Inhibition of muscle insulin-like growth factor I expression by tumor necrosis factor-alpha

Laura Fernández-Celemín, Nevi Pasko, Valérie Blomart, and Jean-Paul Thissen

Unité de Diabétologie et Nutrition, Université catholique de Louvain, B-1200 Brussels, Belgium


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The role of TNF-alpha in muscle catabolism is well established, but little is known about the mechanisms of its catabolic action. One possibility could be that TNF-alpha impairs the production of local growth factors like IGF-I. The aim of this study was to investigate whether TNF-alpha can directly inhibit IGF-I gene and protein expression in muscle. First, we investigated whether the acute inflammation induced by endotoxin injection changes IGF-I and TNF-alpha mRNA in rat tibialis anterior muscle. Endotoxin rapidly increased TNF-alpha mRNA (7-fold at 1 h, P < 0.001) and later decreased IGF-I mRNA (-73% at 12 h, P < 0.001). Furthermore, in a model of C2C12 myotubes, TNF-alpha strongly inhibited IGF-I mRNA and protein (-73 and -47% after 72 h, P < 0.001 and P < 0.01, respectively). Other proinflammatory cytokines failed to inhibit IGF-I mRNA. The effect of TNF-alpha on IGF-I mRNA was not mediated by nitric oxide, and the activation of NF-kappa B was insufficient to inhibit IGF-I expression. Taken together, our data suggest that TNF-alpha induced in muscle after LPS injection can locally inhibit IGF-I expression. The inhibition of muscle IGF-I production could contribute to the catabolic effect of TNF-alpha .

skeletal muscle; lipopolysaccharide; C2C12 cells


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A DECREASE IN LEAN BODY MASS is commonly observed in pathological conditions such as sepsis, cancer, AIDS, or chronic inflammatory diseases. This change in body composition may lead to functional alterations that increase mortality and hamper a patient's rehabilitation. The loss of lean mass is mainly due to a loss of skeletal muscle (4). The mechanisms of muscle catabolism involve complex interactions among several mediators and remain nowadays only partially unraveled. Hence, a better understanding of these interactions is important for developing successful therapies able to prevent the decline in lean body mass and reduce morbidity and mortality.

Tumor necrosis factor (TNF)-alpha is a proinflammatory cytokine that is essential for a successful response against invading pathogens (19, 41, 43). On the other hand, TNF-alpha is considered to play a major role in muscle catabolism. First, circulating levels of TNF-alpha are markedly increased in catabolic states (3). Second, enhanced protein degradation and muscle loss have been observed in TNF-alpha -transgenic animals (29) after chronic administration of the cytokine (23, 54) or in animals bearing TNF-alpha -producing tumors (12). Furthermore, administration of anti-TNF-alpha antibodies or of TNF-alpha -soluble receptor attenuates the catabolic reaction (28, 42).

Catabolic actions of TNF-alpha in skeletal muscle can be exerted in different ways. One possibility is that TNF-alpha acts directly on skeletal muscle to induce muscle catabolism. Indeed, TNF-alpha has been reported to inhibit protein synthesis and myogenesis in myoblasts (22, 25, 34). Recent observations indicate that TNF-alpha can also stimulate the proteolysis of myosin heavy chains in C2C12 myotubes by activating the ubiquitin-proteasome pathway (36). Besides, TNF-alpha increases apoptotic cell death in skeletal muscle (39). Alternatively, TNF-alpha can act indirectly to stimulate skeletal muscle catabolism by modifying hormones that regulate protein turnover (15, 53), by increasing the production of other cytokines (53), or by inducing anorexia (54).

Insulin-like growth factor I (IGF)-I is an anabolic growth factor responsible for normal growth and development (18). Autocrine IGF-I production has been demonstrated to play a crucial role in muscle growth (47, 56). In skeletal muscle, IGF-I acts as an anabolic growth factor stimulating protein synthesis (5) as well as proliferation and differentiation of satellite cells (18). IGF-I has also been reported to suppress proteolysis (21, 27) and to inhibit the ubiquitin-proteasome system (11). Finally, IGF-I exerts antiapoptotic effects in muscle, favoring the survival of differentiated cells (35).

Sepsis and endotoxin (LPS) injection, a classical model of acute catabolism, are associated with an increase in circulating TNF-alpha levels (3) and a decrease in muscle IGF-I (16). Taking into consideration the opposite actions of these two molecules in the control of muscle mass, we hypothesized that the catabolic effects of TNF-alpha might result from a direct inhibition of muscle IGF-I gene expression. The aims of this study were first to assess whether the acute inflammation caused by LPS is associated with a reduction in muscle IGF-I mRNA and an increase in muscle TNF-alpha mRNA and second to investigate the role of TNF-alpha in the decrease of muscle IGF-I caused by LPS.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. For in vivo experiments, lipopolysaccharide of Escherichia coli (LPS, serotype 0127:B8) was obtained from Sigma Chemical (St. Louis, MO) and was diluted to 1 mg/ml in sterile, endotoxin-free saline. For in vitro experiments, recombinant murine TNF-alpha , interleukin-1beta , and interferon-gamma (rmTNF-alpha , rmIL-1beta , rmIFN-gamma ) and recombinant rat IL-6 were purchased through R&D Systems (Abingdon, UK). NUNCLON plastic dishes were purchased from NUNC (Roskilde, Denmark), and Dulbecco's modified Eagle's medium (DMEM), fetal calf serum, horse serum (lot: 3010240D), nonessential amino acids, and L-glutamine were purchased from GIBCO-BRL Life Technologies (Paisley, UK).

In vivo experiment. Male Wistar rats (Katholieke Universiteit of Leuven, Leuven, Belgium) ~8 wk of age (218 ± 9.2 g; mean ± SD) were maintained for 1 wk under standardized conditions of light (12:12-h light-dark cycle) and temperature (22 ± 2°C). Access to animal chow was available only between 6:00 PM and 9:00 AM, whereas access to water was unrestricted. The morning of the 8th day, rats were injected intraperitoneally with LPS (750 µg/100 g body wt) and killed at different times after the injection. Tibialis anterior muscles were dissected, flash-frozen in liquid nitrogen, and stored at -80°C. The choice of tibialis anterior, a fast muscle, was based on the observation that fast muscles, composed mainly of white fiber and weakly resistant to fatigue, are more sensitive than slow muscles to catabolic stimuli (26, 17).

Muscle incubations. Male Wistar rats (221 ± 12 g, 7 wk old, n = 4; Janvier Breeding, Le Genest-Saint-Isle, France) were anesthetized with a mixture of Ketalar/Rompum (75:15 mg/kg body wt ip), and the extensor digitorum longus (EDL) muscles were gently dissected with intact tendons and mounted on supports at approximate resting length. Individual muscles were preincubated for 30 min and then incubated for 3 h in 20 ml of oxygenated Krebs-Ringer bicarbonate solution at 25°C in the presence or absence of LPS (10 µg/ml). Previous studies had demonstrated that, at 25°C, the histochemical and mechanical properties of muscles are better conserved than at temperatures above 25°C (45). After the 3-h incubation, muscles were deep-frozen in liquid nitrogen, and RNA extraction and Northern blot were performed as described in RNA extraction and Northern blot analysis.

C2C12 cell culture. Myoblasts from the muscle-derived C2C12 cell line were obtained from American Type Culture Collection (Manassas, VA). The seeding density used throughout the experiments was 0.4 × 106 cellules/plate of 10 mm diameter. Undifferentiated cells were grown in DMEM supplemented with 10% fetal calf serum, 1% penicillin-streptomycin (100 U · 100 µg-1 · ml-1), 1% nonessential amino acids, and 2% L-glutamine at 37°C in the presence of 5% CO2. This medium will be referred to as growth medium (GM). When cells reached 90-100% confluence (after 3 days), 10% heat-inactivated fetal calf serum was replaced by 2% heat-inactivated horse serum to induce myogenic differentiation. This medium will be referred to as differentiation medium (DM). Muscle cells were examined for evidence of myotube formation and growth by use of an inverted Olympus IMT microscope (Olympus Optical, Hamburg, Germany). The medium was changed every 48 h, and differentiation was allowed to continue for 96 h (4 days) before the experimentation period. To preserve the characteristics of the C2C12 cell line, the splitting of cells was done up to a maximum of seven times. The experiments lasted from 24 to 72 h, and the cytokines or other molecules used were added to the DM every 24 h at the doses indicated in the figure legends.

