucose transporter (GLUT)-4 at the plasma membrane and also glucose uptake in the L6 muscle cell line stably expressing GLUT4 tagged with the c-myc epitope. Regardless of the state of differentiation of the L6 cells, TNF{alpha} did not affect the rate of proliferation or of apoptosis. The stimulatory effects of TNF{alpha} on cell surface GLUT4 and glucose uptake were blocked by nuclear factor-{kappa}B and p38MAPK pathway specific inhibitors (Bay 11-7082 and SB220025), and these two pathways were stimulated by TNF{alpha}. Furthermore, although TNF{alpha} increased IL-6 mRNA and protein expression, IL-6 did not mediate the effects of TNF{alpha} on cell surface GLUT4 levels, which also did not require de novo protein synthesis. The results indicate that TNF{alpha} can stimulate glucose uptake in L6 muscle cells by inducing GLUT4 translocation to the plasma membrane, possibly through activation of the nuclear factor-{kappa}B and p38MAPK signaling pathways and independently of the production of IL-6.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OVER THE LAST years, type 2 diabetes has increasingly been considered a chronic inflammatory state, during which the levels in plasma of cytokines such as TNF{alpha}, IL-6, or IL-1 are increased (1). In particular, TNF{alpha} levels can rise from 60 pg/ml in healthy individuals to 3–6 ng/ml in individuals with pathologies such as rheumatoid arthritis or cancer or to 100 pg/ml in patients with type 2 diabetes (2, 3). Since the initial reports on the constitutive adipocyte expression of TNF{alpha}, there is increasing evidence to suggest that skeletal muscle has the cellular machinery suitable to respond to TNF{alpha} (TNF receptors 1 and 2) (4), that the muscle itself can produce different cytokines (5, 6), and that TNF{alpha} has an important effect on skeletal muscle metabolism, mostly stimulating catabolism (2). The study of glucose transporters in both skeletal muscle and adipose tissue are important for our understanding of the type 2 diabetes etiology because they are responsible (mostly skeletal muscle because of its mass) for the peripheral glucose uptake in response to insulin. It is well known that acute insulin treatment stimulates glucose transport into myocytes and adipocytes by stimulating glucose transporter (GLUT)-4 translocation from intracellular compartments to the plasma membrane (7). Different groups have explored the effects of TNF{alpha} on insulin-stimulated glucose uptake, and controversial data have emerged. An impairment of insulin-mediated glucose uptake by TNF{alpha} has been observed in skeletal muscle and adipose cellular models (8, 9), muscle primary cell cultures (10), and TNF{alpha} infusion studies in humans (11, 12). Moreover, TNF{alpha} can block insulin action in adipocytes by altering the expression of signaling molecules and glucose transporters including GLUT4 (13). Inhibition of insulin-mediated glucose transport by TNF{alpha} has been associated to an insulin-resistant state, as demonstrated also by the protection against developing insulin resistance observed in TNF{alpha}- and TNF{alpha} receptor-deficient mice (14). The proposed mechanism for the induction of insulin resistance by TNF{alpha} involves 307Ser phosphorylation of insulin receptor substrate-1, which in turn acts as an inhibitor of insulin receptor tyrosine kinase activity and impairs downstream signaling through Akt/phosphatidylinositol 3-kinase (PI3K) (15, 16).

On the other hand, different observations have assessed that TNF{alpha} is able to stimulate basal glucose uptake up to 3-fold in L6 myotubes (17), chondrocytes (18), and 3T3 adipocytes (19). However, in isolated rat skeletal muscle, TNF{alpha} does not affect insulin mediated glucose uptake and does not activate or impair insulin signaling through insulin receptor substrate-1 (20). All these data together suggest that the effects of TNF{alpha} can be mediated by different mechanisms depending on the differentiation state, the cellular type, the magnitude, and time of stimuli or on insulin mediation. In fact, TNF{alpha} is a cytokine with multiple faces that can act as a proliferative or pro- or antiapoptotic as well as a pro- or anti-inflammatory factor stimulating different pathways including nuclear factor-{kappa}B (NF{kappa}B), c-jun terminal kinase, or p38MAPK (21), but the complex mechanisms by which TNF{alpha} mediates these diverse cell responses are not fully understood.

The aims of this study were, first, to perform a detailed study of the effects of TNF{alpha} on glucose uptake and GLUT4 translocation to the plasma membrane in rat skeletal muscle cells; second, to establish which cellular pathways are activated by TNF{alpha} in L6 cells; and lastly, to determine whether these pathways are involved in GLUT4 translocation to plasma membrane. Our results indicate that TNF{alpha}, through activation of the NF{kappa}B and p38MAPK signaling pathways, is able to increase the levels of GLUT4 at the plasma membrane and, as a result, causes an increase in glucose uptake in L6 cells. Furthermore, TNF{alpha} stimulates the mRNA expression and secretion of IL-6 by L6 cells but IL-6 does not appear to be involved in mediating the effects of TNF{alpha} on GLUT4 cell surface levels.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
{alpha}-MEM and fetal bovine serum (FBS) was from Invitrogen (Prat del Llobregat, Spain). Blasticidin S, SB220025, and Bay 11-7082 were from Calbiochem (La Jolla, CA). All other tissue culture reagents, propidium iodide-staining solution, 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT), and human recombinant TNF{alpha} were purchased from Sigma (Tres Cantos, Madrid, Spain). Recombinant IL-6 was from R&D Systems (Minneapolis, MN). Plasticware for cell culture was from BD Biosciences (Madrid, Spain). Human insulin (Humulin R) was from Lilly (Alcobendas, Madrid, Spain). Polyclonal anti-myc (sc-789), p38MAPK (sc-535), antiphospho-Akt, and NF{kappa}B inhibitor (I{kappa}B; sc-371) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antiphospho-p38MAPK (3D7) Anti-phospho-signal transducer and activator of transcription (STAT)-3 and anti-STAT3 were from Cell Signaling (Barcelona, Spain). Anti-IL-6 antibody was from R&D Systems. Antimyogenin antibody and antiratGLUT4 antibody were kindly donated by Dr. Marta Camps (Universitat de Barcelona). Horseradish peroxidase (HRP)-conjugated goat antirabbit IgG was from Jackson ImmunoResearch (Soham, UK). 2-Deoxy-D-[2,6-3H]glucose (2-DG) was purchased from Amersham Biosciences (Barcelona, Spain). Bio-Rad protein assay and IgG were obtained from Bio-Rad (Prat del Llobregat, Spain).

L6 cell culture
L6 myoblasts stably expressing rat-GLUT4myc (22, 23, 24) were used in the present study. L6-GLUT4myc cells were maintained in {alpha}-MEM supplemented with 10% FBS, blasticidin S (2 µg/ml), and 1% antibiotic-antimycotic solution (10,000 U/ml penicillin G, 10 mg/ml streptomycin, 25 µg/ml amphotericin B) in a humidified atmosphere of air and 5% CO2 at 37 C. For experiments with myoblasts only, L6 cells were seeded in medium containing 10% FBS and used at confluence 2 d after seeding. L6 cells were differentiated in medium supplemented with 2% FBS into myotubes within 6–7 d after seeding.

