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The Journal of Immunology, 2004, 172: 7713-7720.
Copyright © 2004 by The American Association of Immunologists

IL-1{beta} Impairs Insulin-Like Growth Factor I-Induced Differentiation and Downstream Activation Signals of the Insulin-Like Growth Factor I Receptor in Myoblasts1

Suzanne R. Broussard2,*, Robert H. McCusker*, Jan E. Novakofski{dagger}, Klemen Strle*, Wen Hong Shen*, Rodney W. Johnson{ddagger}, Robert Dantzer§ and Keith W. Kelley*

* Laboratories of Immunophysiology, {dagger} Developmental Endocrinology, and {ddagger} Integrative Biology, Department of Animal Sciences, University of Illinois, Urbana-Champaign, Urbana, IL 61801; and § Integrative Neurobiology, FRE, Centre National de la Recherche Scientifique, Unité Mixte de Recherche Institut National de la Recherche Agronomique, Institut François Magendie des Neurosciences, Bordeaux, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Proinflammatory cytokines are elevated in disorders characterized by muscle wasting and weakness, such as inflammatory myopathies and AIDS wasting. We recently demonstrated that TNF-{alpha} impairs the ability of insulin-like growth factor (IGF)-I to promote protein synthesis in muscle precursor cells. In this study we extend these findings by showing that low concentrations of IL-1{beta} impair IGF-I-dependent differentiation of myoblasts, as assessed by expression of the muscle specific protein, myosin heavy chain. In the absence of exogenous IGF-I, IL-1{beta} (1 ng/ml) did not impair muscle cell development. However, in the presence of IGF-I, 100-fold lower concentrations of IL-1{beta} (0.01 ng/ml) significantly suppressed myoblast differentiation, protein synthesis, and myogenin expression. Increasing IL-1{beta} to 1 ng/ml completely blocked the anabolic actions of IGF-I in murine C2C12 myoblasts. Similarly, IL-1{beta} inhibited IGF-I-stimulated protein synthesis in primary porcine myoblasts. IL-1{beta} impaired the actions of IGF-I at a point distal to the IGF receptor, and this was not due to IL-1{beta}-induced cell death. Instead, IL-1{beta} inhibited the ability of IGF-I to phosphorylate tyrosine residues on both of its downstream docking proteins, insulin receptor substrate 1 and insulin receptor substrate 2. These data establish that physiological concentrations of IL-1{beta} block the ability of IGF-I to promote protein synthesis, leading to reduced expression of the myogenic transcription factor, myogenin, and the subsequent development of more mature differentiated cells that express myosin heavy chain. Collectively, the results are consistent with the notion that very low concentrations of IL-1{beta} significantly impair myogenesis, but they are unable to do so in the absence of the growth factor IGF-I.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The two major proinflammatory cytokines, IL-1{beta} and TNF-{alpha}, are expressed in muscle as well as in the inflammatory cellular infiltrates of all inflammatory myopathies (reviewed in Ref.1). This heterogeneous group of muscle disorders is defined by a progressive loss in muscle mass and weakness. Elevated expression of proinflammatory cytokines is considered to play a major role in muscle wasting. Administration of either IL-1{beta} (2) or TNF-{alpha} (3) to rats in vivo significantly reduces synthesis of the major skeletal muscle proteins. Satellite myoblast cells are likely to be critical targets for proinflammatory cytokines. Myoblasts are myogenic progenitor cells that are located between the sheath of the external lamina and the mature muscle fiber. Myoblasts are responsible for regeneration of skeletal muscle and for promoting myofiber repair by fusing with adjacent myofibers to replenish nuclei (reviewed in Ref.4). The importance of myoblasts in maintaining muscle mass is highlighted by their failure to differentiate in patients with Duchene muscular dystrophy, which leads to a progressive failure in myoregeneration (5). Indeed, it has recently been established that both TNF-{alpha} and IL-1{beta} are elevated before the onset of muscular dystrophy in the murine model of this disease (6). We (7) and others (8, 9, 10, 11) have shown that TNF-{alpha} can act on myoblasts to block their fusion into mature muscle cells.

Growth, development, and regeneration of muscle fibers is largely controlled by the potent anabolic hormone, insulin-like growth factor (IGF)3 I (reviewed in Refs.12, 13). Mice that overexpress IGF-I specifically in skeletal muscle display an increase in both muscle mass and regeneration capacity (14 and reviewed in Ref.15). Additionally, IGF-I treatment in vivo increases skeletal muscle regeneration (16) and ameliorates muscle wasting (17) in murine models of muscular dystrophy. The critical role of IGF-I in promoting protein synthesis (18, 19) and differentiation (9, 20) can be mimicked in vitro with myoblasts of both human and murine origin. Recent experiments using small interfering RNA to eliminate endogenous IGF blocks spontaneous fusion of immature myoblasts into more mature, multinucleated myotubes (21). The biological actions of IGF occur through ligand activation of the IGF-I receptor, which uses adaptor docking proteins to transmit downstream signals. In the absence of a functional IGF-I receptor, myoblasts are unable to differentiate into mature muscle cells in vitro (22). When examined in vivo by using IGF-I (23) or IGF-I receptor (24) knockout mice, deficient pups have grossly underdeveloped skeletal muscle.

Despite an absolute requirement for the IGF-I receptor in muscle differentiation, little attention has been given to the possibility that proinflammatory cytokines act by interfering with the actions of IGF-I. We recently reported that TNF-{alpha} blocks IGF-I-induced protein synthesis in myoblasts (7). Additionally, TNF-{alpha} interferes with the ability of IGF-I to promote passage of breast cancer epithelial cells through the G1/S checkpoint of the cell cycle (25, 26). We recently established that TNF-induced cytostasis occurs by impairing synthesis of the E2F-1 transcription factor, thereby inhibiting expression of cyclin A (25). In both these systems, TNF-{alpha} impairs the actions of IGF-I by a post-IGF-I receptor mechanism (27). We now provide evidence that physiological concentrations of the other major proinflammatory cytokine, IL-1{beta}, acts on myoblasts to block IGF-I stimulation of de novo protein synthesis, expression of myogenin and development of more mature muscle cells expressing myosin heavy chain. In the absence of the critical IGF-I hormonal peptide, IL-1{beta} does not affect any of these biological events. These results establish that IL-1{beta} acts on muscle progenitor cells to impair their responsiveness to IGF-I, therefore leading to IGF resistance.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Culture of myoblasts

C2C12 murine myoblasts obtained from the American Type Culture Collection (Manassas, VA) were cultured in DMEM supplemented with 3.7 g/l sodium bicarbonate, 4.5 g/l glucose, 100 µg/ml streptomycin, and 100 U/ml penicillin G (all from Sigma-Aldrich, St. Louis, MO). The DMEM was supplemented with 10% heat-inactivated FBS purchased from HyClone Laboratories (Logan, UT) containing nondetectable concentrations of endotoxin, as determined by the Limulus amebocyte lysate assay (28). Murine myoblasts were incubated in a modified atmosphere of 7% CO2 at 37°C, and all experiments were conducted when cells reached 70–80% confluency.