RNA extraction and Northern blot analysis. For in vivo experiments, RNA was extracted from tibialis anterior muscle with TRIzol reagent (Life Technologies, Paisley, UK) following the instructions of the manufacturer. Briefly, 100 mg of tibialis anterior muscle, previously pestled in liquid nitrogen, were homogenized with Ultraturrax (IKA-labortechnik, Staufen, Germany) in 1 ml of TRIzol reagent and centrifuged at 12,000 g for 10 min at 4°C. The supernatant was then added with chloroform, thoroughly vortexed, incubated for 10 min at room temperature, and centrifuged at 12,000 g for 15 min at 4°C. The aqueous phase containing the RNA was retained, and RNA was precipitated with isopropanol. The RNA pellet was washed with 75% ethanol, dried, resuspended in diethyl pyrocarbonate-treated water, and stored at -80°C. The concentration of total RNA was determined by spectrophotometry. For in vitro experiments, the cells were washed once with 1× PBS, pH 7.4, and then each plate was collected in 1.5 ml of TRIzol and processed as described for the in vivo experiments.

Twenty micrograms of total RNA from each sample were denatured in formaldehyde-MOPS and subjected to electrophoresis on 1% agarose gels. Homogeneity of the loading was assessed by UV transillumination of the gels after staining with ethidium bromide. The RNA was transferred to nylon membranes (Hybond N; Amersham, Buckinghamshire, UK) by capillary overnight blotting, and levels of IGF-I, IGF-II, or TNF-alpha mRNA were determined by hybridization with specific probes. For IGF-I, a 194-bp AvaII-HinfI rat IGF-I exon 4 cDNA (15) was inserted into the plasmid vector Bluescript pBSM13+ (Stratagene, La Jolla, CA) and linearized with HindIII. This probe includes only the coding region for the mature IGF-I peptide and detects all IGF-I mRNAs. For IGF-II, a 275-bp RSA1 rat IGF-II exon 3 (coding region of IGF-II) cDNA was ligated into the pBS+ (Stratagene) and linearized with EcoRI. Specific 32P-labeled RNA antisense probes were generated from linearized plasmids with uridine 5'-[alpha -32P]triphosphate (800 Ci/mmol; Amersham) by use of T7 (IGF-I) or T3 (IGF-II) RNA polymerase. Levels of TNF-alpha mRNA were determined by hybridization with a random-primed [32P]dCTP-labeled TNF-alpha probe (p6r TNF-alpha ) encompassing the rat TNF-alpha coding sequence (amino acids 44-231) (40). To verify uniform loading, control hybridization was performed with a 23-mer 18S oligonucleotide end-labeled with adenosine [gamma -32P]triphosphate by T4 polynucleotide kinase (Amersham).

For IGF-I and IGF-II, blots were prehybridized for 3-4 h at 65°C in buffer containing NaCl (0.8 M), Na phosphate (50 nM), EDTA (0.5 mM), Denhardt's (2 g/l each of bovine serum albumin, Ficoll, and polyvinylpyrrolidone), salmon sperm DNA (200 µg/ml), and formamide (50%). For TNF-alpha , blots were prehybridized for 4 h at 50°C in buffer containing NaCl (0.75 M), Na citrate (75 mM), SDS (0.25%), Denhardt's (2 g/l each of bovine serum albumin, Ficoll, and polyvinylpyrrolidone), salmon sperm DNA (200 µg/ml), and formamide (50%). They were then hybridized with the radiolabeled antisense RNA probe or cDNA probe (1 × 106 cpm/ml of buffer) for 16-18 h in a shaking water bath at 65°C (for IGF-I or IGF-II) and at 50°C (for TNF-alpha ). After hybridization, blots were washed several times. For IGF-I or IGF-II, blots were washed rapidly once with 300 ml of washing buffer (in mM: 20 Na phosphate, 50 NaCl, and 1 EDTA and 0.1% SDS) at room temperature and then more extensively twice for 30 min in a shaking waterbath at 70°C. For TNF-alpha , blots were washed twice for 20 min with 300 ml of washing buffer I (0.3 M NaCl, 30 mM Na citrate, 0.1% SDS) at room temperature and then twice with 300 ml of washing buffer II (0.03 M NaCl, 3 m Na citrate M, 0.25% SDS) for 20 min in a shaking water bath at 55°C. Membranes were finally exposed to XAR-5 Kodak film for several hours to several days at -80°C. The mRNA levels were quantified by densitometric scanning of the hybridization signal (LKB Ultroscan XL laser densitometry; LKB, Bromma, Sweden) with the use of software (Gel Scan, Pharmacia & Upjohn, Brussels, Belgium). IGF-I mRNA transcripts, as visualized by Northern blot analysis, presented a complex picture consisting of a large (7.5 kb) transcript, a group of transcripts ranging from 0.8 to 1.2 kb, and two additional minor transcripts of 1.7 and 4.7 kb. Because all of these transcripts may potentially be translated into IGF-I precursors, we performed a densitometric analysis of the four bands visible on the blot. For IGF-II, we considered 1.5- to 2.2-kb transcripts and a 4.6-kb transcript for the densitometric analysis. Densitometric values were normalized by assigning to the mRNA levels observed after 96 h postdifferentiation (D4, day on which experiments were initiated) an arbitrary value of 100%.

Cytoplasmic and nuclear extracts. After cells were washed twice with ice-cold PBS and harvested in 500 µl of cold buffer A (10 mM HEPES-KOH, pH 7.9, 5% glycerol, 100 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 1 µg/ml leupeptin, and 2 µg/ml aprotinin), cytoplasmic extracts were prepared by subjecting the cells to three cycles of freezing-thawing. Supernatants were then collected after samples were centrifuged for 10 s in a microcentrifuge. Nuclear extracts were prepared by resuspending the pellets in cold buffer B (20 mM HEPES-KOH, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 1 µg/ml leupeptin, and 2 µg/ml aprotinin). The resuspended pellets were mixed and incubated at 4°C for 20 min and centrifuged for 10 min in a microcentrifuge at 4°C. Supernatants were then collected and stored at -80°C. Protein content of the cytoplasmic and nuclear extracts was determined using Bradford's protein assay (Bio-Rad, Munich, Germany).

Electrophoretic mobility shift assay. For the electrophoretic mobility shift assay (EMSA), preannealed, chromatography-purified, double-stranded oligonucleotides containing the consensus DNA-binding sequence for NF-kappa B (5'-TCGAGGGCTGGGGATTCCCCATCTC-3') were radiolabeled with [gamma -32P]-ATP, and EMSA was performed as previously described (48). Briefly, 10 µg of nuclear proteins (prepared as described) were incubated with radiolabeled DNA probe for 20 min at room temperature and then electrophoresed on a 5% polyacrylamide gel. To verify the specificity of the binding reaction, an excess of unlabeled oligonucleotide was incubated with the nuclear extracts for 5 min at room temperature before the addition of the radiolabeled probe and then incubated for 20 min. For supershift, 4 µl (0.8 µg/ml) of anti-p50- or anti-p65-specific polyclonal antibody for the NF-kappa B subunits were incubated with the extract samples for 5 min before the addition of the labeled probe. NF-kappa B p65 and p50 antibodies are, respectively, rabbit and goat affinity-purified polyclonal antibodies raised against a peptide mapping the carboxy terminus of NF-kappa B p65 or p50 of human origin and identical to the corresponding mouse sequence (Santa Cruz Biotechnology, Santa Cruz, CA). Gels were dried and exposed for 20 h to a Hyperfilm MP (Amersham).