SB220025 and Bay 11-7082 inhibitors were administered in dimethylsulfoxide (DMSO), and the maximum concentration of the vehicle did not exceed 0.05% (vol/vol). This concentration of vehicle was without effect on any of the parameters measured (data not shown). Inhibitors were added to the culture medium 60 min before TNF{alpha} stimulation and maintained throughout the 18 h treatment with TNF{alpha}.

Determination of the proportion of GLUT4myc at the cell surface
L6 cells were stimulated with TNF{alpha} in serum-deprived medium ({alpha}-MEM 0.1% FBS) for 18 h and subsequently stimulated, or not, with 100 nM insulin for 20 min. Medium was then removed by washing (three times) in ice-cold PBS (154 mM NaCl, 5.6 mM Na2HPO4, 1.1 mM KH2PO4) supplemented with 1 mM CaCl2 and 1 mM MgCl2 at 4 C [PBS+ (pH 7.4)]. To label cell surface GLUT4myc in intact L6 myoblasts, cells were blocked in 5% goat serum in PBS+ for 15 min and then incubated with {alpha}-myc antibody solution (1.0 µg/ml in PBS+ with 5% goat serum) for 1 h at 4 C. After labeling, excess anti-myc antibodies were removed by extensive washing in ice-cold PBS+. Cells were then fixed in 4% paraformaldehyde in PBS+ for 30 min and quenched in 100 mM glycine in PBS+ for 10 min, all at 4 C. Cell surface GLUT4-bound anti-myc antibodies were probed by HRP-conjugated secondary antibodies followed by detection of bound HRP by o-phenylenediamide assay, as previously described (24). The fraction of GLUT4myc at the cell surface, measured in triplicate, was expressed as fold induction with respect to unstimulated cells and normalized to total protein.

Glucose uptake measurements
Determination of 2-DG uptake in L6 cells was performed as described by Huang et al. (25) with some modifications. Glucose uptake was assessed for 5 min in HEPES-buffered saline [140 mM NaCl, 20 mM HEPES, 5 mM KCl, 2.5 mM MgSO4, 1 mM CaCl2 (pH 7.4)] containing 10 µM 2-DG (0.5 µCi/ml 2-[3H]DG) at room temperature. Subsequently cells were rinsed three times with an ice-cold solution containing 0.9% NaCl and 20 mM D-glucose. To quantify the radioactivity incorporated, cells were lysed with 0.05 N NaOH and lysates were counted with scintillation liquid in a β-counter. Nonspecific uptake was carried out in the presence of cytochalasin B (50 µM) during the assay, and these values were subtracted from all other values. Glucose uptake measured in triplicate and normalized to total protein was expressed as fold induction with respect to unstimulated cells.

Cellular viability and proliferation
Myoblast viability and proliferation after TNF{alpha} treatment were assayed by propidium iodide (PI) staining and analyzed by fluorescence-activated cell sorter (FACS). Briefly, for viability assay cells were trypsinized, washed with PBS, and stained with PI in PBS for 15 min at room temperature. For proliferation assay, trypsinized cells were fixed in ethanol 70% at –20 C and stained overnight at 4 C in PBS, 0.1% Nonidet P-40. After staining, cells were incubated with RNase A (20 µg/ml) 15 min at 37 C and analyzed by FACS (6000 cells). Myotube viability was assayed with MTT. Briefly, after TNF{alpha} treatment, cells were incubated with MTT (0.5 mg/ml) for 3 h at 37 C and washed with PBS. Three hundred microliters DMSO were added to each well, and absorbance of converted dye was measured at a wavelength of 540 nm in a microplate reader (Tecan Infinite 200, Mannedorf, Switzerland). To discard that different methodological approaches could give us different viability percentages, we also measured myoblast viability by the MTT method and obtained comparable viability results to the FACS method (data not shown).

Western blot analysis and ELISA
For Western blot analysis, cells were lysed in 2.5x Laemmli buffer supplemented with NaOV3 (1 mM), sonicated (three 15-sec bursts) and centrifuged at 12,000 x g for 10 min. Supernatants were boiled for 5 min and subjected to 12% SDS-PAGE. After electrophoresis, proteins were transferred to a polyvinylidene difluoride membrane using Miniprotean III electrophoresis and blotting system (Bio-Rad). The membrane was blocked with blocking buffer (Tris-buffered saline containing 0.1% Tween 20 and 5% nonfat dry milk) for 1 h and subsequently incubated with antiphospho-p38, antiphospho-Akt, anti-I{kappa}B{alpha}, and antiphospho-STAT3 antibodies diluted 1:1000 in blocking buffer with 2.5% nonfat dry milk at 4 C overnight under continuous shaking. After three washes the membrane was incubated with HRP-conjugated secondary antibody diluted 1:10,000 for 1 h at room temperature. Immunoreactive bands were visualized by chemiluminescence, scanned, and quantified with an image analyzer (GS-700 and QuantityOne software; Bio-Rad). After visualization membranes were stripped 1 h at room temperature with stripping buffer [1% SDS, 0.2 M glycine (pH 2.5)] and incubated with p38MAPK, Akt, or anti-STAT3 antibody (1:1000) and HRP-conjugated secondary antibody diluted 1:10,000 for 1 h at room temperature.

Conditioned medium from L6-GLUT4myc cells stimulated with TNF{alpha} (10 ng/ml) was collected at various time points and frozen at –20 C until assay. Rat IL-6 was measured with a sandwich ELISA of two antibodies and streptavidin-conjugated peroxidase (Invitrogen). Undiluted conditioned medium from four independent experiments was assayed in duplicate.

RNA isolation and real-time PCR
RNA isolation from L6-GLUT4myc cells was performed with Nucleospin RNA II extraction kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions. RNA was quantified with RiboGreen RNA assay (Quant-IT; Invitrogen) and reverse transcription was carried out with SuperScript III (Invitrogen) and oligodT (Promega, Madison, WI) with 2 µg of total RNA. Primers for IL-6 and glyceraldehyde phosphate dehydrogenase (GAPDH) were: IL-6 forward, 5'-CCC CAA CTT CCA ATG CTC TCC and IL-6 reverse, 5'-TGC CGA GTA GAC CTC ATA GTG ACC; GAPDH forward, 5'-CCA TGG AGA AGG CTG GGG and GAPDH reverse, 5'-CAA AGT TGT CAT GGA TGA CC. Real-time PCRs were carried out in a 25-µl reaction with SYBR Green I (Bio-Rad) using a 1:25 dilution of the cDNAs and at 250 nM of primers. Quantitative PCR was performed using a MyiQ instrument (Bio-Rad) and quantification was done according to Pfaffl method corrected for efficiency for each primer set (26).