Primary skeletal muscle myoblasts were isolated from 1-day-old piglets obtained from the Swine Research Center, University of Illinois, Urbana, IL. Piglets were euthanized and the longissimus muscle was immediately removed, minced, and washed twice with HBSS to remove erythrocytes. A single cell suspension was generated by digesting minced muscle for 2 h with 1 mg/ml collagenase, 0.2 mg/ml elastase, and 0.5 mg/ml hyaluronidase, passage through 20-µm screens and cultured in DMEM (20% FBS). Primary skeletal muscle myoblasts were enriched by preincubating single cell suspensions for 3 h in polystyrene petri dishes (Falcon 3003) to remove adherent fibroblasts. Porcine myoblasts were then transferred to 24-well tissue culture plates coated with gelatin (type A gelatin from porcine skin, Sigma-Aldrich) for 5–7 days until they reached >70% confluence. As we have previously reported (7), these 5–7 day porcine cultures consisted of >90% myoblasts as determined with indirect immunofluorescence with a myoblast-specific mAb (5-1H11; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) using a Cytomation MoFlo cytometer. All animal procedures were approved by the University of Illinois Institutional Laboratory Animal Care Advisory Committee.

De novo protein synthesis

A constant number (5 x 105 cells) of either primary porcine or murine C2C12 myoblasts were seeded into each well of 24-well polystyrene plates (Costar, Corning, NY) and cultured to 70–80% confluency. Growth factors were removed from cells by washing three times in serum-free DMEM, culturing for an additional 5 h and replacement with fresh DMEM (0.5 ml). Primary porcine and C2C12 murine myoblasts were then treated for 1 h with either recombinant human (10 ng/ml) or murine (0.01, 0.1, 1, or 10 ng/ml) IL-{beta} (Intergen, Purchase, NY), respectively. Myoblasts were then treated with an optimal concentration of IGF-I (50 ng/ml) for an additional 5 h in the presence of 1.5 µCi of [3H]phenylalanine (L-2,3,4,5,6-[3H]phenylalanine; Amersham Pharmacia Biotech, Piscataway, NJ). Cells were then placed in the –80°C until analysis using a PHD cell harvester (Cambridge Technology, Watertown, MA). Incorporation of [3H]phenylalanine into protein was determined by harvesting cells onto glass fiber filters (Whatman International, Maidstone, U.K.) and removing unincorporated [3H]phenylalanine by washing four times. The amount of [3H]phenylalanine-labeled cellular protein on the glass fiber filters was quantified using a LS 6000IC Beckman Coulter Scintillation Counter (Fullerton, CA). Radioactivity for each individual treatment was divided by that of the medium control and presented as standardized fold-induction of protein synthesis.

Live and apoptotic cell populations

Myoblasts were treated with IL-1{beta} and IGF-I as described for the de novo protein synthesis experiments. C2C12 myoblasts at 70–80% confluency were washed three times and cultured in DMEM for 2 h. The medium was then replaced with fresh DMEM, followed by pretreatment with or without IL-1{beta} (0.1, 1, or 10 ng/ml) for 1 h. Cells were subsequently incubated an additional 5 h in the presence or absence IGF-I (50 ng/ml). The culture supernatant and trypsinized (0.25%) adherent cells were pooled and suspended at room temperature in DMEM supplemented with 10% FBS. Early apoptotic cells were detected by flow cytometry with a Cytomation MoFlo flow cytometer as Hoechst 33342 positive, propidium iodide-negative cells, as previously described (7, 28), following addition of Hoechst 33342 (7 ng/ml; Sigma-Aldrich) to myoblasts for 2 min. Live cells were designated as those impermeable to both propidium iodide and Hoechst 33342, whereas dying cells were considered those staining positive with only propidium iodide.

Western blotting

Differentiation of C2C12 myoblasts was initiated after the cells were 70–80% confluent by washing three times and subsequently incubating them for 5 h in serum-free DMEM. Fresh medium was added and the cells were treated for 1 h with 0.01, 0.1, or 1 ng/ml IL-1{beta} or control medium. Myoblasts were subsequently cultured for an additional 24 to 30 h in the presence or absence of IGF-I (50 ng/ml). Adherent cells were lysed in RIPA buffer (1% Nonidet P-40, 1 mM PMSF, 0.1% SDS, 0.5% sodium-deoxycholate, 1x PBS, 2 µg/ml pepstatin, 2 µg/ml aprotinin, and 40 nM leupeptin) and centrifuged at 16,000 x g at 4°C for 15 min. Total protein in the supernatant was quantified using the Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA). Seventy-five micrograms of protein were resolved in 7 and 12% SDS-PAGE gels and then transferred to Trans-Blot polyvinylidene difluoride membranes (Bio-Rad). The blots were then probed with murine mAbs specific to myogenin (M-225; Santa Cruz Biotechnology, Santa Cruz, CA), myosin heavy chain (F1.652 developed by Dr. H. M. Blau at Stanford University School of Medicine and obtained from the Developmental Studies Hybridoma Bank maintained at the Department of Biological Sciences, University of Iowa, Iowa City, IA) or {alpha}-tubulin (B512; Sigma-Aldrich). Myogenin and myosin heavy chain expression were calculated by dividing their respective densitometric intensities by the {alpha}-tubulin loading control in each lane, and data are presented as standardized "fold induction."

Immunoprecipitation

Washed C2C12 myoblasts were cultured in DMEM for 5 h before treatment with rIL-1{beta} (0.1, 1, or 10 ng/ml) or control medium for 1 h. Cells were subsequently stimulated for 3 min with IGF-I (50 ng/ml) and immediately lysed in ice-cold buffer (1% Nonidet P-40, 100 mM NaCl, 25 mM benzamidine, 1 mM PMSF, 2 µg/ml aprotinin, 40 nM leupeptin, 2 µg/ml pepstatin, 50 mM NaF, 1 mM DTT, 2 mM sodium orthovanadate, and 50 mM Tris, pH 7.4). The lysate was centrifuged at 16,000 x g for 10 min to remove insoluble cellular debris. Specific proteins were immunoprecipitated from the cell lysate supernatant by rotating at 4°C overnight in the presence of excess (1 µg) amounts of either rabbit anti-IGF-I receptor {beta}-chain (C-20; Santa Cruz Biotechnology), rabbit anti-insulin receptor substrate (IRS)-1 (06-248; Upstate Biotechnology, Lake Placid, NY) or rabbit anti-IRS-2 (06-506; Upstate Biotechnology) Abs and 25 µl of protein G-Sepharose beads (Pharmacia Biotech, Uppsala, Sweden), as we previously described (7). Nonspecific proteins were removed from immunocomplexes by washing three times in cold lysis buffer. Laemmli buffer was added to the samples, followed by heating at 100°C for 7 min. The proteins were separated in 7% SDS-PAGE gels and then immobilized on polyvinylidene difluoride membranes and blotted with the appropriate Ab. A murine monoclonal PY-20 phosphotyrosine Ab (PY20; Transduction Laboratories, Lexington, KY) was used to detect tyrosine phosphorylated proteins. Blots were stripped using 0.1 M 2-ME, as described by the manufacturer (Bio-Rad), before detection of the total protein mass by blotting with either IGF-I receptor {beta}-chain, IRS-1 or IRS-2 Abs. The Abs were detected by incubation with either a secondary sheep anti-mouse or donkey anti-rabbit IgG HRP-linked Abs (both from Amersham Pharmacia Biotech). The polyvinylidene difluoride membranes were developed using ECL (Amersham, Arlington Heights, IL) and exposed to autoradiographic film (Eastman Kodak, Rochester, NY). Densitometric values of autoradiograms were quantified by scanning with a Duoscan T1200 densitometer equipped with AGFA Fotolook 3.00 software, as we have previously described (29). Data were calculated as the ratio of the densitometric density of the mass of tyrosine phosphorylated protein divided by the total mass of the same protein in that lane and presented as standardized fold induction.