Creatine kinase activity. Myogenic differentiation was assessed biochemically on cytoplasmic extracts (prepared as described) via determination of muscle creatine kinase (CK) activity with a SYNCHRON LX Systems reagent kit (Beckman Coulter) following manufacturer's instructions. Specific CK activity was calculated after correction for total protein.

Nitrite determination. Nitrite (NO<UP><SUB>2</SUB><SUP>−</SUP></UP>) is a stable end product used extensively as an indicator of NO production by cultured cells. In our experimental conditions, nitrite accumulation was assayed by the Griess reaction, according to the method described previously (20). Briefly, 400 µl of cell culture medium were mixed with three times the amount of Griess reagent (1% sulfanilamide, 0.1% naphthylenediamine, 2.5% H3PO4). Samples were incubated at room temperature for 10 min, and absorbance was subsequently read at 543 nm by a spectrophotometer. Nitrite concentrations were calculated compared with a sodium nitrite (NaNO2) standard curve.

End point PCR of TNF-alpha receptors. For PCR assay, 1 µg of total RNA from C2C12 myotubes was reverse transcribed into cDNA in the presence of 1× RT-PCR buffer (Superscript kit no. 18053-07, Life Technologies), 4.44 mM of dNTPs (no. 1581295, Roche Diagnostics), 88.8 pM of random hexamers (Clinical Molecular Biology Unit, St Luc Hospital, Brussels, Belgium), 11 mM DTT, and 50 U of superscript reverse transcriptase (Superscript Kit, Life Technologies) per reaction. The final volume was 18 µl, and cDNA synthesis was performed in the GeneAmp PCR system 2400 (Perkin-Elmer, Foster City, CA) as follows: 22°C for 10 min, 42°C for 1 h, and 99°C for 5 min.

End point PCR amplification was then carried out on 3 µl of the cDNA product, with 1× PCR buffer (Perkin-Elmer kit N808-0161), 1 mM MgCl2, 0.8 mM dNTP (no. 1581295; Roche Diagnostics), 0.5 mM of specific primers, and 3.5 U/reaction of Taq DNA polymerase (PWO-expand; Roche Diagnostics); the final volume was 50 µl. End point PCR was performed in the GeneAmp PCR system 2400 (Perkin-Elmer) as follows: 94°C for 1 min followed by 45 cycles [94°C for 1 min; 60°C (p60) and 58°C (p80) for 2 min; 72°C for 3 min]. The primer pairs used to amplify TNF-alpha Rp60 and Rp80 cDNA (GenBank nos. M59377 and M59378, respectively) are shown in Table 1. Negative control reverse transcription and amplification reactions were also run without RNA input or cDNA input. A portion (20 µl) of RT-PCR product was electrophoresed in 1% agarose gel in 1× Tris-borate-EDTA buffer (89 mM Tris base, 89 mM boric acid, 200 mM EDTA), together with positive controls (mouse spleen and WEG-1 cell cDNAs) and DNA molecular weight markers from 8 to 587 bp (Roche, Mannheim, Germany). The gel was stained with ethidium bromide and photographed under UV transillumination.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Summary of primer sequences

IGF-I RIA. Culture medium concentrations of IGF-I were measured by RIA after acid extraction of medium samples with 0.5 N HCl in silicic acid columns (C18 Sep-Pak cartridges from Waters, Milford, MA), as described previously (13). The rabbit IGF-I antiserum (UB-487) was used at a dilution of 1:40,000 and was a kind gift from Dr. L. E. Underwood (Chapel Hill, NC).

Statistical analysis. Statistical significance between samples was determined by one-way analysis of variance (ANOVA) followed by the Newman-Keuls multiple comparisons test. For in vivo experiments, each point is the mean ± SE of seven or eight animals. For in vitro experiments, each point is the mean ± SE of values obtained in three or six separate experiments, each performed in one new culture of C2C12 cells. Statistical significance was set at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Regulation of muscle expression of IGF-I and TNF-alpha by LPS. In a first step, we investigated the changes produced by LPS injection in muscle TNF-alpha mRNA and IGF-I mRNA.

Basal TNF-alpha gene expression was very low in tibialis anterior muscle of intact animals. LPS administration induced a rapid peak of TNF-alpha mRNA between 1 and 3 h, with a 7-fold increase at 1 and 2 h and a 5.5-fold increase at 3 h (P < 0.001 vs. time-matched saline group). Afterward, TNF-alpha transcripts decreased to reach basal values by 12 h (Fig. 1A). Animals continued to display symptoms of infection (gastric dilatation, decreased mobility, piloerection) until 12 h. Furthermore, LPS injection resulted in a progressive inhibition of tibialis anterior IGF-I gene expression that became significant after 6 and 12 h (61 and 73% reduction, respectively, P < 0.001 vs. time-matched saline group; Fig. 1B). This reduction was not due to the anorectic effect of LPS, as LPS-injected and saline-treated animals fasted throughout the day of the experiment.


View larger version (39K):
[in this window]
[in a new window]
 
Fig. 1.   Effect of LPS injection on TNF-alpha (A) and IGF-I (B) mRNA in rat tibialis anterior muscle. Male Wistar rats were injected ip with either 750 µg LPS/100 g body wt (n = 7-8) or saline (n = 4) and killed 1, 2, 3, 6, or 12 h after the injection. After tibialis anterior muscle RNA was extracted, 10 µg of total RNA were electrophoresed in each lane, and Northern blot analysis was performed as indicated in MATERIALS AND METHODS. Top: autoradiography; bottom: densitometric analysis. Data are expressed as means ± SE. *** P < 0.001 vs. time-matched saline.

Although LPS significantly decreased IGF-I gene expression in skeletal muscle, it did not seem to affect IGF-II expression (data not shown).

Muscle TNF-alpha and IGF-I mRNA levels in saline-injected animals were not different from those in noninjected controls (time 0 h), indicating that the injection procedure had no effect by itself on TNF-alpha and IGF-I gene expression (Fig. 1).

In vitro experiments with EDL muscles incubated in the presence or absence of LPS demonstrated that LPS is capable of rapidly inducing TNF-alpha gene expression in skeletal muscle (2-fold increase vs. control, P < 0.001; Fig. 2).


View larger version (49K):
[in this window]
[in a new window]
 
Fig. 2.   Induction of TNF-alpha mRNA in extensor digitorum longus (EDL) muscles incubated with LPS. Individual EDL muscles from normal rats were incubated for 3 h in the presence or absence of LPS (10 µg/ml). After the incubation, muscles were collected, and total RNA was extracted. Total RNA (20 µg) was electrophoresed in each lane, and Northern blot analysis was performed as indicated in MATERIALS AND METHODS. Top: autoradiography; bottom: densitometric analysis. Data are expressed as means ± SE of 4 separate experiments. *** P < 0.001 vs. control.

Regulation of myotube expression of IGF-I by TNF-alpha . Initial experiments served to characterize the time frame of C2C12 cell differentiation after replacement of GM with DM. This was done through analysis of cell morphology and muscle-specific differentiation markers. By day 4 of differentiation, myotubes were long and multinucleated and displayed contractile properties. The activity of CK, total cytoplasmic protein content, and myosin heavy-chain (MHC) content increased with differentiation (34). Because all of these parameters were stable between day 4 and day 8 of differentiation, experimental procedures were performed from day 4.

Inhibition of IGF-I gene and protein expression by TNF-alpha . Because in vivo results suggested a causal relationship between the increase in TNF-alpha expression and the reduction of IGF-I mRNA levels, we began by investigating the effect of this proinflammatory cytokine on the IGF-I gene expression in the C2C12 myotubes. In this model, TNF-alpha has been reported to exert a catabolic action leading to decrease in MHC content (37).