IL-6 immunoneutralization assay
L6 myoblasts were incubated in {alpha}-MEM 10% FBS for 3 h with anti-IL-6 antibody (100 ng/ml). After incubation cells were washed with {alpha}-MEM 0.1% FBS and incubated with 10 ng/ml TNF{alpha} plus 100 ng/ml of IL-6 antibody for 18 h. Finally, cells were stimulated, or not, with 100 nM insulin for 20 min, and determination of the proportion of GLUT4myc at the cell surface was performed as previously described. As a positive control, short-term incubations with recombinant IL-6 (1 ng/ml) were carried out for 2 h in the presence or not of 100 ng/ml of anti-IL-6 antibody. As a negative control for the antibody, 100 ng/ml of goat IgG was added to the control wells.

Statistical analysis
Values are given as mean ± SE. Analysis of statistical significance of differences in measurements between samples was done by one-way ANOVA with Newman-Keul’s posttest and Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TNF{alpha} increases the amount of GLUT4myc at the plasma membrane and does not impair the insulin-mediated increase of cell surface GLUT4myc in L6 cells
The levels of GLUT4myc present at the plasma membrane were measured in L6 myoblasts and myotubes that stably express GLUT4myc. We performed dose-response experiments with TNF{alpha} for 18 h in culture due to the lack of effects of TNF{alpha} on the amount of GLUT4myc at the cell surface after a short-time incubation (60 min) in preliminary experiments. TNF{alpha}, at doses ranging from 1 to 50 ng/ml, caused a significant increase in the levels of GLUT4myc at the plasma membrane in L6 myoblasts (1.36- to 1.4-fold). This effect was comparable in magnitude with that observed after a 20-min treatment with insulin under these conditions of prolonged preincubation with low serum (Fig. 1AGo). Similar results were obtained with L6 myotubes in which the stimulation of GLUT4myc cell surface levels due to TNF{alpha} was between 1.63- and 1.76-fold (Fig. 1BGo), again comparable with that observed with insulin. In the presence of TNF{alpha}, insulin increased the cell surface levels of GLUT4myc to a maximum of 1.75-fold in myoblasts (Fig. 1AGo) and 2.1-fold in myotubes (Fig. 1BGo), which is indicative of an additive effect of both activating inputs.


Figure 1
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  		FIG. 1. GLUT4myc at the cell surface in L6 myoblasts and myotubes in response to TNF{alpha} and insulin. The proportion of GLUT4myc at the cell surface relative to control was determined in L6 myoblasts (A) or myotubes (B) stably expressing rat GLUT4myc, and stimulated with TNF{alpha} (0, 1, 10, or 50 ng/ml) in the absence (white bars) or presence (black bars) of 100 nM insulin, as described in Materials and Methods. Results are expressed as fold stimulation above the control group, which was set to 1. Results shown are the means ± SE of five independent experiments, each performed in triplicate. **, P < 0.001, relative to the rat-GLUT4myc basal condition; #, P < 0.05, relative to the rat-GLUT4myc insulin control.

 
TNF{alpha} stimulates glucose uptake in L6-GLUT4myc cells
To assess the effects of TNF{alpha} on glucose uptake in L6 myoblasts and myotubes that stably express GLUT4myc, cells were incubated with different concentrations of TNF{alpha} for 18 h in the absence or presence of insulin for 20 min. TNF{alpha} significantly stimulated basal glucose uptake at a dose as low as 1 ng/ml in both myoblasts (Fig. 2AGo) and myotubes (Fig. 2BGo), although the response to TNF{alpha} was comparatively higher in myoblasts than myotubes (1.4- and 1.18-fold at 50 ng/ml TNF{alpha}, respectively). Addition of insulin resulted in a further stimulation of glucose uptake that again was more pronounced in L6 myoblasts (1.37-fold at 0 ng/ml TNF{alpha} and 2.16-fold at 50 ng/ml TNF{alpha}, Fig. 2AGo) than in myotubes (1.52-fold at 0 ng/ml TNF{alpha} and 1.74-fold at 50 ng/ml TNF{alpha}, Fig. 2BGo). The lower response of myotubes to TNF{alpha} did not appear to be due to higher basal glucose uptake in myotubes because myotubes had lower basal glucose uptake levels than myoblasts (2.3 vs. 7 pmol/mg·min, respectively) in accordance to previous reports (27).


Figure 2
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  		FIG. 2. Glucose uptake in GLUT4myc L6 myoblasts and myotubes in response to TNF{alpha} and insulin. In L6 myoblasts (A) and myotubes (B), 2-DG uptake was determined in response to TNF{alpha} (0, 1, 10, or 50 ng/ml) in the absence (white bars) or presence (black bars) of 100 nM insulin, as described in Materials and Methods. Results are expressed as fold stimulation above the control group, which was set to 1. Results shown are the means ± SE of five independent experiments, each performed in triplicate. *, P < 0.05, relative to the rat-GLUT4myc basal condition; #, P < 0.05, relative to the rat-GLUT4myc insulin control.

 
TNF{alpha} does not affect proliferation or viability in L6-GLUT4myc cells
To discard the possibility that increases in GLUT4 translocation in response to TNF{alpha} were due to changes in the total amount of cells, viability, and proliferation of L6-GLUT4myc myoblasts and viability of L6-GLUT4myc myotubes were assessed. No significant changes in L6 myoblast viability (Fig. 3AGo) or proliferation (Fig. 3CGo) were detected due to the TNF{alpha} treatment at any dose or time used in our experiments. The viability of L6 myoblasts was not affected by the 18-h incubation period in the presence of TNF{alpha} and remained at 95 ± 1% (Fig. 3AGo). In addition, the proliferation rate of TNF{alpha}-treated myoblasts was also not affected (90 ± 5% G0/G1 and 9 ± 0.1% G2/M; Fig. 3CGo). Similarly, the viability of L6 myotubes was not affected by TNF{alpha} at any dose and remained at approximately 100% (Fig. 3BGo).


Figure 3
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  		FIG. 3. Proliferation and viability of GLUT4myc-L6 myoblasts and viability of GLUT4myc-L6 myotubes is not affected by TNF{alpha}. Viability of myoblasts (A) and myotubes (B) was analyzed by FACS and MTT staining, respectively. Results are expressed as a percentage of viable cells. C, Myoblast proliferation was analyzed by PI staining followed by FACS analysis. The results are expressed as percentage of cells in G0/G1 and G2/M. Results shown are the means ± SE of three independent experiments, each performed in triplicate.