Statistical analysis

All experiments were independently replicated three times unless otherwise specified in the figure, and data were summarized as mean ± SEM. All experiments were analyzed as completely randomized designs using standard ANOVA procedures with the Statistical Analysis System for Windows (30). Treatment differences were detected using Duncan’s New Multiple Range test (30). Two-sided values of p < 0.05 or p < 0.01 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IL-1{beta} blocks IGF-I-dependent differentiation

IL-1{beta}, at concentrations ranging from 1 to 100 ng/ml, has been reported to act on muscle cells cultured in fetal bovine or horse sera by blocking development to a more mature phenotype (10, 31), but the mechanism remains unknown. Because activation of the IGF-I receptor is required for differentiation of myoblasts (22), we hypothesized that IL-1{beta} acts by inhibiting the ability of IGF-I to promote differentiation of precursors into more mature myotubes. To test this possibility, serum-derived growth factors were removed from C2C12 myoblasts (70 to 80% confluent) to facilitate differentiation. Cells were then pretreated with or without IL-1{beta} for 1 h and allowed to differentiate in the presence or absence of recombinant IGF-I for 24 to 30 h. Differentiation was assessed by measuring the mass of the sarcomere myosin heavy chain, a protein specifically expressed in myotubes, but not in myoblasts (21). As expected, recombinant IGF-I nearly doubled the mass of myosin (Fig. 1). Very low concentrations of IL-1{beta} impaired the ability of IGF-I to increase expression of myosin heavy chain in a dose-dependent manner. As little as 0.01 ng/ml IL-1{beta} significantly inhibited by 47% the ability of IGF-I to increase myosin expression. When the concentration of IL-1{beta} was increased to 1 ng/ml, it completely blocked IGF-I-dependent expression of myosin heavy chain. In the absence of IGF-I, even the highest concentration of IL-1{beta} failed to affect the mass of either myosin or {alpha}-tubulin. Taken together, these data establish that IL-1{beta}, at very low concentrations, blocks myoblast differentiation by abrogating the actions of IGF-I.



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  		FIGURE 1. IL-1{beta} inhibits myoblast differentiation by interfering with the myogenic actions of IGF-I. A, A representative Western blot in which whole cell lysates were probed with Abs that specifically recognize myosin heavy chain or the loading control protein, {alpha}-tubulin. B, Graph summarizes three independent replications and is presented as the densitometric values of myosin heavy chain relative to the intensity of the {alpha}-tubulin loading control in the same lane. C2C12 myoblasts were treated with or without increasing concentrations of IL-1{beta} for 1 h and then stimulated either in the presence or absence of IGF-I for 24 to 30 h. In the absence of IL-1{beta}, IGF-I significantly (p < 0.01) increased myoblast differentiation, as assessed by nearly a doubling in expression of the muscle-specific protein, myosin heavy chain. In the absence of IGF-I, IL-1{beta} alone did not affect myosin expression. However, in the presence of both IGF-I and IL-1{beta}, as little as 0.01 ng/ml IL-1{beta} significantly impaired the ability of IGF-I to promote expression of myosin heavy chain by 47% (**, p < 0.01; n = 3). Increasing IL-1{beta} concentration to 1 ng/ml resulted in a complete inhibition of IGF-I-stimulated myosin expression (**, p < 0.01). None of the treatments, alone or in combination, affected expression of the control protein, {alpha}-tubulin (p > 0.1).

 
IL-1{beta} inhibits IGF-I-induced de novo protein synthesis

Differentiation and fusion of myoblasts requires synthesis of several muscle-specific proteins (20, 32). In primary human myoblasts, as little as 2 ng/ml TNF-{alpha} significantly impairs IGF-I-induced de novo protein synthesis by ~35%, and 100 ng/ml TNF-{alpha} fully blocks IGF-I-induced protein synthesis (18). We therefore speculated that IL-1{beta} impairs the ability of IGF-I to induce protein synthesis. To test this possibility, we conducted experiments similar to those with primary human myoblasts (18) by incubating primary porcine myoblasts with similar concentrations of recombinant human IL-1{beta} in the presence or absence of an optimal concentration of IGF-I (50 ng/ml). IGF-I (50 ng/ml) increased (p < 0.01) de novo protein synthesis to ~1.5-fold in these primary cells (Fig. 2A). However, pretreatment of porcine myoblasts with 10 ng/ml IL-1{beta} completely blocked (p < 0.01) the ability of IGF-I to induce protein synthesis, and similar results were observed with 50 ng/ml IL-1{beta} (data not shown). In the absence of IGF-I, IL-1{beta} did not reduce protein synthesis.



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  		FIGURE 2. IL-1{beta} interferes with the ability of IGF-I to promote de novo protein synthesis. A, IGF-I (50 ng/ml) caused a ~50% increase (p < 0.01) in de novo protein synthesis in primary porcine myoblasts, as assessed using a 5-h pulse with [3H]phenylalanine. IL-1{beta} (10 ng/ml) completely blocked IGF-I stimulation of protein synthesis in these primary cells (**, p < 0.01; n = 3). No change in protein synthesis was detected in porcine myoblasts treated with IL-1{beta} alone (p > 0.1). B, C2C12 myoblasts were very sensitive to IGF-I, and 1 ng/ml IGF-I significantly (*, p < 0.05; n = 2) increased protein synthesis by 20%. Increasing the concentration of IGF-I to 50 ng/ml resulted in optimal protein synthesis (**, p < 0.01). C, Using this optimal concentration of IGF-I (50 ng/ml), murine myoblasts that were treated with IL-1{beta} for 1 h before IGF-I treatment displayed a significant and dose-dependent reduction in protein synthesis. As little as 0.01 ng/ml IL-1{beta} impaired IGF-I-stimulated protein synthesis in C2C12 myoblasts (**, p < 0.01, n = 5). Increasing IL-1{beta} to 1 ng/ml completely blocked (**, p < 0.01) the ability of IGF-I to increase de novo synthesis of protein. No significant change in basal protein synthesis was detected in murine myoblasts in the presence of ≤1 ng/ml IL-1{beta} (p > 0.1).

 
We then used the well-characterized clonal population of C2C12 murine myoblasts to determine the dose response to IGF-I. IGF-I (1 ng/ml) significantly (p > 0.05) induced protein synthesis (Fig. 2B), and increasing the concentration of IGF-I to 50 ng/ml resulted in optimal protein synthesis (p > 0.01). Increasing the concentration of IGF-I to 100 ng/ml did not result in a higher incorporation of [3H]phenylalanine into protein compared with 50 ng/ml IGF-I (data not shown). By using this optimal concentration of IGF-I to promote de novo protein synthesis, we next found that very low concentrations of IL-1{beta} impaired the anabolic actions of IGF-I (Fig. 2C). As little as 0.01 ng/ml IL-1{beta} significantly inhibited (p > 0.01) IGF-I-stimulated protein synthesis by 33%. Increasing the concentration of IL-1{beta} continued to reduce IGF-I-stimulated protein synthesis in a dose-dependent manner. IL-1{beta} at 0.1 ng/ml impaired protein synthesis by 53%, and 1 ng/ml completely blocked IGF-I-induced synthesis of new protein. Because we used an optimal concentration of IGF-I, it is possible that even more sensitive inhibition by IL-1{beta} would occur at suboptimal concentrations of IGF-I. No change in basal protein synthesis was detected even at the highest concentration of IL-1{beta} (p > 0.10). These experiments confirm the hypothesis that IL-1{beta} inhibits the ability of IGF-I to promote de novo protein synthesis in progenitor muscle cells of two different species.