Four-day-differentiated myotubes were incubated for 2, 4, 6, 8, or 10 h in the presence or absence of TNF-alpha (5 ng/ml). TNF-alpha resulted in a progressive decrease of IGF-I mRNA levels to 48% of the time-matched control values at 8 h and to 62% at 10 h (Fig. 3A). A longer exposure of myotubes to TNF-alpha emphasized its inhibitory effect, further diminishing IGF-I mRNA levels by 66% at 24 h (P < 0.05), 69% at 48 h (P < 0.001), and 73% at 72 h (P < 0.001) vs. time-matched control expression (Fig. 3B). As expected from the decrease in IGF-I mRNA levels, exposure of myotubes to TNF-alpha for 72 h reduced IGF-I medium concentrations by 47% (control: 1.55 ± 0.25 ng/ml vs. TNF-alpha : 0.82 ± 0.08 ng/ml; P < 0.01, n = 6).


View larger version (51K):
[in this window]
[in a new window]
 
Fig. 3.   Time-dependent effect of TNF-alpha on IGF-I mRNA in C2C12 myotubes. After 4 days of differentiation (D4), C2C12 myotubes were incubated for a short-term period (A) or a long-term period (B) with TNF-alpha (5 ng/ml). D5, D6, and D7, 24, 48, and 72 h of incubation, respectively. At each time point, cells were collected, and total RNA was extracted. Total RNA (20 µg) was electrophoresed in each lane, and Northern blot analysis was performed as indicated in MATERIALS AND METHODS. Top: autoradiography; bottom: densitometric analysis. Data are expressed as means ± SE of 3 (A) or 6 (B) separate experiments. * P < 0.05 and *** P < 0.001 vs. time-matched control.

TNF-alpha decreased IGF-I mRNA levels in a dose-dependent manner. Incubation of 4-day-differentiated myotubes with 1 ng/ml of the cytokine decreased IGF-I mRNA by 27% compared with control values (P < 0.05). A dose of 5 ng/ml, which was used throughout the experiments unless otherwise specified in the figure legends, resulted in a 70% reduction of IGF-I transcripts (P < 0.001 vs. control). Higher doses of TNF-alpha (<= 50 ng/ml) did not inhibit further IGF-I gene expression (Fig. 4 and data not shown).


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 4.   Dose-dependent effect of TNF-alpha on IGF-I mRNA in C2C12 myotubes. After 4 days of differentiation (D4), C2C12 myotubes were incubated for 24 h with 1, 5, or 10 ng/ml of TNF-alpha . At each point, cells were collected and total RNA was extracted. Total RNA (20 µg) was electrophoresed in each lane, and Northern blot analysis was performed as indicated in MATERIALS AND METHODS. Top: autoradiography; bottom: densitometric analysis. Data are expressed as means ± SE of 3 separate experiments. * P < 0.05 and *** P < 0.001 vs. control (0 ng/ml TNF-alpha ).

Because mature C2C12 myotubes continue to express IGF-II, we studied whether TNF-alpha was able to cause IGF-II gene expression to decrease. Exposure of myotubes to 5 ng/ml TNF-alpha for 24, 48, or 72 h did not affect IGF-II transcripts, suggesting that TNF-alpha effect on IGF-I mRNA did not result from a generalized inhibitory effect on gene expression (Fig. 5).


View larger version (40K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of TNF-alpha on IGF-II mRNA in C2C12 myotubes. After 4 days of differentiation (D4), C2C12 myotubes were incubated for 24 (D5), 48 (D6), or 72 h (D7) with TNF-alpha (5 ng/ml). At each time point, cells were collected, and total RNA was extracted. Total RNA (20 µg) was electrophoresed in each lane, and Northern blot analysis was performed as indicated in MATERIALS AND METHODS. Top: autoradiography; bottom: densitometric analysis. Data are expressed as means ± SE of 3 separate experiments.

Reversibility and relation to differentiation state of the inhibition of IGF-I gene expression by TNF-alpha . The inhibition of IGF-I mRNA levels induced by 24-h exposure to TNF-alpha (5 ng/ml) was totally reversed by removal of the cytokine from the conditioned medium. Indeed, IGF-I mRNA values were restored to normal 24 h after TNF-alpha was removed (Fig. 6). This result indirectly indicates that the TNF-alpha inhibitory effect on IGF-I mRNA is independent of a cytotoxic effect of the cytokine.


View larger version (43K):
[in this window]
[in a new window]
 
Fig. 6.   Reversibility of TNF-alpha effect on IGF-I mRNA in C2C12 myotubes. After 4 days of differentiation (D4), C2C12 myotubes were incubated for 24 h (D5) with TNF-alpha (5 ng/ml). After 24 h, medium was changed to control medium, and cells were incubated for another 24 (D6) or 48 h (D7) without the cytokine. At each time point, cells were collected, and total RNA was extracted. Total RNA (20 µg) was electrophoresed in each lane, and Northern blot analysis was performed as indicated in MATERIALS AND METHODS. Top: autoradiography; bottom: densitometric analysis. Data are expressed as means ± SE of 3 separate experiments. ** P < 0.01 vs. time-matched control.

After 72 h of TNF-alpha treatment (5 ng/ml), myotube CK activity was slightly but significantly decreased (P < 0.01 vs. control; Fig. 7), suggesting a modest inhibitory effect of TNF-alpha on cell differentiation.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 7.   Effect of TNF-alpha on creatine kinase (CK) activity of C2C12 myotubes. After 4 days of differentiation (D4), C2C12 myotubes were incubated for 24 (D5), 48 (D6), or 72 h (D7) with TNF-alpha (5 ng/ml). CK activity was measured in cytoplasmic extracts, as indicated in MATERIALS AND METHODS, and normalized for total protein. Data are expressed as means ± SE of 3 separate experiments. ** P < 0.01 vs. time-matched control.

Effect of other proinflammatory cytokines and LPS on IGF-I gene expression. To determine whether other proinflammatory cytokines might be involved in the decrease of muscle IGF-I mRNA observed in vivo in response to LPS, we tested the effect of IL-1beta , IL-6, and IFN-gamma (5 ng/ml) on 4-day-differentiated myotubes. Although, as expected, TNF-alpha decreased IGF-I mRNA levels at all times tested (P < 0.05 at 24 h, P < 0.01 at 48 h, and P < 0.001 at 72 h vs. time-matched control group), none of the other cytokines (IL-1beta , IL-6, or IFN-gamma ) modified consistently the IGF-I mRNA levels in muscle cells (Fig. 8A). All together, these observations indicate that, of all the cytokines tested, TNF-alpha is the only one to significantly inhibit IGF-I mRNA.


View larger version (59K):
[in this window]
[in a new window]
 
Fig. 8.   Effect of different proinflammatory cytokines and LPS on IGF-I mRNA in C2C12 myotubes. After 4 days of differentiation (D4), C2C12 myotubes were incubated for 24 (D5), 48 (D6), or 72 h (D7) with 5 ng/ml of TNF-alpha , IL-1beta , IL-6, or IFN-gamma (A) or for 24 h (D5) with 2 µg/ml of LPS (B). At each time point, cells were collected, and total RNA was extracted. Total RNA (20 µg) was electrophoresed in each lane, and Northern blot analysis was performed as indicated in MATERIALS AND METHODS. Top: autoradiography; bottom: densitometric analysis. Data are expressed as means ± SE of 3 separate experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001 vs. time-matched control.

Because LPS injection resulted in decreased IGF-I mRNA levels in vivo, we studied whether LPS could directly influence IGF-I mRNA in differentiated myotubes. After 24 h of exposure to LPS (2 µg/ml), IGF-I gene expression was slightly decreased, albeit in a nonsignificant way (Fig. 8B).

Effect of the combination of TNF-alpha and IFN-gamma on IGF-I gene expression. Because IFN-gamma can potentiate some TNF-alpha actions (50), we investigated the effect of the combination of these two cytokines on IGF-I gene expression in C2C12 myotubes. Four-day-differentiated myotubes were treated for 24 h with TNF-alpha (3 ng/ml) and IFN-gamma (5 ng/ml) alone or in combination. As expected, IFN-gamma did not significantly modify IGF-I mRNA (29% decrease, P > 0.05), whereas TNF-alpha alone caused a 74% decrease of IGF-I mRNA levels (P < 0.01 vs. control). When myotubes were challenged with the combination of TNF-alpha and IFN-gamma , the IGF-I mRNA levels were further decreased to 90% of the control values (P < 0.001 vs. control and P < 0.05 vs. TNF-alpha alone), suggesting more an additive action of both cytokines than a potentiation (Fig. 9).