 
Inhibitors of the p38MAPK and NF{kappa}B pathways impair the stimulation of GLUT4myc cell surface levels by TNF{alpha} in L6 cells
The p38MAPK and NF{kappa}B pathways signaling pathways are known to transduce the effects of TNF{alpha} (21). Hence, we explored their participation in the stimulatory effects of TNF{alpha} on GLUT4myc cell surface levels in L6 cells. L6-GLUT4myc myoblasts or myotubes were preincubated with SB220035 (1 µM) or Bay 11-7082 (1 µM), specific inhibitors of the p38MAPK and NF{kappa}B pathways, respectively, or with DMSO (vehicle) for 60 min, followed by TNF{alpha} treatment (10 ng/ml) for 18 h in the presence of the inhibitors. The TNF{alpha}-induced increase in GLUT4myc cell surface levels in L6 myoblasts was inhibited by 84% with Bay-117082 and 88.5% with SB220035 (Fig. 4AGo). Similar results were obtained with L6 myotubes, namely 83.9% inhibition of the TNF{alpha}-induced increase in GLUT4myc cell surface levels with Bay 11-7082 and 80.3% with SB220035 (Fig. 4BGo). The inhibitors used were able to impair the TNF{alpha}-induced increase in GLUT4myc cell surface levels in L6 myoblasts and myotubes at doses from 1 to 5 µM of Bay 11-7082 and doses from 0.5 to 10 µM of SB220035 (data not shown). In addition, we explored the ability of these inhibitors to prevent the stimulation of glucose uptake by TNF{alpha} and indeed detected a significant inhibition of glucose uptake at 5 µM of SB220035 (83% inhibition) and at 1 µM Bay 11-7082 (86% inhibition) (data not shown).


Figure 4
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  		FIG. 4. Inhibition of GLUT4myc translocation in L6 myoblasts and myotubes by p38MAPK and NF{kappa}B inhibitors. Cells were pretreated with SB220025 or Bay 11-7082 for 60 min before TNF{alpha} stimulation (10 ng/ml for 18 h), as described in Materials and Methods, and the levels of GLUT4myc at the cell surface in myoblasts (A) and myotubes (B) were measured. Results are expressed as fold stimulation or inhibition with respect to basal levels. Results shown are the means ± SE of four independent experiments, each performed in triplicate. *, P < 0.05 and **, P < 0.001, relative to the rat-GLUT4myc insulin control.

 
TNF{alpha} activates p38MAPK and NF{kappa}B signaling in L6-GLUT4myc cells
To confirm that the p38MAPK and NF{kappa}B signaling pathways could be involved in mediating the effects of TNF{alpha}, we tested the activation of these two pathways in response to TNF{alpha} in L6 myoblasts and myotubes. Activation of p38MAPK was followed by the phosphorylation of a consensus sequence (TGY) on p38MAPK using an antiphospho-p38 antibody (28). On the other hand, NF{kappa}B activation was followed by the degradation of I{kappa}B that, in nonstimulated conditions, masks the nuclear localization signal on NF{kappa}B, keeping this transcription factor in the cytosol and therefore inactive (21). In L6-GLUT4myc myoblasts and myotubes incubated with TNF{alpha} (10 ng/ml), a time-dependent activation of NF{kappa}B and p38MAPK was detected (Fig. 5Go). In myoblasts treated with TNF{alpha}, the degradation of I{kappa}B was apparent at 15 min, reaching a maximum at 60 min and recovering its levels at 6 h after TNF{alpha} stimulation (Fig. 5AGo), indicating that NF{kappa}B activation occurred between 15 min and 6 h after incubation with TNF{alpha}. In contrast, in myotubes treated with TNF{alpha}, I{kappa}B degradation was first observed at 60 min and continued until 18 h after TNF{alpha} stimulation (Fig. 5BGo), indicating that TNF{alpha} activated NF{kappa}B after 60 min of treatment. A different time course was observed for p38MAPK phosphorylation in response to TNF{alpha} between L6 myoblasts and myotubes. Whereas L6 myoblasts had two peaks of activation in response to TNF{alpha} (at 15 min and 18 h), L6 myotubes reached the maximum level of activation at 15 min after TNF{alpha} stimulation and maintained a similar level of activation of p38MAPK until 18 h (Fig. 5Go, A and B). Interestingly, myoblasts presented a higher level of basal phosphorylation of p38MAPK than myotubes, as shown previously (29).


Figure 5
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  		FIG. 5. Time course of the activation of p38MAPK and NF{kappa}B in response to TNF{alpha} in L6 myoblasts and myotubes. Total extracts of L6 cells were obtained at indicated times after TNF{alpha} stimulation (10 ng/ml). Protein extracts were submitted to SDS-PAGE, transferred onto polyvinyl difluoride membranes and probed with different antibodies as described in Materials and Methods. A, L6 myoblasts were probed with anti-I{kappa}B{alpha}, anti-phospho-p38 (P_p38) and anti-p38MAPK antibodies. B, L6 myotubes were probed with anti-I{kappa}B{alpha}, antiphospho-p38 (P_p38), and anti-p38MAPK. C, L6 myoblasts were probed with antiphospho-Akt (P_Akt) and anti-Akt. In the right panel, L6 myoblasts were incubated in the absence or presence of 100 nM insulin for 20 min and loaded on the same gel. D, L6 myotubes were probed with antiphospho-Akt (P_Akt) and anti-Akt. Representative immunoblots of three independent experiments are shown.

 
It is well known that the TNF{alpha} and insulin signaling pathways can cross talk (30). To explore the possibility that TNF{alpha} exerted its effects on GLUT4 translocation and glucose uptake through the insulin signaling pathway, we examined the phosphorylation of Akt. TNF{alpha} did not cause activation of Akt in myoblasts (Fig. 5CGo) or myotubes (Fig. 5DGo). On the other hand, insulin (100 nM, 20 min) strongly activated Akt (Fig. 5CGo). We can also discard an effect of TNF{alpha} up-regulating GLUT4 expression because expression levels of GLUT4 (endogenous and myc tagged) tested with an anti-GLUT4 antibody did not reveal any changes in the amount of GLUT4 protein (data not shown). Moreover, there was no difference in total GLUT4myc content measured by the o-phenylenediamide assay, whereas there was the expected increase on surface GLUT4myc (data not shown).

Myoblast differentiation involves the activation of p38MAPK in C2C12 cells (31), which occurs 12 h after transfer to a differentiation medium, reaching a maximum at 48 h. To assess whether incubation in low serum medium (0.1% FBS) for 18 h was inducing myoblast differentiation and therefore activating p38MAPK, we tested for myogenin expression by Western blotting. Myogenin is an early response myogenic factor that is induced when myoblasts initiate their differentiation program after approximately 12 h (32) and can be considered an early differentiation marker. However, no induction of myogenin was observed in myoblasts incubated with low serum medium for 18 h (data not shown), and therefore, we can discard that p38MAPK activation was due to differentiation instead of TNF{alpha} per se.