IL-1{beta} does not kill myoblasts

IL-1{beta} has been reported to induce apoptosis in a variety of cells (33), whereas IGF-I promotes survival of murine (28) and human (34) promyeloid cells as well as progenitor muscle cells (35, 36). To test the possibility that IL-1{beta} impairs IGF-I-stimulation of protein synthesis by simply killing the cell, we tested C2C12 myoblasts in the presence or absence of both IGF-I and IL-1{beta} (Table I). IGF-I minimally, but significantly (p < 0.01), reduced cell death in serum-free medium from 7% to 4%, as determined by the proportion of propidium iodide-positive cells. Similar results were obtained when the proportion of early apoptotic cells was measured with Hoechst 33342. These experiments demonstrated that IGF-I reduced the proportion of apoptotic myoblasts from 13% to 5% (p < 0.001) during this same time frame. However in the absence of IGF-I, 10 ng/ml IL-1{beta} did not further increase the proportion of cells that were dead (positive for propidium iodide; 7% vs 7%) or apoptotic (positive for Hoechst 33342 and negative for propidium iodide, 13% vs 15%; Table I; p > 0.10) Additionally, pretreatment of the myoblasts with even the highest concentration IL-1{beta} did not impair the ability of IGF-I to promote cell survival. Collectively, these data establish that IL-1{beta} does not promote or suppress cell death in either the absence or presence of IGF-I.


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  		Table I. Inhibition of IGF-I-stimulated protein synthesis by IL-1{beta} is not due to cell deatha

 
IGF-I-stimulated tyrosine phosphorylation of IRS docking proteins is impaired by IL-{beta}

Activation of the IGF-I receptor is well known to induce tyrosine phosphorylation of the two high m.w. docking proteins, IRS-1, and IRS-2 (37). TNF-{alpha} impairs the biological activity of IGF-I at least in part by inhibiting tyrosine phosphorylation of these docking proteins in both cancer cells (26) and murine myoblasts (7). To explore the possibility that IL-1{beta} also targets the IRS docking proteins in muscle cells, C2C12 myoblasts were treated with increasing concentrations of IL-1{beta} in the presence or absence of an optimal concentration of IGF-I (50 ng/ml). As expected, IGF-I increased (p < 0.01) tyrosine phosphorylation of IRS-1 by nearly 7-fold (Fig. 3, A and B) and that of IRS-2 ~4-fold (Fig. 3, C and D). At concentrations as low as 0.1 and 1 ng/ml, IL-1{beta} significantly impaired (p < 0.01) IGF-I stimulation of IRS-1 tyrosine phosphorylation by 38% and 57% reduction, respectively. No further reduction in IGF-I-stimulated tyrosine phosphorylation of IRS-1 was detected when the concentration of IL-1{beta} was increased to 10 ng/ml. Similarly, IL-1{beta} impaired the ability of IGF-I to tyrosine phosphorylate IRS-2 (Fig. 3, C and D, p < 0.01). The mass of IRS-1 and IRS-2 remained unaffected (p > 0.10) by any concentration of IL-1{beta}, confirming the ability of very low concentrations of IL-1{beta} to specifically inhibit tyrosine phosphorylation of both IRS-1 and IRS-2 following stimulation with IGF-I.



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  		FIGURE 3. IL-1{beta} impairs IGF-I-induced tyrosine phosphorylation of IRS-1 and IRS-2 docking proteins. C2C12 myoblasts were pretreated with IL-1{beta} for 1 h before stimulation with IGF-I for 3 min. IRS-1 (A and B) and IRS-2 (C and D) were immunoprecipitated from whole cell lysates, separated in 7% SDS-PAGE gels and the proteins were transferred to polyvinylidene difluoride membranes. Tyrosine phosphorylation was detected using a phospho-specific Ab (PY) followed by quantification of total IRS protein. A, A representative autoradiogram showed that IGF-I induced substantial tyrosine phosphorylation of IRS-1, and this increase was significantly (**, p < 0.01) reduced by pretreatment with increasing concentrations of IL-1{beta}. B, A summary of three independent experiments established that IGF-I caused a 7-fold increase (p < 0.01) in tyrosine phosphorylation of IRS-1, and as little as 0.1 ng/ml IL-1{beta} inhibited (**, p < 0.01) this event. C, C2C12 myoblasts were treated as described in (A) and IRS-2 was precipitated from whole cell lysates. As expected, IL-1{beta} alone did not induce tyrosine phosphorylation of IRS-2. However, in the presence of IGF-I, IL-1{beta} inhibited the ability of IGF-I to phosphorylate IRS-2. D, A densitometric summary confirmed that in the absence of IGF-I, IL-1{beta} (10 ng/ml) did not affect (p > 0.10, n = 4) tyrosine phosphorylation of IRS-2. However, as little as 0.1 ng/ml IL-1{beta} impaired (**, p < 0.01) the ability of IGF-I to induce tyrosine phosphorylation of IRS-2.

 
Autophosphorylation of the IGF-I receptor is not affected by IL-1{beta}

One logical explanation for the effect of IL-1{beta} is that it impairs the intrinsic tyrosine kinase activity of the IGF-IR, which is necessary to induce tyrosine phosphorylation of both IRS-1 and IRS-2 (37). To test this hypothesis, C2C12 myoblasts were stimulated with 50 ng/ml IGF-I, which increased autophosphorylation of the {beta}-chain of the IGF-I receptor by ~4-fold (Fig. 4). However, IL-1{beta} had no discernible effect on the IGF-I receptor, either in the presence or absence of IGF-I. IL-1{beta}, at a concentration as high as 10 ng/ml, did not interfere with the ability of IGF-I to induce tyrosine phosphorylation of the IGF-I receptor. Similar to results with human breast cancer cells (26), these data establish that IL-1{beta} does not impair intrinsic tyrosine kinase activity of its receptor.



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  		FIGURE 4. Autophosphorylation of the IGF-I receptor is not impaired by IL-1{beta}. C2C12 myoblasts were treated for 1 h with IL-1{beta} before IGF-I stimulation for 3 min. Tyrosine phosphorylation of the type I IGF-I receptor was quantified from immunoprecipitated {beta}-chains on Western blots that were probed with either a phosphotyrosine (PY) or the immunoprecipitating Ab. A, A representative autoradiogram showed that IL-1{beta} did not affect autophosphorylation of the IGF-I receptor, either in the absence or presence of IGF-I. B, A densitometric summary of four independent experiments established that IGF-I stimulated a 6-fold increase (p < 0.01, n = 4) in tyrosine phosphorylation of IGF-I receptor {beta}-chains. However, even the highest concentration of IL-1{beta}, 10 ng/ml, did not affect the intrinsic tyrosine kinase activity of the IGF-I receptor (p > 0.10).