View larger version (35K):
[in this window]
[in a new window]
 
Fig. 9.   Effect of TNF-alpha and IFN-gamma on IGF-I mRNA in C2C12 myotubes. After 4 days of differentiation (D4), C2C12 myotubes were incubated for 24 h (D5) with TNF-alpha (3 ng/ml), IFN-gamma (5 ng/ml), or their combination. Cells were collected, and total RNA was extracted. Total RNA (20 µg) was electrophoresed in each lane, and Northern blot analysis was performed as indicated in MATERIALS AND METHODS. Top: autoradiography; bottom: densitometric analysis. Data are expressed as means ± SE of 3 separate experiments. ** P < 0.01, *** P < 0.001 vs. control, and ° P < 0.05 vs. TNF-alpha alone.

Expression of TNF-alpha receptors on C2C12 myotubes. To assess the nature of TNF-alpha receptor isoforms expressed in C2C12 myotubes, total cDNA was prepared and amplified for transcripts encoding the different TNF-alpha receptor isoforms p60 (TNFR1) and p80 (TNFR2). Both types of TNF-alpha receptors (TNFR1 and TNFR2) were found to be expressed at the mRNA level in this cell line (Fig. 10).


View larger version (59K):
[in this window]
[in a new window]
 
Fig. 10.   Presence of TNF-alpha receptor isoforms in C2C12 myotubes. After 4 days of differentiation (D4), C2C12 myotubes were incubated for 24 h in the presence or absence of TNF-alpha (5 ng/ml). Then, myotube total RNA was extracted, and RT-PCR was performed on total RNA for the expression of TNF-alpha receptors TNFR1 (p60) and TNFR2 (p80), as described in MATERIALS AND METHODS. Positive controls included mouse spleen and WEG-1 cells. Amplicon size (MW) markers are indicated in the left margin; p60 and p80 amplicon sizes are indicated in the right margin.

Stimulation of NO production by TNF-alpha and IFN-gamma . Inducible nitric oxide synthase (iNOS) plays an important role in the transduction of TNF-alpha signal in catabolic states (7). Hence, we examined the possibility that TNF-alpha inhibition of the IGF-I gene would be mediated through NO production. Myotubes were stimulated for 24 h with TNF-alpha (3 ng/ml), IFN-gamma (5 ng/ml), or their combination. Neither TNF-alpha nor IFN-gamma exposure increased nitrite production in C2C12 myotubes. However, marked nitrite production occurred only when myotubes were stimulated with the combination of TNF-alpha and IFN-gamma (47-fold the control values; P < 0.001; Table 2). This result clearly shows a synergy of action between TNF-alpha and IFN-gamma on NO production in our model.

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Cytokine induction of nitrite production by C2C12 myotubes

Activation of NF-kappa B by TNF-alpha and IL-1beta . Because NF-kappa B activation is essential for the inflammatory response (51), we assessed whether this transcription factor plays a role in the inhibitory effect of TNF-alpha on IGF-I gene expression. Our data show that exposure of 4-day-differentiated myotubes for 30 min to TNF-alpha (3 ng/ml) activates NF-kappa B (Fig. 11). The incubation of nuclear extracts with specific antibodies demonstrated that the signal was indeed NF-kappa B and that it was composed mainly of the subunits p50 and p65. Further incubation of the nuclear extracts with an excess of cold, homologous probe or with a heterologous Sp1 probe allowed us to confirm the specificity of the NF-kappa B oligonucleotide complex formed (Fig. 11).


View larger version (53K):
[in this window]
[in a new window]
 
Fig. 11.   Activation of NF-kappa B by TNF-alpha in C2C12 myotubes. After 4 days of differentiation (D4), C2C12 myotubes were incubated for 30 min with TNF-alpha (3 ng/ml). Gel shift assay and supershift, using p50 and p65 specific antibodies, were performed on nuclear extracts, as described in MATERIALS AND METHODS. These results are from 1 of 3 separate experiments.

To assess the requirement of NF-kappa B activation for the inhibition of IGF-I mRNA by TNF-alpha , we tested whether IL-1beta , another proinflammatory cytokine that was unable to inhibit IGF-I expression, activated NF-kappa B transcription factor. Incubation of myotubes for 30 min with IL-1beta (3 ng/ml) was, in fact, capable of NF-kappa B activation (Fig. 12). Because both TNF-alpha and IL-1beta activated NF-kappa B but only TNF-alpha inhibited IGF-I gene expression, we can conclude that NF-kappa B activation by TNF-alpha is insufficient to inhibit IGF-I gene expression.


View larger version (40K):
[in this window]
[in a new window]
 
Fig. 12.   Activation of NF-kappa B by TNF-alpha and IL-1beta in C2C12 myotubes. After 4 days of induced differentiation (D4), C2C12 myotubes were incubated for 30 min with TNF-alpha or IL-1beta (3 ng/ml). Gel shift assay was performed on nuclear extracts as described in MATERIALS AND METHODS. These results are from 1 of 3 separate experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our results show that LPS injection induces a rapid increase of TNF-alpha expression and a later inhibition of IGF-I expression in skeletal muscle, suggesting the possibility of a causal relationship between these two changes. This hypothesis was confirmed in a model of differentiated C2C12 myotubes, where we observed that TNF-alpha inhibits IGF-I gene and protein expression.

In our in vivo model, TNF-alpha mRNA levels are induced rapidly and transiently in tibialis anterior after LPS administration. This indicates that the stimulation of TNF-alpha gene expression by LPS is not restricted to diaphragm tissue, about which it was originally described (46), but is probably a generalized response of skeletal muscles. These observations also support the hypothesis that the catabolic action of TNF-alpha in skeletal muscle is exerted in an autocrine/paracrine way. Our data do not allow us to identify the nature of the cells expressing TNF-alpha in skeletal muscle. Although the possibility exists that TNF-alpha is produced, at least partially, by the myocytes (15, 44), in our model of C2C12 myotubes, LPS failed to induce significant TNF-alpha expression (data not shown). In contrast, TNF-alpha expression was stimulated in whole muscles incubated in the presence of LPS. From these observations, we speculate that cells other than myocytes, probably immune cells, are the main contributor of TNF-alpha production in muscles exposed to LPS.

The mechanisms for increased TNF-alpha expression in skeletal muscle after LPS injection are still unknown. This effect could result from a direct effect of LPS on skeletal muscle, or it could be mediated by circulating TNF-alpha produced principally by the liver in response to LPS. The first possibility is strongly supported by our observation that LPS can act directly on muscle to increase TNF-alpha expression. On the other hand, the second possibility is suggested by the fact that TNF-alpha induces its own expression in vitro, both in immune cells and in myocytes (1, 15, 53, 57). However, the concomitant induction of TNF-alpha in liver and muscle after LPS injection favors a major direct role of LPS in the induction of muscle TNF-alpha .

LPS injection caused a decrease in muscle IGF-I mRNA levels. This is in agreement with previous work showing that LPS injection decreases immunoreactive IGF-I and IGF-I mRNA in gastrocnemius muscle (31). Because gastrocnemius is a mixed-fiber muscle and tibialis anterior a fast-fiber muscle, the decrease in IGF-I gene expression after LPS does not seem to be restricted to a specific type of muscle.

The time course of TNF-alpha induction and IGF-I inhibition caused by LPS in skeletal muscle suggests a causal relationship between these two changes. This hypothesis is strengthened by the parallelism with the liver situation, where LPS increases TNF-alpha and decreases IGF-I production (14) and by the fact that TNF-alpha inhibits IGF-I mRNA in hepatocytes (52). Because the time courses of the IGF-I mRNA decrease coincide in muscle and in liver, it is likely that the mechanism of IGF-I gene inhibition by LPS is the same in both tissues. Therefore, the decrease in muscle IGF-I expression might be secondary to the muscle TNF-alpha induction by LPS.