TNF{alpha} up-regulates IL-6 mRNA expression and IL-6 secretion in L6-GLUT4myc myoblasts
In view of the known stimulatory effects of IL-6 on GLUT4 translocation in L6 cells (33) and the effects of TNF{alpha} stimulating IL-6 expression (34), we explored the hypothesis that the observed stimulatory effects of TNF{alpha} on GLUT4myc translocation in L6 cells might be mediated by IL-6. First, we examined the time-related response of IL-6 mRNA and protein expression to TNF{alpha}. L6-GLUT4myc myoblasts were incubated with TNF{alpha} (10 ng/ml) for various times (0, 6, 12, 24, and 48 h), and IL-6 mRNA expression was measured by real-time PCR and IL-6 secretion by ELISA. The results indicate that TNF{alpha} caused a significant up-regulation of IL-6 mRNA expression, reaching a maximum (2.19-fold increase) 12 h after stimulation (Fig. 6AGo). In addition, a significant increase in IL-6 secretion was detected 24 h after TNF{alpha} stimulation (Fig. 6BGo). The basal IL-6 values found in the conditioned medium were between 20 and 50 pg/ml, depending on the experiment and after treatment with TNF{alpha} reached a maximum value of 113 pg/ml at 24 h. These results indicate that TNF{alpha} increases the expression of IL-6 at the mRNA and protein levels in L6 myoblasts and that IL-6 production takes place between 12 and 24 h after TNF{alpha} treatment.


Figure 6
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  		FIG. 6. Effects of TNF{alpha} on IL-6 mRNA and protein expression. L6 myoblasts were treated with TNF{alpha} (10 ng/ml) for 6, 12, 24, and 48 h; culture medium was used to evaluate IL-6 secretion, and total RNA was extracted for real-time PCR assays. A, The time-course of IL-6 mRNA expression in L6 myoblasts assayed by real-time PCR is shown. Results are expressed as fold increase with respect to the control group (0 ng/ml TNF{alpha}), normalizing each time point against GAPDH expression, used as a housekeeping gene. B, The time-course of IL-6 secretion in L6 myoblasts assayed by ELISA is shown. Results are expressed as fold stimulation with respect to the control group (0 ng/ml TNF{alpha}). *, P < 0.05, relative to the control group.

 
To determine the implication of the NF{kappa}B and p38MAPK signaling pathways on TNF{alpha}-induced IL-6 mRNA expression, IL-6 mRNA levels were measured in the presence of inhibitors (SB220035 and BAY 11-7082) and TNF{alpha}. As shown in supplemental Fig. 1, published as supplemental data on The Endocrine Society’s Journals Online Web site at http://endo.endojournals.org, the inhibitors, alone or in combination, did not affect the basal nor the effects of TNF{alpha} on IL-6 mRNA expression, suggesting that the stimulation of IL-6 expression by TNF{alpha} does not take place through the NF{kappa}B and p38MAPK signaling pathways.

IL-6 does not mediate the stimulatory effects of TNF{alpha} on GLUT4 cell surface levels
To test the possibility that IL-6 might mediate the stimulatory effects of TNF{alpha} on the cell surface levels of GLUT4, we neutralized IL-6 through an IL-6 antibody. As shown in Fig. 7AGo, neutralization of IL-6 did not affect the TNF{alpha} (1 ng/ml)-stimulated increase in GLUT4myc cell surface levels in the presence or absence of insulin. In addition, TNF{alpha} did not stimulate the phosphorylation of STAT3 during the same culture conditions, whereas IL-6, used as a positive control, induced a strong phosphorylation of STAT3 after a 2-h incubation that was blocked by the IL-6 neutralizing antibody (Fig. 7BGo). Moreover, small interfering RNA gene-silencing of IL-6 did not show any effect on TNF{alpha} mediated increase on GLUT4myc cell surface levels (supplemental Fig. 3). Finally, a decrease in GLUT4myc cell surface levels was observed in response to 1 ng/ml recombinant IL-6 in the absence of insulin (basal state) that was reverted by immunodepletion with the anti-IL-6 antibody (Fig. 7CGo).


Figure 7
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  		FIG. 7. TNF{alpha}-mediated GLUT4 redistribution to the cell surface does not require the action of IL-6. L6 myoblasts were grown in 24-well plates (A and C) or six-well plates (B) followed by treatments in {alpha}-MEM supplemented with 0.1% FBS. A, TNF{alpha} (10 ng/ml) treatment was for 18 h in the presence of nonrelated mouse IgG (IgG, 100 ng/ml) or IL-6 neutralizing antibodies (IL-6 nAb; 100 ng/ml) in the absence (white bars) or presence (black bars) of 100 nM insulin. GLUT4myc cell surface density was measured as described in Materials and Methods. Results shown are the means of four independent experiments. *, P < 0.05, compared with basal (control). #, P < 0.05, compared with basal within a group. B, TNF{alpha} (10 ng/ml) treatment was for 18 h in the presence of nonrelated mouse IgG (IgG, 100 ng/ml) or IL-6 neutralizing antibodies (IL-6 nAb; 100 ng/ml) or cells were incubated for 2 h IL-6 (10 ng/ml) with/without IL-6 neutralizing antibodies (10 µg/ml). Cells were lysed and prepared for immunoblot analysis using pY705-STAT3 as described in Materials and Methods. The same blot was probed for β-actin to determine equal sample loading. Shown are representative gels. Densitometric readings of the exposed autoradiographs were expressed as a ratio of Y705-STAT3 to β-actin and plotted relative to the value in untreated samples. Results shown are the means of three independent experiments. *, P < 0.05, compared with control. C, TNF{alpha} (10 ng/ml) treatment was for 18 h, with or without the addition of IL-6 (1 ng/ml) during the final 2 h of incubation in the absence (white bars) or presence (black bars) of 100 nM insulin. GLUT4myc cell surface density was measured as described in Materials and Methods. Shown are the means of four independent experiments. *, P < 0.05, compared with basal (control). #, P < 0.05, compared with basal (IL-6).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
TNF{alpha} is a proinflammatory cytokine generally believed to promote insulin resistance by impairing the insulin-mediated glucose transport (8). However, there is a certain controversy in the literature regarding the action of TNF{alpha} because TNF{alpha} has been reported to stimulate glucose uptake, through a process not mediated by insulin, in L6 cells, 3T3 adipocytes and human chondrocytes (17, 18, 19), whereas other studies have reported a lack of effects of TNF{alpha} on basal or insulin-mediated glucose transport in L6 cells (35, 36) or an impairment of basal and insulin-mediated glucose uptake by TNF{alpha} in L6 cells (37). Differences in the reported effects of TNF{alpha} on glucose uptake in L6 cells among the various studies most likely arise due to variations in the experimental conditions. Therefore, the metabolic effects of TNF{alpha} on skeletal muscle are still controversial, and it has been suggested that the study of TNF{alpha} in isolation from other adipokines could make our understanding on the biological function of adipokines more difficult (38).