 
IL-1{beta} impairs the ability of IGF-I to promote expression of the myogenin transcription factor

Myoblast differentiation requires expression of myogenin to transactivate muscle-specific gene expression by interacting with the consensus nucleotide motif designated the E box (32). Myogenin has recently been shown to induce expression of the muscle structural gene myosin heavy chain in Xenopus laevis (38). IGF-I is well known to promote expression of myogenin in myoblasts (20). These findings led us to test the hypothesis that IL-1{beta} acts to impair myogenesis, as demonstrated by reduced expression of myosin heavy chain (Fig. 1), by blocking the ability of IGF-I to promote expression of myogenin. As expected, stimulation of C2C12 myoblasts with IGF-I for 24 to 30 h significantly increased myogenin expression by ~2-fold (Fig. 5; p < 0.01). Similar to results with both myosin heavy chain expression and de novo protein synthesis, as little as 0.01 ng/ml IL-1{beta} significantly impaired myogenin expression (24%; p > 0.01) in the presence of IGF-I. Increasing the concentration of IL-1{beta} to 0.1 ng/ml further inhibited IGF-I-stimulated myogenin expression by 67%, and 1 ng/ml IL-1{beta} completely blocked it. In the absence of IGF-I, IL-1{beta} affected neither the mass of myogenin nor the loading control protein {alpha}-tubulin. These results demonstrate that IL-1{beta}, at picogram concentrations, inhibits the ability of IGF-I to induce expression of the critical transcription factor myogenin.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Proinflammatory cytokines are implicated as an etiological agent in a variety of muscle pathologies ranging from wasting that is associated with AIDS (39) and terminal cancer (40), the sarcopenia of aging (41), and inflammatory myopathies (1). In this study, we explored the hypothesis that IL-1{beta} acts by impairing the biological activity of the IGF-I receptor, which is required for progenitor cells to differentiate into myofibers and then more mature muscle cells. These experiments demonstrate that IL-1{beta} acts directly on myoblasts by impairing the ability of IGF-I to promote their differentiation into more mature cells that then express the sarcomere protein, myosin heavy chain (Fig. 1). As little as 0.01 ng/ml IL-1{beta} significantly suppresses IGF-I-stimulation of protein synthesis by C2C12 myoblasts (Fig. 2). Similarly, IL-1{beta} at 0.01 ng/ml significantly impairs, and at 1 ng/ml it completely blocks IGF-I-induced expression of myogenin (Fig. 5), which is required for the differentiation of mononucleated myoblasts into multinucleated myotubes. In the absence of IGF-I, none of these inhibitory properties of IL-1{beta} are detectable. Importantly, analysis of apoptosis and cell death established that the inhibition by IL-1{beta} is not simply due to killing the cells or by preventing IGF-I from increasing cell survival (Fig. 3). Instead, IL-1{beta} suppresses the ability of IGF-I to tyrosine phosphorylate both of the major IRS docking proteins that are required to transduce downstream signals (Fig. 3). Not even the highest concentration of IL-1{beta} (10 ng/ml) inhibits intrinsic tyrosine kinase activity of the IGF-I receptor {beta}-chain (Fig. 4). Collectively, these data confirm that IL-1{beta} inhibits muscle cell differentiation, but that it does so by inhibiting the biological actions of a potent anabolic hormone, IGF-I. These new findings are consistent with the emerging idea that proinflammatory cytokines induce IGF resistance, much as they do in insulin resistance in type II diabetes (reviewed in Ref.42).



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  		FIGURE 5. Expression of the key myogenic transcription factor myogenin in response to IGF-I is blocked by IL-1{beta}. C2C12 myoblasts induced to differentiate with 50 ng/ml IGF-I were first pretreated with IL-1{beta} for 1 h. Myogenin expression was detected by Western blot analysis of whole cell lysates 24 to 30 h later. IGF-I-induced abundant expression of myogenin, as shown in both a representative Western blot (A) and a densitometric summary of three independent experiments (B). IL-1{beta} significantly reduced IGF-I-induced expression of myogenin in a dose-dependent manner, with as little as 0.01 ng/ml IL-1{beta} causing significant inhibition (**, p < 0.01). In the absence of IGF-I, IL-1{beta} did not significantly impair expression of basal levels of either myogenin or the loading control protein {alpha}-tubulin (p > 0.10).

 
Extensive studies have been performed in whole animals to demonstrate that proinflammatory cytokines are important mediators of muscle atrophy. There are several reviews that provide extensive citations of animal studies showing that proinflammatory cytokines induce muscle wasting (1, 39, 43, 44). Chronic administration of IL-{beta} to rats reduces protein synthesis and muscle mass (2), whereas administration of the IL-1 receptor antagonist to septic mice prevents the decrease in protein synthesis, translational efficiency and muscle mass (2). Interference with myoblast differentiation could also have profound effects in patients with inflammatory myopathies (5). Although proinflammatory cytokines reduce muscle mass, the possibility that IL-1{beta} acts by inhibiting the actions of a growth factor that is required for muscle development has not been explored. In this study we provide the first evidence that IL-1{beta} blocks protein synthesis and myogenesis by inducing a state of IGF-I resistance. Treatment of either primary porcine or murine C2C12 myoblasts with an optimal concentration of IGF-I causes a 50% increase in protein synthesis (Fig. 2) over the relatively short time frame of 5 h. In the presence of 1–10 ng/ml IL-1{beta}, the anabolic actions of IGF-I are completely blocked. Even 0.01 ng/ml IL-1{beta} significantly impairs IGF-I stimulation of protein synthesis. It is also possible that IL-1{beta} may have even a more pronounced inhibition of protein synthesis at suboptimal IGF-I concentrations. In the absence of IGF-I, IL-1{beta} does not affect basal protein synthesis.

IL-1{beta} may act by simply killing myoblasts and decreasing the number of live cells capable of synthesizing protein in response to IGF-I. A variety of stimuli induce a controlled process of cell death referred to as apoptosis (45). For example, in pancreatic {beta} cells, IL-1{beta} induces apoptosis (46), whereas IGF-I acts as a survival factor (47). Surprisingly, we found that IGF-I modestly, but significantly, increases survival of myoblasts during a short 5 h time frame. However, treatment of murine myoblasts with IL-1{beta} alone, at concentrations ranging from 0.1 to 10 ng/ml, does not induce apoptosis or increase the number of dead cells. Furthermore, IL-1{beta} does not impair the minimal, but significant, increase in cell survival that is induced by IGF-I. It is therefore highly unlikely that the ability of proinflammatory cytokines to inhibit the anabolic actions of IGF-I occurs by a cytotoxic mechanism. These data establish that very low physiological concentrations of IL-1{beta} act to block the critical anabolic actions of IGF-I in progenitor muscle cells that control muscle mass.