To demonstrate this hypothesis, we assessed the effects of TNF-alpha on IGF-I gene and protein expression in muscle cells in vitro. We show here that TNF-alpha inhibits IGF-I gene and protein expression in differentiated C2C12 myotubes. The use of differentiated myotubes rather than myoblasts strengthens the relevance of our observations, as it is more likely for the same effects to be found in mature muscle (32).

Although muscle IGF-I mRNA concentrations are decreased in vivo by LPS and in vitro by TNF-alpha , IGF-II mRNA is not affected by these stimuli. Because IGFs share many biological properties, one could question the physiological consequences on muscle mass of a local IGF-I decline. However, several lines of evidence indicate that the respective actions of IGF-I and IGF-II on the muscle are not "interchangeable." igf1-Null mice present a generalized skeletal muscle hypoplasia that occurs despite normal IGF-II expression (8). Furthermore, although igf1-transgenic mice are characterized by an increase in muscle growth, igf2-transgenic mice have normal muscle mass and body growth (18, 49). Taken together, these data support a different role for IGF-I and IGF-II regarding the control of muscle mass and imply that the decline in muscle IGF-I production can impair muscle mass despite normal IGF-II expression.

Although TNF-alpha can lead to cell death in different cell systems, this was not the case in our model. First, the morphological aspect of myotubes exposed for 72 h to TNF-alpha was similar to that of controls. Second, the decrease in IGF-I expression caused by TNF-alpha was rapidly reversed by removal of the cytokine from the culture medium, thus confirming that IGF-I inhibition is not secondary to cell death. Moreover, several research groups have reported that C2C12 myotubes are resistant to the apoptotic action of TNF-alpha , even after 72 h of incubation (37). Although induction of TNF-soluble receptors can block TNF-alpha action, this seems not to be the case in our model, as the TNF-alpha inhibitory effect on IGF-I was sustained for >= 72 h.

The inhibition of IGF-I mRNA by TNF-alpha could result from dedifferentiation of myotubes caused by the cytokine (34). Indeed, TNF-alpha clearly impairs the differentiation of myoblasts into myotubes by inhibiting several myogenic markers (25). In the present study, TNF-alpha treatment significantly decreased CK activity, a marker of differentiation, but this occurred only after 72 h of treatment. Others have reported similar late effects of TNF-alpha on the myogenic differentiation of myotubes (34). However, we cannot exclude the possibility that changes in CK activity detected at 72 h reflect early changes in steady-state levels of CK mRNA. These observations suggest that the rapid inhibitory action of TNF-alpha on IGF-I gene expression is probably independent of myotube dedifferentiation, although this phenomenon could be involved at later stages.

The TNF-alpha concentration capable of causing a decrease in IGF-I expression can be considered as "physiological," i.e., in the range of those found in inflammatory states or after LPS injection (3, 38) and close to the affinity of the TNF-alpha receptor for its ligand (30). Besides, when possible paracrine interaction between the cytokine-producing cells and myocytes is considered, local concentrations could be even higher than those found in circulation after LPS injection or in inflammatory states. In addition, the kinetics of the IGF-I mRNA decrease after TNF-alpha treatment of myotubes are close to those observed in vivo after LPS injection. All of these observations plead for a direct autocrine/paracrine effect of TNF-alpha , produced by LPS, on IGF-I in skeletal muscle.

Whereas TNF-alpha is not the sole cytokine capable of inducing a catabolic state (38) and whereas some actions of TNF-alpha have been shown to be mediated by other cytokines (2), we studied whether IL-1beta , IL-6, or IFN-gamma were capable of inhibiting IGF-I expression. All of these cytokines fail to decrease IGF-I mRNA levels significantly in C2C12 myotubes. However, our data allow us to demonstrate the responsiveness of C2C12 myotubes to IL-1beta and IFN-gamma , as the former activated NF-kappa B and the latter potentiated the TNF-alpha effect on nitrite production. A similar conclusion can be drawn for IL-6, as C2C12 myotube sensitivity to IL-6 has recently been reported (2). Our data suggest that the TNF-alpha effect on IGF-I expression is not mediated by IL-1, IL-6, or IFN-gamma . Nevertheless, the mechanism by which TNF-alpha exerts its inhibitory effect on IGF-I expression might still be an indirect one, needing other mediators or a combination of different cytokines (15, 57).

LPS administration did not result in a significant inhibition of IGF-I expression in differentiated myotubes. This apparent insensitivity might result from the absence of Toll-like receptors capable of LPS recognition in C2C12 myotubes. However, this is unlikely, as other biological responses to LPS have been described in the same model (24). Therefore, the lack of effect of LPS is most likely due to its failure to induce significant TNF-alpha expression in C2C12 myotubes.

Because most of the TNF-alpha catabolic actions are mediated through TNFR1, we searched for the presence of this receptor in C2C12 myotubes. This cell line actually expresses both TNF-alpha receptor isoforms, TNFR1 and TNFR2. We cannot, therefore, affirm the nature of the TNF-alpha receptor isoform whose activation causes IGF-I gene expression to decrease.

Although NO induction seems to play a role in some catabolic effects of TNF-alpha , the role of the NO pathway in the IGF-I gene inhibition by TNF-alpha can be excluded, as TNF-alpha alone fails to increase NO production in C2C12 myotubes. This is not surprising, because previous work showed that iNOS activation in C2C12 myocytes required stimulation by a combination of cytokines, among which IFN-gamma appears to be essential (55). This seems to be also the case in our model, where only the combination of TNF-alpha and IFN-gamma resulted in a dramatic NO production, as assessed by nitrite concentration in the medium. The essential role of IFN-gamma in inducing NO production might result from its ability to upregulate TNF-alpha receptor expression (9) or from a synergy operated at the transcriptional level (10). A synergistic action between TNF-alpha and IFN-gamma was not observed on IGF-I mRNA. Both cytokines together decreased IGF-I gene expression more than each one did alone, supporting an additive effect rather than a potentiation.

Activation of NF-kappa B is a crucial step for many TNF-alpha actions (51). Our data show that TNF-alpha indeed activates NF-kappa B in myotubes, confirming the results reported by Li et al. (37). Nevertheless, our observations extend their findings by showing that NF-kappa B is composed mainly of p50 and p65 subunits, which constitute the main heterodimer activated in the inflammatory response (51). The rapid activation of NF-kappa B by TNF-alpha might suggest that this transcription factor plays a role in the inhibition of IGF-I expression caused by TNF-alpha . However, IL-1beta also activates NF-kappa B in C2C12 myotubes, similarly to TNF-alpha but without inhibiting IGF-I expression. Activation of NF-kappa B is therefore not sufficient to inhibit IGF-I gene expression.

Taken together, our data indicate that TNF-alpha induced by LPS in skeletal muscle may exert its catabolic effects in an autocrine/paracrine manner by reducing IGF-I gene and protein expression.


    ACKNOWLEDGEMENTS

We gratefully acknowledge Josiane Verniers, Pascale Lause, Ivo Vanderheyden, and Prof. Marianne Philippe for expert technical assistance.


    FOOTNOTES

This work was supported by grants from the Fund for Scientific Medical Research (Belgium), the Fonds Spéciaux de Recherche (Université Catholique de Louvain), and the Danone Institute (Brussels).

Address for reprint requests and other correspondence: J.-P. Thissen, Unité de Diabétologie et Nutrition, UCL/DIAB 5474, Ave. Hippocrate, 54, B-1200 Brussels, Belgium (E-mail: thissen{at}diab.ucl.ac.be).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

July 2, 2002;10.1152/ajpendo.00054.2002

Received 7 February 2002; accepted in final form 20 June 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alvarez, B, Quinn LS, Busquets S, Lopez-Soriano FJ, and Argiles JM. TNF-alpha modulates cytokine and cytokine receptors in C2C12 myotubes. Cancer Lett 175: 181-185, 2002[ISI][Medline].