The results from this study indicate that TNF{alpha} increases the amount of GLUT4myc at the plasma membrane in L6 myoblasts and myotubes and as a result, increases both basal and insulin-mediated glucose uptake. TNF{alpha} caused a 40% increase in basal glucose uptake in GLUT4myc-expressing L6 myoblasts, in accordance with previous studies with L6 myoblasts (17, 19), and a 20% increase in myotubes. The lower magnitude of glucose uptake stimulation by TNF{alpha} found in myotubes, in view of the similar response of L6 myoblasts and myotubes to the effects of TNF{alpha} on GLUT4 cell surface levels, is intriguing and could perhaps be attributed, at least in part, to possible differences in the activity of diverse glucose transporters or of hexokinase, depending on the differentiation state of the cells. Although there are various reports on the effects of TNF{alpha} on glucose uptake, our results provide the first demonstration of an effect of TNF{alpha} on the amount of GLUT4 at the plasma membrane in both myoblasts and myotubes.

In the present study, we did not observe any impairment in the insulin-mediated increase of cell surface GLUT4 levels or glucose uptake by TNF{alpha}, but instead we observed an additive effect on those processes in the presence of insulin plus TNF{alpha}. The additive effect of TNF{alpha} does not appear to be related to an activation of the insulin-signaling pathway by TNF{alpha} because we did not observe increased Akt phosphorylation in response to TNF{alpha}. We postulate that the observed stimulatory effects of TNF{alpha} on GLUT4 cell surface levels and glucose uptake may take place through other cellular signaling pathways. It is well known that TNF{alpha} is a pleiotropic cytokine that can signal through diverse signaling pathways, the p38MAPK and NF{kappa}B being among the best characterized (21). To investigate the involvement of the p38MAPK and NF{kappa}B signaling pathways in the action of TNF{alpha} in L6 cells, we used SB220025 and Bay 11-7082, which are inhibitors of p38MAPK activity and NF{kappa}B translocation (inhibits irreversibly the TNF{alpha}-inducible phosphorylation of I{kappa}B{alpha}), respectively. The SB220025 inhibitor is a potent (60 nM IC50) and more specific inhibitor of p38MAPK when compared with SB203580 (39). We measured the ability of SB220025 (1 µM) to inhibit the activation of p38MAPK induced by hyperosmolarity (0.5 M NaCl and 0.45 M sucrose), and we obtained inhibitions close to 70% (data not shown), indicating that SB220025 can effectively block well-known p38MAPK-dependent processes in L6 cells. With the use of these inhibitors, we have provided evidence that blocking the p38MAPK and NF{kappa}B signaling pathways results in a significant decrease in the ability of TNF{alpha} to increase the levels of GLUT4 at the cell surface and glucose uptake in L6 cells. These results, coupled with our demonstration of the activation of the p38MAPK and NF{kappa}B signaling pathways by TNF{alpha} by Western blotting, strongly suggest that these two signaling pathways are involved in mediating the biological actions of TNF{alpha} on GLUT4 cell surface levels and glucose uptake in L6 cells. Therefore, it appears that TNF{alpha} and insulin increase the cell surface levels of GLUT4 and glucose uptake through different pathways.

TNF{alpha} has also been described as a pro- or antiapoptotic factor depending on whether it binds to TNFR1 or TNFR2, respectively (21), and can also act as a growth factor by promoting proliferation in human skeletal muscle and C2C12 cells (40, 41). L6 cells express the two types of TNF{alpha} receptors in myoblasts and in myotubes (4, 17). However, we did not detect any effect on proliferation or viability of L6 myoblasts or myotubes after 18 h of incubation in the presence of TNF{alpha}. Moreover, the rise in surface GLUT4mc was not accompanied by a generalized increase in total GLUT4myc levels, nor was it prevented by inhibition of protein synthesis with cycloheximide (supplemental Fig. 2). Therefore, the stimulatory effects of TNF{alpha} on GLUT4 translocation to the plasma membrane and glucose uptake cannot be explained by changes in cell number, total protein content or, specifically, total GLUT4 content, nor by changes in de novo synthesis of regulatory factors. In support of this conclusion, a recent report showed that TNF{alpha} (at 10 ng/ml) does not affect caspase-3 activity and poly(ADP-ribose) polymerase cleavage, both indicators of apoptosis, within the first 48 h of treatment in C2C12 myotubes (42).

Cytokines are typically expressed following an orderly time pattern of expression, with TNF{alpha} expressed early and IL-6 being a secondary expressed cytokine (34). In skeletal muscle cells, TNF{alpha} is known to induce the expression and secretion of IL-6 (43). Our data on the stimulation of IL-6 mRNA expression and secretion by TNF{alpha}, in light of recent reports on the direct stimulatory effects of IL-6 on cell surface levels of GLUT4 and glucose uptake in muscle cells (33, 44), led us to consider the possibility that the stimulatory effects of TNF{alpha} on cell surface levels of GLUT4 could be mediated by IL-6. However, we provide several pieces of evidence indicating that IL-6 does not mediate the stimulatory effects of TNF{alpha} on the levels of GLUT4myc at the cell surface. First, immunoneutralization of IL-6 did not interfere with the ability of TNF{alpha} to increase the levels of GLUT4myc in the cell surface of L6 myoblasts. Second, inhibition of p38MAPK and NF{kappa}B, two signaling molecules shown in the present study to mediate the stimulatory effects of TNF{alpha} on GLUT4myc levels in the cell surface, did not block the effects of TNF{alpha} on the mRNA expression of IL-6, suggesting that TNF{alpha} may increase GLUT4myc cell surface levels and IL-6 expression through different signaling pathways. Third, TNF{alpha} did not increase the activity of STAT3 or AMP-activated protein kinase (data not shown), a target of IL-6, in L6 myoblasts. Fourth, inhibition of protein synthesis by cycloheximide did not alter the stimulatory effects of TNF{alpha} on GLUT4myc cell surface levels (supplemental Fig. 2), suggesting that TNF{alpha} does not require the synthesis of new proteins (including IL-6) to exert its stimulatory effects on GLUT4myc cell surface levels. In fact, incubation of L6 myoblasts with recombinant IL-6 resulted in a significant decrease in GLUT4myc cell surface levels accompanied by an increase in STAT3 phosphorylation, and both effects of IL-6 were reversed by neutralization with an IL-6 antibody. Moreover, in one experiment, knockdown of IL-6 expression via small interfering RNA before and throughout the treatment with TNF{alpha} reduced the levels of the cytokine in the medium by 65% but did not prevent the increase in surface GLUT4myc. These results contrast with a recent report on the stimulation of GLUT4 translocation by IL-6 in L6 cells (33), which could be due to marked differences in experimental conditions between the two studies. However, IL-6 has also been described to exert an inhibitory effect on glucose transport in adipocytes (44) but did not affect glucose transport and insulin signaling when muscle strips were used (22). Overall, these results clearly indicate that IL-6 does not mediate the stimulatory effects of TNF{alpha} on GLUT4myc cell surface levels. However, it is conceivable that in vivo TNF{alpha} acting on muscle could contribute to the generation of IL-6 in muscle that in turn would have deleterious effects on other tissues (i.e. adipose, liver).