Differentiation of myoblasts requires the expression of myogenic transcription factors that bind to specific elements (E-boxes) in the promoter of proteins that are expressed in differentiated myotubes. Myogenin is a myogenic transcription factor that is required for myoblast differentiation. Eliminating myogenin expression in C2C12 myoblasts (48) or embryonic stem cells completely abolishes their ability to differentiate. Myogenin is thought to control differentiation by acting as a critical cotranscription factor for the expression of myosin heavy chain (38) and other muscle specific proteins (49). IGF-I promotes differentiation by inducing the synthesis of myogenin (20). We therefore explored the possibility IL-1{beta} inhibits IGF-I dependent myogenesis by preventing myogenin expression. Indeed, consistent with our protein synthesis data, as little as 0.01 ng/ml IL-1{beta} suppresses the increase in myogenin expression only in the presence of IGF-I (Fig. 5). It remains unknown whether the IGF-I-induced increase in myosin expression, as well as its inhibition by IL-1{beta} (Fig. 1A), is the result of more or less myosin being synthesized per cell, more cells making myosin, or both. Four results suggest the former explanation is correct: 1) The amount of myosin per unit of {alpha}-tubulin is increased by IGF-I (Fig. 1B) because these data were calculated by dividing densitometric intensities of myosin heavy chain by its respective {alpha}-tubulin loading control; 2) the critical transcription factor myogenin is required for differentiation, and myogenin expression per unit of {alpha}-tubulin is increased by IGF-I and reduced by IL-1{beta} (Fig. 5B); 3) expression of both myosin and myogenin was standardized on the basis of a fixed amount of protein (75 µg) per lane rather than simply adding all the protein from a given culture of cells; and 4) because size of the DNA unit is generally accepted to increase with cell differentiation, the {alpha}-tubulin loading control is critical. Expression of both myosin and myogenin per unit of {alpha}-tubulin indicates that independent of the number of myoblasts present or degree of differentiation, IGF-I specifically increases the level of myosin and myogenin on a per cell basis. Collectively, we propose that IL-1{beta} reduces the ability of IGF-I to increase synthesis of myogenin, which subsequently prevents the IGF-I-induced increase in myosin expression. These data suggest that there is substantial cross-talk between proinflammatory cytokines and growth factors. This concept of one receptor influencing the actions of an unrelated receptor is similar in principle to cross-talk between the {beta}2-adrenergic receptor for the neurotransmitter norepinephrine and the B cell receptor (50).

Data in the present experiments provide evidence that signaling through the IGF-I receptor in myoblasts is somehow disrupted by proinflammatory cytokines. We have therefore begun to define the intracellular mechanisms by which IL-1{beta} induces IGF resistance. Activation of the IGF-I receptor results in tyrosine phosphorylation of the IRS-1 and IRS-2 proximal docking proteins that transduce the activation signal to more distal proteins that are well known to regulate protein synthesis (reviewed in Ref.37). TNF-{alpha} induces insulin resistance by impairing the ability of insulin to activate these IRS adaptor proteins (51). In this study, we extend our previous data with TNF-{alpha} to demonstrate the IL-1{beta} also induces a state of IGF-I resistance by targeting IRS-1 and IRS-2 (Fig. 3). IL-1{beta} (1 ng/ml) impairs the ability of IGF-I to tyrosine phosphorylate both IRS-1 and IRS-2 by ~50%. No reduction in tyrosine phosphorylation of the IGF-IR is detected (Fig. 4), providing evidence that IL-1{beta} directly targets the IRS proteins. Currently, the specific contribution of IRS-1 and IRS-2 to myoblast differentiation is not well defined. In vivo, genetically engineered mice that are defective in IRS-2 are insulin resistant, but have minimal growth defects (52). IRS-1 knockout mice display a pronounced reduction in skeletal muscle mass relative to body weight when compared with either IRS-1 homozygous or heterozygous mice (53). Mating transgenic mice that overexpress IGF-I with IRS-1-deficient mice is unable to compensate for the smaller muscle mass of the IRS-1-deficient mice (53). These data indicate that IRS-1 is the major docking protein by which IGF-I regulates muscle mass, but it is likely that IRS-1 and IRS-2 compensate for each other to some extent. For example, IRS-1 knockout mice display an increase in the mass of IRS-2 (54). Importantly, our results demonstrate the IL-1{beta} suppresses IGF-I-induced tyrosine phosphorylation of both major IRS docking proteins in myoblasts.

Increasing IGF-I through administration of growth hormone has been used to increase muscle mass in elderly subjects (55) as well as in patients suffering from muscle wasting due to myopathies (56) and HIV (57). However, increasing IGF-I concentrations in these patients experiencing chronic inflammatory conditions only partially restores muscle mass. Directly blocking the actions of TNF-{alpha} using specific antagonists is only partially effective at preventing muscle loss (reviewed in Ref.58). This is likely due to the redundant actions of TNF-{alpha} and IL-1{beta}, as we have demonstrated in muscle cells (7). Although IGF-I is well known to be required for muscle cells to differentiate, there are no reports of the possibility that IL-1{beta} acts by interfering with the myogenic properties of this critical growth factor. In this study we provide data supporting this concept by demonstrating that IL-1{beta} significantly impairs the actions of IGF-I via a post-IGF-I receptor mechanism by targeting the IRS-1 and IRS-2 proteins. IRS-2 is now a leading candidate for augmenting insulin action in type 2 diabetes (59). It is therefore significant that the inhibitory properties of IL-1{beta} in muscle cell differentiation involve a reduction in tyrosine phosphorylation of IRS-2, supporting the idea that this newly defined interaction between IL-1{beta} and hormones is critical in myogenesis. A clearer understanding of how proinflammatory cytokines impair muscle cell hormone responsiveness should allow for the development of a multifaceted approach to treat muscle wasting, even in the presence of chronic inflammation.


   Footnotes
 
1 This work was supported by Grant AI-50442 from the National Institutes of Health (to K.W.K.). Back

2 Address correspondence and reprint requests to Dr. Suzanne R. Broussard, University of Illinois, Laboratory of Immunophysiology, 207 Edward R. Madigan Laboratory, 1201 West Gregory Drive, Urbana, IL 61801. E-mail address: broussar{at}uiuc.edu Back