2.   Alvarez, B, Quinn LS, Busquets S, Quiles MT, Lopez-Soriano FJ, and Argiles JM. Tumor necrosis factor-alpha exerts interleukin-6-dependent and -independent effects on cultured skeletal muscle cells. Biochim Biophys Acta 1542: 66-72, 2002[ISI][Medline].

3.   Argiles, JM, Lopez-Soriano J, Busquets S, and Lopez-Soriano FJ. Journey from cachexia to obesity by TNF. FASEB J 11: 743-751, 1997[Abstract/Free Full Text].

4.   Baracos, VE. Anabolic and catabolic mediators. Curr Opin Clin Nutr Metab Care 1: 241-244, 1998[Medline].

5.   Bark, TH, McNurlan MA, Lang CH, and Garlick PJ. Increased protein synthesis after acute IGF-I or insulin infusion is localized to muscle in mice. Am J Physiol Endocrinol Metab 275: E118-E123, 1998[Abstract/Free Full Text].

6.   Baumgarten, G, Knuefermann P, Nozaki N, Sivasubramanian N, Mann DL, and Vallejo JG. In vivo expression of proinflammatory mediators in the adult heart after endotoxin administration: the role of toll-like receptor-4. J Infect Dis 183: 1617-1624, 2001[ISI][Medline].

7.   Buck, M, and Chojkier M. Muscle wasting and dedifferentiation induced by oxidative stress in a murine model of cachexia is prevented by inhibitors of nitric oxide synthesis and antioxidants. EMBO J 15: 1753-1765, 1996[Abstract].

8.   Butler, AA, and LeRoith D. Tissue-specific versus generalized gene targeting of the igf1 and igf1r genes and their roles in insulin-like growth factor physiology. Endocrinology 142: 1685-1688, 2001[Abstract/Free Full Text].

9.   Carrel, S, Hartmann F, Salvi S, Albrecht H, Schreyer M, and Rimoldi D. Expression of type A and B tumor necrosis factor (TNF) receptors on melanoma cells can be regulated by dbc-AMP and IFN gamma. Int J Cancer 62: 76-83, 1995[ISI][Medline].

10.   Cheshire, JL, and Baldwin AS, Jr. Synergistic activation of NF-kappaB by tumor necrosis factor alpha and gamma interferon via enhanced I kappaB alpha degradation and de novo I kappaB beta degradation. Mol Cell Biol 17: 6746-6754, 1997[Abstract].

11.   Chrysis, D, and Underwood LE. Regulation of components of the ubiquitin system by insulin-like growth factor I and growth hormone in skeletal muscle of rats made catabolic with dexamethasone. Endocrinology 140: 5635-5641, 1999[Abstract/Free Full Text].

12.   Costelli, P, Carbo N, Tessitore L, Bagby GJ, Lopez Soriano FJ, Argiles JM, and Baccino FM. Tumor necrosis factor-alpha mediates changes in tissue protein turnover in a rat cancer cachexia model. J Clin Invest 92: 2783-2789, 1993[ISI][Medline].

13.   Davenport, ML, Svoboda ME, Koerber KL, Van Wyk JJ, Clemmons DR, and Underwood LE. Serum concentrations of insulin-like growth factor II are not changed by short-term fasting and refeeding. J Clin Endocrinol Metab 67: 1231-1236, 1988[Abstract].

14.   Defalque, D, Brandt N, Ketelslegers JM, and Thissen JP. GH insensitivity induced by endotoxin injection is associated with decreased liver GH receptors. Am J Physiol Endocrinol Metab 276: E565-E572, 1999[Abstract/Free Full Text].

15.   De Rossi, M, Bernasconi P, Baggi F, de Waal MR, and Mantegazza R. Cytokines and chemokines are both expressed by human myoblasts: possible relevance for the immune pathogenesis of muscle inflammation. Int Immunol 12: 1329-1332, 2000[Abstract/Free Full Text].

16.   Fan, J, Molina PE, Gelato MC, and Lang CH. Differential tissue regulation of insulin-like growth factor-I content and binding proteins after endotoxin. Endocrinology 134: 1685-1692, 1994[Abstract].

17.   Fang, CH, James HJ, Ogle C, Fischer JE, and Hasselgren PO. Influence of burn injury on protein metabolism in different types of skeletal muscle and the role of glucocorticoids. J Am Coll Surg 180: 33-42, 1995[ISI][Medline].

18.   Florini, JR, Ewton DZ, and Coolican SA. Growth hormone and the insulin-like growth factor system in myogenesis. Endocr Rev 17: 481-517, 1996[Abstract].

19.   Flynn, JL, Goldstein MM, Chan J, Triebold KJ, Pfeffer K, Lowenstein CJ, Schreiber R, Mak TW, and Bloom BR. Tumor necrosis factor-alpha is required in the protective immune response against Mycobacterium tuberculosis in mice. Immunity 2: 561-572, 1995[ISI][Medline].

20.   Foufelle, F, Girard J, and Ferre P. Glucose regulation of gene expression. Curr Opin Clin Nutr Metab Care 1: 323-328, 1998[Medline].

21.   Frost, RA, and Lang CH. Differential effects of insulin-like growth factor I (IGF-I) and IGF-binding protein-1 on protein metabolism in human skeletal muscle cells. Endocrinology 140: 3962-3970, 1999[Abstract/Free Full Text].

22.   Frost, RA, Lang CH, and Gelato MC. Transient exposure of human myoblasts to tumor necrosis factor-alpha inhibits serum and insulin-like growth factor-I stimulated protein synthesis. Endocrinology 138: 4153-4159, 1997[Abstract/Free Full Text].

23.   Garcia-Martinez, C, Lopez-Soriano FJ, and Argiles JM. Acute treatment with tumour necrosis factor-alpha induces changes in protein metabolism in rat skeletal muscle. Mol Cell Biochem 125: 11-18, 1993[ISI][Medline].

24.   Gath, I, Ebert J, Godtel-Armbrust U, Ross R, Reske-Kunz AB, and Forstermann U. NO synthase II in mouse skeletal muscle is associated with caveolin 3. Biochem J 340: 723-728, 1999[ISI][Medline].

25.   Guttridge, DC, Mayo MW, Madrid LV, Wang CY, and Baldwin AS, Jr. NF-kappaB-induced loss of MyoD messenger RNA: possible role in muscle decay and cachexia. Science 289: 2363-2366, 2000[Abstract/Free Full Text].

26.   Hobler, SC, Wang JJ, Williams AB, Melandri F, Sun XY, Fischer JE, and Hasselgren PO. Sepsis is associated with increased ubiquitin-conjugating enzyme E214k mRNA in skeletal muscle. Am J Physiol Regul Integr Comp Physiol 276: R468-R473, 1999[Abstract/Free Full Text].

27.   Hong, D, and Forsberg NE. Effects of serum and insulin-like growth factor I on protein degradation and protease gene expression in rat L8 myotubes. J Anim Sci 72: 2279-2288, 1994[Abstract/Free Full Text].

28.   Ksontini, R, MacKay SL, and Moldawer LL. Revisiting the role of tumor necrosis factor alpha and the response to surgical injury and inflammation. Arch Surg 133: 558-567, 1998[Abstract/Free Full Text].

29.   Kubota, T, McTiernan CF, Frye CS, Slawson SE, Lemster BH, Koretsky AP, Demetris AJ, and Feldman AM. Dilated cardiomyopathy in transgenic mice with cardiac-specific overexpression of tumor necrosis factor-alpha. Circ Res 81: 627-635, 1997[Abstract/Free Full Text].

30.   Kull, FC, Jr, Jacobs S, and Cuatrecasas P. Cellular receptor for 125I-labeled tumor necrosis factor: specific binding, affinity labeling, and relationship to sensitivity. Proc Natl Acad Sci USA 82: 5756-5760, 1985[Abstract].