In summary, we have demonstrated that TNF{alpha} stimulates both the cell surface levels of GLUT4 and glucose uptake in L6 myoblasts as well as myotubes through a NF{kappa}B- and p38MAPK-dependent mechanism. Furthermore, although TNF{alpha} stimulates both the mRNA expression and secretion of IL-6 by L6 cells, IL-6 does not mediate the stimulatory effects of TNF{alpha} on the cell surface levels of GLUT4.


   Acknowledgments
 
We thank Dr. Marta Camps for her kind gift of antibodies and Dr. Philip J. Bilan for valuable insight and review of this manuscript.


   Footnotes
 
This work was supported by Grant AGL2005-01230 from the Ministerio de Educación y Ciencia (MEC; Spain) (to J.V.P.) and a grant from the Canadian Diabetes Association (to A.K.). N.R. was supported by a Juan de la Cierva postdoctoral fellowship (MEC, Spain), and M.D. was supported by a fellowship from the Departament d’Universitats, Recerca i Societat de la Informació (Generalitat de Catalunya). V.S. was supported by a Restracomp Fellowship (The Hospital for Sick Children, Ontario, Toronto, Canada).

Disclosure Statement: The authors of this manuscript have nothing to declare.

First Published Online December 27, 2007

Abbreviations: 2-DG, 2-Deoxy-D-[2,6-3H]glucose; DMSO, dimethylsulfoxide; FACS, fluorescence-activated cell sorter; FBS, fetal bovine serum; GAPDH, glyceraldehyde phosphate dehydrogenase; GLUT, glucose transporter; HRP, horseradish peroxidase; I{kappa}B, NF{kappa}B inhibitor; MTT, 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide; NF{kappa}B, nuclear factor-{kappa}B; PI, propidium iodide; STAT, signal transducer and activator of transcription.

Received July 31, 2007.