3 Abbreviations used in this paper: IGF, insulin-like growth factor; IRS, insulin receptor substrate. Back

Received for publication February 5, 2004. Accepted for publication April 1, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Figarella-Branger, D., M. Civatte, C. Bartoli, J. F. Pellissier. 2003. Cytokines, chemokines, and cell adhesion molecules in inflammatory myopathies. Muscle Nerve 28:659.[Medline]
  2. Cooney, R. N., G. O. Maish, III, T. Gilpin, M. L. Shumate, C. H. Lang, T. C. Vary. 1999. Mechanism of IL-1 induced inhibition of protein synthesis in skeletal muscle. Shock 11:235.[Medline]
  3. Lang, C. H., R. A. Frost, A. C. Nairn, D. A. MacLean, T. C. Vary. 2002. TNF-{alpha} impairs heart and skeletal muscle protein synthesis by altering translation initiation. Am. J. Physiol. 282:E336.
  4. Snider, L., S. J. Tapscott. 2003. Emerging parallels in the generation and regeneration of skeletal muscle. Cell 113:811.[Medline]
  5. Melone, M. A., G. Peluso, O. Petillo, U. Galderisi, R. Cotrufo. 1999. Defective growth in vitro of Duchenne muscular dystrophy myoblasts: the molecular and biochemical basis. J. Cell. Biochem. 76:118.[Medline]
  6. Kumar, A., A. M. Boriek. 2003. Mechanical stress activates the nuclear factor-{kappa}B pathway in skeletal muscle fibers: a possible role in Duchenne muscular dystrophy. FASEB J. 17:386.[Abstract/Free Full Text]
  7. Broussard, S. R., R. H. McCusker, J. E. Novakofski, K. Strle, W. H. Shen, R. W. Johnson, G. G. Freund, R. Dantzer, K. W. Kelley. 2003. Cytokine-hormone interactions: tumor necrosis factor {alpha} impairs biologic activity and downstream activation signals of the insulin-like growth factor I receptor in myoblasts. Endocrinology 144:2988.[Abstract/Free Full Text]
  8. Miller, S. C., H. Ito, H. M. Blau, F. M. Torti. 1988. Tumor necrosis factor inhibits human myogenesis in vitro. Mol. Cell. Biol. 8:2295.[Abstract/Free Full Text]
  9. Layne, M. D., S. R. Farmer. 1999. Tumor necrosis factor-{alpha} and basic fibroblast growth factor differentially inhibit the insulin-like growth factor-I induced expression of myogenin in C2C12 myoblasts. Exp. Cell Res. 249:177.[Medline]
  10. Langen, R. C., A. M. Schols, M. C. Kelders, E. F. Wouters, Y. M. Janssen-Heininger. 2001. Inflammatory cytokines inhibit myogenic differentiation through activation of nuclear factor-{kappa}B. FASEB J. 15:1169.[Abstract/Free Full Text]
  11. Szalay, K., Z. Razga, E. Duda. 1997. TNF inhibits myogenesis and downregulates the expression of myogenic regulatory factors myoD and myogenin. Eur. J. Cell Biol. 74:391.[Medline]
  12. Frost, R. A., C. H. Lang. 2003. Regulation of insulin-like growth factor-I in skeletal muscle and muscle cells. Minerva Endocrinol. 28:53.[Medline]
  13. Glass, D. J.. 2003. Signalling pathways that mediate skeletal muscle hypertrophy and atrophy. Nat. Cell Biol. 5:87.[Medline]
  14. Musaro, A., K. McCullagh, A. Paul, L. Houghton, G. Dobrowolny, M. Molinaro, E. R. Barton, H. L. Sweeney, N. Rosenthal. 2001. Localized Igf-1 transgene expression sustains hypertrophy and regeneration in senescent skeletal muscle. Nat. Genet. 27:195.[Medline]
  15. Grounds, M. D.. 2002. Reasons for the degeneration of ageing skeletal muscle: a central role for IGF-1 signalling. Biogerontology 3:19.[Medline]
  16. Barton, E. R., L. Morris, A. Musaro, N. Rosenthal, H. L. Sweeney. 2002. Muscle-specific expression of insulin-like growth factor I counters muscle decline in mdx mice. J. Cell Biol. 157:137.[Abstract/Free Full Text]
  17. Lynch, G. S., S. A. Cuffe, D. R. Plant, P. Gregorevic. 2001. IGF-I treatment improves the functional properties of fast- and slow-twitch skeletal muscles from dystrophic mice. Neuromuscul. Disord. 11:260.[Medline]
  18. Frost, R. A., C. H. Lang, M. C. Gelato. 1997. Transient exposure of human myoblasts to tumor necrosis factor-{alpha} inhibits serum and insulin-like growth factor-I stimulated protein synthesis. Endocrinology 138:4153.[Abstract/Free Full Text]
  19. Palmer, R. M., M. G. Thompson, R. M. Knott, G. P. Campbell, A. Thom, K. S. Morrison. 1997. Insulin and insulin-like growth factor-I responsiveness and signalling mechanisms in C2C12 satellite cells: effect of differentiation and fusion. Biochim. Biophys. Acta 1355:167.[Medline]
  20. Florini, J. R., D. Z. Ewton, S. L. Roof. 1991. Insulin-like growth factor-I stimulates terminal myogenic differentiation by induction of myogenin gene expression. Mol. Endocrinol. 5:718.[Abstract/Free Full Text]
  21. Wilson, E. M., M. M. Hsieh, P. Rotwein. 2003. Autocrine growth factor signaling by insulin-like growth factor-II mediates MyoD-stimulated myocyte maturation. J. Biol. Chem. 278:41109.[Abstract/Free Full Text]
  22. Cheng, Z. Q., S. Adi, N. Y. Wu, D. Hsiao, E. J. Woo, E. H. Filvaroff, T. A. Gustafson, S. M. Rosenthal. 2000. Functional inactivation of the IGF-I receptor delays differentiation of skeletal muscle cells. J. Endocrinol. 167:175.[Abstract]
  23. Powell-Braxton, L., P. Hollingshead, C. Warburton, M. Dowd, S. Pitts-Meek, D. Dalton, N. Gillett, T. A. Stewart. 1993. IGF-I is required for normal embryonic growth in mice. Genes Dev. 7:2609.[Abstract/Free Full Text]
  24. Liu, J. P., J. Baker, A. S. Perkins, E. J. Robertson, A. Efstratiadis. 1993. Mice carrying null mutations of the genes encoding insulin-like growth factor I (Igf-1) and type 1 IGF receptor (Igf1r). Cell 75:59.[Medline]
  25. Shen, W. H., Y. Yin, S. R. Broussard, R. H. McCusker, G. G. Freund, R. Dantzer, K. W. Kelley. 2004. TNF{alpha} inhibits cyclin A expression and RB hyperphosphorylation triggered by IGF-I induction of new E2F-1 synthesis. J. Biol. Chem. 279:7438.[Abstract/Free Full Text]
  26. Shen, W. H., J. H. Zhou, S. R. Broussard, G. G. Freund, R. Dantzer, K. W. Kelley. 2002. Proinflammatory cytokines block growth of breast cancer cells by impairing signals from a growth factor receptor. Cancer Res. 62:4746.[Abstract/Free Full Text]
  27. Kelley, K. W.. 2004. Norman Cousins Memorial Lecture, from hormones to immunity: the physiology of immunology. Brain Behav. Immun. 18:95.[Medline]
  28. Minshall, C., S. Arkins, G. G. Freund, K. W. Kelley. 1996. Requirement for phosphatidylinositol 3'-kinase to protect hemopoietic progenitors against apoptosis depends upon the extracellular survival factor. J. Immunol. 156:939.[Abstract]
  29. Strle, K., J. H. Zhou, S. R. Broussard, H. D. Venters, R. W. Johnson, G. G. Freund, R. Dantzer, K. W. Kelley. 2002. IL-10 promotes survival of microglia without activating Akt. J. Neuroimmunol. 122:9.[Medline]