31.   Lang, CH, Frost RA, Jefferson LS, Kimball SR, and Vary TC. Endotoxin-induced decrease in muscle protein synthesis is associated with changes in eIF2B, eIF4E, and IGF-I. Am J Physiol Endocrinol Metab 278: E1133-E1143, 2000[Abstract/Free Full Text].

32.   Lang, CH, Nystrom GJ, and Frost RA. Tissue-specific regulation of IGF-I and IGF-binding proteins in response to TNFalpha. Growth Horm IGF Res 11: 250-260, 2001[ISI][Medline].

33.   Lang, CH, Silvis C, Nystrom G, and Frost RA. Regulation of myostatin by glucocorticoids after thermal injury. FASEB J 15: 1807-1809, 2001[Abstract/Free Full Text].

34.   Langen, RC, Schols AM, Kelders MC, Wouters EF, and Janssen-Heininger YW. Inflammatory cytokines inhibit myogenic differentiation through activation of nuclear factor-kappaB. FASEB J 15: 1169-1180, 2001[Abstract/Free Full Text].

35.   Lawlor, MA, and Rotwein P. Insulin-like growth factor-mediated muscle cell survival: central roles for akt and cyclin-dependent kinase inhibitor. Mol Cell Biol 20: 8983-8995, 2000[Abstract/Free Full Text].

36.   Li, YP, and Reid MB. NF-kappaB mediates the protein loss induced by TNF-alpha in differentiated skeletal muscle myotubes. Am J Physiol Regul Integr Comp Physiol 279: R1165-R1170, 2000[Abstract/Free Full Text].

37.   Li, YP, Schwartz RJ, Waddell ID, Holloway BR, and Reid MB. Skeletal muscle myocytes undergo protein loss and reactive oxygen-mediated NF-kappaB activation in response to tumor necrosis factor alpha. FASEB J 12: 871-880, 1998[Abstract/Free Full Text].

38.   Matthys, P, and Billiau A. Cytokines and cachexia. Nutrition 13: 763-770, 1997[ISI][Medline].

39.   Meadows, KA, Holly JM, and Stewart CE. Tumor necrosis factor-alpha-induced apoptosis is associated with suppression of insulin-like growth factor binding protein-5 secretion in differentiating murine skeletal myoblasts. J Cell Physiol 183: 330-337, 2000[ISI][Medline].

40.   Pampfer, S, Vanderheyden I, Wuu YD, Baufays L, Maillet O, and De Hertogh R. Possible role for TNF-alpha in early embryopathy associated with maternal diabetes in the rat. Diabetes 44: 531-536, 1995[Abstract].

41.   Pfeffer, K, Matsuyama T, Kundig TM, Wakeham A, Kishihara K, Shahinian A, Wiegmann K, Ohashi PS, Kronke M, and Mak TW. Mice deficient for the 55 kd tumor necrosis factor receptor are resistant to endotoxic shock, yet succumb to L monocytogenes infection. Cell 73: 457-467, 1993[ISI][Medline].

42.   Remick, DG, Call DR, Ebong SJ, Newcomb DE, Nybom P, Nemzek JA, and Bolgos GE. Combination immunotherapy with soluble tumor necrosis factor receptors plus interleukin 1 receptor antagonist decreases sepsis mortality. Crit Care Med 29: 473-481, 2001[ISI][Medline].

43.   Rothe, J, Lesslauer W, Lotscher H, Lang Y, Koebel P, Kontgen F, Althage A, Zinkernagel R, Steinmetz M, and Bluethmann H. Mice lacking the tumour necrosis factor receptor 1 are resistant to TNF-mediated toxicity but highly susceptible to infection by Listeria monocytogenes. Nature 364: 798-802, 1993[ISI][Medline].

44.   Saghizadeh, M, Ong JM, Garvey WT, Henry RR, and Kern PA. The expression of TNF by human muscle---relationship to insulin resistance. J Clin Invest 97: 1111-1116, 1996[Abstract/Free Full Text].

45.   Segal, SS, and Faulkner JA. Temperature-dependent physiological stability of rat skeletal muscle in vitro. Am J Physiol Cell Physiol 248: C265-C270, 1985[Abstract].

46.   Shindoh, C, Hida W, Ohkawara Y, Yamauchi K, Ohno I, Takishima T, and Shirato K. TNF-alpha mRNA expression in diaphragm muscle after endotoxin administration. Am J Respir Crit Care Med 152: 1690-1696, 1995[Abstract].

47.   Sjögren, K, Liu JL, Blad K, Skrtic S, Vidal O, Wallenius V, LeRoith D, Törnell J, Isaksson OG, Jansson JO, and Ohlsson C. Liver-derived insulin-like growth factor I (IGF-I) is the principal source of IGF-I in blood but is not required for postnatal body growth in mice. Proc Natl Acad Sci USA 96: 7088-7092, 1999[Abstract/Free Full Text].

48.   Stärkel, P, Horsmans Y, Sempoux C, De Saeger C, Wary J, Lause P, Maiter D, and Lambotte L. After portal branch ligation in rat, nuclear factor kappaB, interleukin-6, signal transducers and activators of transcription 3, c-fos, c-myc, and c-jun are similarly induced in the ligated and nonligated lobes. Hepatology 29: 1463-1470, 1999[ISI][Medline].

49.   Stewart, CE, and Rotwein P. Growth, differentiation, and survival: multiple physiological functions for insulin-like growth factors. Physiol Rev 76: 1005-1026, 1996[Abstract/Free Full Text].

50.   Suk, K, Kim S, Kim YH, Kim KA, Chang I, Yagita H, Shong M, and Lee MS. IFN-gamma/TNF-alpha synergism as the final effector in autoimmune diabetes: a key role for STAT1/IFN regulatory factor-1 pathway in pancreatic beta cell death. J Immunol 166: 4481-4489, 2001[Abstract/Free Full Text].

51.   Tak, PP, and Firestein GS. NF-kappaB: a key role in inflammatory diseases. J Clin Invest 107: 7-11, 2001[Free Full Text].

52.   Thissen, JP, and Verniers J. Inhibition by interleukin-1beta and tumor necrosis factor-alpha of the insulin-like growth factor I messenger ribonucleic acid response to growth hormone in rat hepatocyte primary culture. Endocrinology 138: 1078-1084, 1997[Abstract/Free Full Text].

53.   Tracey, KJ, and Cerami A. Tumor necrosis factor, other cytokines and disease. Annu Rev Cell Biol 9: 317-343, 1993[ISI].

54.   Tracey, KJ, Morgello S, Koplin B, Fahey TJ, III, Fox J, Aledo A, Manogue KR, and Cerami A. Metabolic effects of cachectin/tumor necrosis factor are modified by site of production. Cachectin/tumor necrosis factor-secreting tumor in skeletal muscle induces chronic cachexia, while implantation in brain induces predominantly acute anorexia. J Clin Invest 86: 2014-2024, 1990[ISI][Medline].

55.   Williams, G, Brown T, Becker L, Prager M, and Giroir BP. Cytokine-induced expression of nitric oxide synthase in C2C12 skeletal muscle myocytes. Am J Physiol Regul Integr Comp Physiol 267: R1020-R1025, 1994[Abstract/Free Full Text].

56.   Yakar, S, Liu JL, Stannard B, Butler A, Accili D, Sauer B, and LeRoith D. Normal growth and development in the absence of hepatic insulin-like growth factor I. Proc Natl Acad Sci USA 96: 7324-7329, 1999[Abstract/Free Full Text].

57.   Zhang, Y, Pilon G, Marette A, and Baracos VE. Cytokines and endotoxin induce cytokine receptors in skeletal muscle. Am J Physiol Endocrinol Metab 279: E196-E205, 2000[Abstract/Free Full Text].


Am J Physiol Endocrinol Metab 283(6):E1279-E1290
0193-1849/02 $5.00 Copyright © 2002 the American Physiological Society