Accepted for publication December 17, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Dandona P, Aljada A, Bandyopadhyay A 2004 Inflammation: the link between insulin resistance, obesity and diabetes. Trends Immunol 25:4–7[CrossRef][Medline]
  2. Li YP, Reid MB 2001 Effect of tumor necrosis factor-{alpha} on skeletal muscle metabolism. Curr Opin Rheumatol 13:483–487[CrossRef][Medline]
  3. Lu L, Pu LJ, Xu XW, Zhang Q, Zhang RY, Zhang JS, Hu J, Yang ZK, Lu AK, Ding FH, Shen J, Chen QJ, Lou S, Fang DH, Shen WF 2007 Association of serum levels of glycated albumin, C-reactive protein and tumor necrosis factor-alpha with the severity of coronary artery disease and renal impairment in patients with type 2 diabetes mellitus. Clin Biochem 40:810–816[CrossRef][Medline]
  4. Zhang Y, Pilon G, Marette A, Baracos VE 2000 Cytokines and endotoxin induce cytokine receptors in skeletal muscle. Am J Physiol Endocrinol Metab 279:E196–E205
  5. Chen SE, Jin B, Li YP 2007 TNF-{alpha} regulates myogenesis and muscle regeneration by activating p38 MAPK. Am J Physiol Cell Physiol 292:C1660–C1671
  6. Penkowa M, Keller C, Keller P, Jauffred S, Pedersen BK 2003 Immunohistochemical detection of interleukin-6 in human skeletal muscle fibers following exercise. FASEB J 17:2166–2168[Abstract/Free Full Text]
  7. Thong FS, Dugani CB, Klip A 2005 Turning signals on and off: GLUT4 traffic in the insulin-signaling highway. Physiology (Bethesda) 20:271–284[CrossRef][Medline]
  8. del Aguila LF, Claffey KP, Kirwan JP 1999 TNF-{alpha} impairs insulin signaling and insulin stimulation of glucose uptake in C2C12 muscle cells. Am J Physiol 276:E849–E855
  9. Teruel T, Hernandez R, Lorenzo M 2001 Ceramide mediates insulin resistance by tumor necrosis factor-{alpha} in brown adipocytes by maintaining Akt in an inactive dephosphorylated state. Diabetes 50:2563–2571[Abstract/Free Full Text]
  10. de Alvaro C, Teruel T, Hernandez R, Lorenzo M 2004 Tumor necrosis factor {alpha} produces insulin resistance in skeletal muscle by activation of inhibitor {kappa}B kinase in a p38 MAPK-dependent manner. J Biol Chem 279:17070–17078[Abstract/Free Full Text]
  11. Bouzakri K, Zierath JR 2007 MAP4K4 gene silencing in human skeletal muscle prevents tumor necrosis factor-{alpha}-induced insulin resistance. J Biol Chem 282:7783–7789[Abstract/Free Full Text]
  12. Plomgaard P, Bouzakri K, Krogh-Madsen R, Mittendorfer B, Zierath JR, Pedersen BK 2005 Tumor necrosis factor-{alpha} induces skeletal muscle insulin resistance in healthy human subjects via inhibition of Akt substrate 160 phosphorylation. Diabetes 54:2939–2945[Abstract/Free Full Text]
  13. Lumeng CN, Deyoung SM, Saltiel AR 2007 Macrophages block insulin action in adipocytes by altering expression of signaling and glucose transport proteins. Am J Physiol Endocrinol Metab 292:E166–E174
  14. Uysal KT, Wiesbrock SM, Marino MW, Hotamisligil GS 1997 Protection from obesity-induced insulin resistance in mice lacking TNF-{alpha} function. Nature 389:610–614[CrossRef][Medline]
  15. Nakamori Y, Emoto M, Fukuda N, Taguchi A, Okuya S, Tajiri M, Miyagishi M, Taira K, Wada Y, Tanizawa Y 2006 Myosin motor Myo1c and its receptor NEMO/IKK-{gamma} promote TNF-{alpha}-induced serine307 phosphorylation of IRS-1. J Cell Biol 173:665–671[Abstract/Free Full Text]
  16. Valverde AM, Benito M, Lorenzo M 2005 The brown adipose cell: a model for understanding the molecular mechanisms of insulin resistance. Acta Physiol Scand 183:59–73[CrossRef][Medline]
  17. Yamasaki H, Yamaguchi Y, Takino H, Matsuo H, Matsumoto K, Uotani S, Akazawa S, Yamashita S, Nagataki S 1996 TNF-{alpha} stimulates glucose uptake in L6 myoblasts. Diabetes Res Clin Pract 32:11–18[CrossRef][Medline]
  18. Shikhman AR, Brinson DC, Valbracht J, Lotz MK 2001 Cytokine regulation of facilitated glucose transport in human articular chondrocytes. J Immunol 167:7001-7008[Abstract/Free Full Text]
  19. Ranganathan S, Davidson MB 1996 Effect of tumor necrosis factor-{alpha} on basal and insulin-stimulated glucose transport in cultured muscle and fat cells. Metabolism 45:1089–1094[CrossRef][Medline]
  20. Nolte LA, Hansen PA, Chen MM, Schluter JM, Gulve EA, Holloszy JO 1998 Short-term exposure to tumor necrosis factor-{alpha} does not affect insulin-stimulated glucose uptake in skeletal muscle. Diabetes 47:721-726[Abstract]
  21. Baud V, Karin M 2001 Signal transduction by tumor necrosis factor and its relatives. Trends Cell Biol 11:372–377[CrossRef][Medline]
  22. Kishi K, Muromoto N, Nakaya Y, Miyata I, Hagi A, Hayashi H, Ebina Y 1998 Bradykinin directly triggers GLUT4 translocation via an insulin-independent pathway. Diabetes 47:550–558[Abstract]
  23. Ueyama A, Yaworsky KL, Wang Q, Ebina Y, Klip A 1999 GLUT-4myc ectopic expression in L6 myoblasts generates a GLUT-4-specific pool conferring insulin sensitivity. Am J Physiol 277:E572–E578
  24. Wang Q, Khayat Z, Kishi K, Ebina Y, Klip A 1998 GLUT4 translocation by insulin in intact muscle cells: detection by a fast and quantitative assay. FEBS Lett 427:193–197[CrossRef][Medline]
  25. Huang C, Thirone AC, Huang X, Klip A 2005 Differential contribution of insulin receptor substrates 1 versus 2 to insulin signaling and glucose uptake in l6 myotubes. J Biol Chem 280:19426–19435[Abstract/Free Full Text]
  26. Pfaffl MW 2001 A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45
  27. Klip A, Guma A, Ramlal T, Bilan PJ, Lam L, Leiter LA 1992 Stimulation of hexose transport by metformin in L6 muscle cells in culture. Endocrinology 130:2535–2544[Abstract/Free Full Text]
  28. Ashwell JD 2006 The many paths to p38 mitogen-activated protein kinase activation in the immune system. Nat Rev Immunol 6:532–540[CrossRef][Medline]
  29. Niu W, Huang C, Nawaz Z, Levy M, Somwar R, Li D, Bilan PJ, Klip A 2003 Maturation of the regulation of GLUT4 activity by p38 MAPK during L6 cell myogenesis. J Biol Chem 278:17953–17962[Abstract/Free Full Text]
  30. Steinberg GR 2007 Inflammation in obesity is the common link between defects in fatty acid metabolism and insulin resistance. Cell Cycle 6:888–894[Medline]
  31. Perdiguero E, Ruiz-Bonilla V, Gresh L, Hui L, Ballestar E, Sousa-Victor P, Baeza-Raja B, Jardi M, Bosch-Comas A, Esteller M, Caelles C, Serrano AL, Wagner EF, Munoz-Canoves P 2007 Genetic analysis of p38 MAP kinases in myogenesis: fundamental role of p38{alpha} in abrogating myoblast proliferation. EMBO J 26:1245–1256[CrossRef][Medline]
  32. Lluis F, Perdiguero E, Nebreda AR, Munoz-Canoves P 2006 Regulation of skeletal muscle gene expression by p38 MAP kinases. Trends Cell Biol 16:36–44[CrossRef][Medline]
  33. Carey AL, Steinberg GR, Macaulay SL, Thomas WG, Holmes AG, Ramm G, Prelovsek O, Hohnen-Behrens C, Watt MJ, James DE, Kemp BE, Pedersen BK, Febbraio MA 2006 Interleukin-6 increases insulin-stimulated glucose disposal in humans and glucose uptake and fatty acid oxidation in vitro via AMP-activated protein kinase. Diabetes 55:2688–2697[Abstract/Free Full Text]
  34. Bonizzi G, Karin M 2004 The two NF-{kappa}B activation pathways and their role in innate and adaptive immunity. Trends Immunol 25:280–288[CrossRef][Medline]
  35. Khanna S, Roy S, Packer L, Sen CK 1999 Cytokine-induced glucose uptake in skeletal muscle: redox regulation and the role of {alpha}-lipoic acid. Am J Physiol 276:R1327–R1333
  36. Patiag D, Gray S, Idris I, Donnelly R 2000 Effects of tumour necrosis factor-{alpha} and inhibition of protein kinase C on glucose uptake in L6 myoblasts. Clin Sci (Lond) 99:303–307[Medline]
  37. Begum N, Ragolia L 1996 Effect of tumor necrosis factor-{alpha} on insulin action in cultured rat skeletal muscle cells. Endocrinology 137:2441–2446[Abstract]
  38. Dyck DJ, Heigenhauser GJ, Bruce CR 2006 The role of adipokines as regulators of skeletal muscle fatty acid metabolism and insulin sensitivity. Acta Physiol 186:5–16[CrossRef]
  39. Jackson JR, Bolognese B, Hillegass L, Kassis S, Adams J, Griswold DE, Winkler JD 1998 Pharmacological effects of SB 220025, a selective inhibitor of P38 mitogen-activated protein kinase, in angiogenesis and chronic inflammatory disease models. J Pharmacol Exp Ther 284:687–692[Abstract/Free Full Text]
  40. Foulstone EJ, Huser C, Crown AL, Holly JM, Stewart CE 2004 Differential signalling mechanisms predisposing primary human skeletal muscle cells to altered proliferation and differentiation: roles of IGF-I and TNF{alpha}. Exp Cell Res 294:223–235[CrossRef][Medline]
  41. Girgenrath M, Weng S, Kostek CA, Browning B, Wang M, Brown SA, Winkles JA, Michaelson JS, Allaire N, Schneider P, Scott ML, Hsu YM, Yagita H, Flavell RA, Miller JB, Burkly LC, Zheng TS 2006 TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal muscle regeneration. EMBO J 25:5826–5839[CrossRef][Medline]
  42. Tolosa L, Morla M, Iglesias A, Busquets X, Llado J, Olmos G 2005 IFN-{gamma} prevents TNF-{alpha}-induced apoptosis in C2C12 myotubes through down-regulation of TNF-R2 and increased NF-{kappa}B activity. Cell Signal 17:1333–1342[CrossRef][Medline]
  43. Efthimiou P 2006 Tumor necrosis factor-{alpha} in inflammatory myopathies: pathophysiology and therapeutic implications. Semin Arthritis Rheum 36:168–172[CrossRef][Medline]
  44. Pedersen BK 2007 IL-6 signalling in exercise and disease. Biochem Soc Trans 35:1295–1297[CrossRef][Medline]



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