  30. SAS Institute 2000. Statistical Analysis System/STAT Release 8.1 SAS, Cary, NC.
  31. Ji, S. Q., S. Neustrom, G. M. Willis, M. E. Spurlock. 1998. Proinflammatory cytokines regulate myogenic cell proliferation and fusion but have no impact on myotube protein metabolism or stress protein expression. J. Interferon Cytokine Res. 18:879.[Medline]
  32. Myer, A., E. N. Olson, W. H. Klein. 2001. MyoD cannot compensate for the absence of myogenin during skeletal muscle differentiation in murine embryonic stem cells. Dev. Biol. 229:340.[Medline]
  33. O’Neill, L. A.. 2000. The interleukin-1 receptor/Toll-like receptor superfamily: signal transduction during inflammation and host defense. Sci. STKE. 2000:RE1.
  34. Liu, Q., D. Schacher, C. Hurth, G. G. Freund, R. Dantzer, K. W. Kelley. 1997. Activation of phosphatidylinositol 3'-kinase by insulin-like growth factor-I rescues promyeloid cells from apoptosis and permits their differentiation into granulocytes. J. Immunol. 159:829.[Abstract]
  35. Crown, A. L., X. L. He, J. M. Holly, S. L. Lightman, C. E. Stewart. 2000. Characterisation of the IGF system in a primary adult human skeletal muscle cell model, and comparison of the effects of insulin and IGF-I on protein metabolism. J. Endocrinol. 167:403.[Abstract]
  36. Stewart, C. E., P. Rotwein. 1996. Insulin-like growth factor-II is an autocrine survival factor for differentiating myoblasts. J. Biol. Chem. 271:11330.[Abstract/Free Full Text]
  37. Rhodes, C. J., M. F. White. 2002. Molecular insights into insulin action and secretion. Eur. J. Clin. Invest. 32:(Suppl 3):3.
  38. Charbonnier, F., B. D. Gaspera, A. S. Armand, W. J. Van der Laarse, T. Launay, C. Becker, C. L. Gallien, C. Chanoine. 2002. Two myogenin-related genes are differentially expressed in Xenopus laevis myogenesis and differ in their ability to transactivate muscle structural genes. J. Biol. Chem. 277:1139.[Abstract/Free Full Text]
  39. Baracos, V. E.. 2000. Regulation of skeletal-muscle-protein turnover in cancer-associated cachexia. Nutrition 16:1015.[Medline]
  40. Belec, L., D. Meillet, A. Hernvann, G. Gresenguet, R. Gherardi. 1994. Differential elevation of circulating interleukin-1{beta}, tumor necrosis factor {alpha}, and interleukin-6 in AIDS-associated cachectic states. Clin. Diagn. Lab. Immunol. 1:117.[Abstract/Free Full Text]
  41. Hepple, R. T.. 2003. Sarcopenia: a critical perspective. Sci. Aging Knowl. Environ. 46:pe31.
  42. Wellen, K. E., G. S. Hotamisligil. 2003. Obesity-induced inflammatory changes in adipose tissue. J. Clin. Invest. 112:1785.
  43. Argiles, J. M., S. H. Meijsing, J. Pallares-Trujillo, X. Guirao, F. J. Lopez-Soriano. 2001. Cancer cachexia: a therapeutic approach. Med. Res. Rev. 21:83.[Medline]
  44. Lundberg, I. E., P. Nyberg. 1998. New developments in the role of cytokines and chemokines in inflammatory myopathies. Curr. Opin. Rheumatol. 10:521.[Medline]
  45. Franklin, R. A., J. A. McCubrey. 2000. Kinases: positive and negative regulators of apoptosis. Leukemia 14:2019.[Medline]
  46. Nikulina, M. A., N. Sandhu, Z. Shamim, N. A. Andersen, A. Oberson, P. Dupraz, B. Thorens, A. E. Karlsen, C. Bonny, T. Mandrup-Poulsen. 2003. The JNK binding domain of islet-brain 1 inhibits IL-1 induced JNK activity and apoptosis but not the transcription of key proapoptotic or protective genes in insulin-secreting cell lines. Cytokine 24:13.[Medline]
  47. Gallaher, B. W., R. Hille, K. Raile, W. Kiess. 2001. Apoptosis: live or die-hard work either way!. Horm. Metab. Res. 33:511.[Medline]
  48. Dedieu, S., G. Mazeres, P. Cottin, J. J. Brustis. 2002. Involvement of myogenic regulator factors during fusion in the cell line C2C12. Int. J. Dev. Biol. 46:235.[Medline]
  49. Biesiada, E., Y. Hamamori, L. Kedes, V. Sartorelli. 1999. Myogenic basic helix-loop-helix proteins and Sp1 interact as components of a multiprotein transcriptional complex required for activity of the human cardiac {alpha}-actin promoter. Mol. Cell. Biol. 19:2577.[Abstract/Free Full Text]
  50. Kohm, A. P., A. Mozaffarian, V. M. Sanders. 2002. B cell receptor- and {beta}2-adrenergic receptor-induced regulation of B7-2 (CD86) expression in B cells. J. Immunol. 168:6314.[Abstract/Free Full Text]
  51. Hirosumi, J., G. Tuncman, L. Chang, C. Z. Gorgun, K. T. Uysal, K. Maeda, M. Karin, G. S. Hotamisligil. 2002. A central role for JNK in obesity and insulin resistance. Nature 420:333.[Medline]
  52. Withers, D. J., J. S. Gutierrez, H. Towery, D. J. Burks, J. M. Ren, S. Previs, Y. Zhang, D. Bernal, S. Pons, G. I. Shulman, et al 1998. Disruption of IRS-2 causes type 2 diabetes in mice. Nature 391:900.[Medline]
  53. Pete, G., C. R. Fuller, J. M. Oldham, D. R. Smith, A. J. D’Ercole, C. R. Kahn, P. K. Lund. 1999. Postnatal growth responses to insulin-like growth factor I in insulin receptor substrate-1-deficient mice. Endocrinology 140:5478.[Abstract/Free Full Text]
  54. Araki, E., M. A. Lipes, M. E. Patti, J. C. Bruning, B. Haag, III, R. S. Johnson, C. R. Kahn. 1994. Alternative pathway of insulin signalling in mice with targeted disruption of the IRS-1 gene. Nature 372:186.[Medline]
  55. Butterfield, G. E., J. Thompson, M. J. Rennie, R. Marcus, R. L. Hintz, A. R. Hoffman. 1997. Effect of rhGH and rhIGF-I treatment on protein utilization in elderly women. Am. J. Physiol. 272:E94.
  56. Cittadini, A., L. Ines Comi, S. Longobardi, V. Rocco Petretta, C. Casaburi, L. Passamano, B. Merola, E. Durante-Mangoni, L. Sacca, L. Politano. 2003. A preliminary randomized study of growth hormone administration in Becker and Duchenne muscular dystrophies. Eur. Heart J. 24:664.[Abstract/Free Full Text]
  57. McNurlan, M. A., P. J. Garlick, R. T. Steigbigel, K. A. DeCristofaro, R. A. Frost, C. H. Lang, R. W. Johnson, A. M. Santasier, C. J. Cabahug, J. Fuhrer, M. C. Gelato. 1997. Responsiveness of muscle protein synthesis to growth hormone administration in HIV-infected individuals declines with severity of disease. J. Clin. Invest. 100:2125.[Medline]
  58. von Haehling, S., S. Genth-Zotz, S. D. Anker, H. D. Volk. 2002. Cachexia: a therapeutic approach beyond cytokine antagonism. Int. J. Cardiol. 85:173.[Medline]
  59. White, M. F.. 2003. Insulin signaling in health and disease. Science 302:1710.[Abstract/Free Full Text]



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K. Strle, S. R. Broussard, R. H. McCusker, W.-H. Shen, R. W. Johnson, G. G. Freund, R. Dantzer, and K. W. Kelley
Proinflammatory Cytokine Impairment of Insulin-Like Growth Factor I-Induced Protein Synthesis in Skeletal Muscle Myoblasts Requires Ceramide
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