Unité de Diabétologie et Nutrition, Université catholique de Louvain, B-1200 Brussels, Belgium
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ABSTRACT |
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The role of TNF- in muscle catabolism
is well established, but little is known about the mechanisms of its
catabolic action. One possibility could be that TNF-
impairs the
production of local growth factors like IGF-I. The aim of this study
was to investigate whether TNF-
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-
mRNA in rat tibialis anterior muscle. Endotoxin rapidly increased
TNF-
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-
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-
on IGF-I mRNA was not mediated by
nitric oxide, and the activation of NF-
B was insufficient to inhibit
IGF-I expression. Taken together, our data suggest that TNF-
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-
.
skeletal muscle; lipopolysaccharide; C2C12 cells
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INTRODUCTION |
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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)- is a proinflammatory cytokine that is
essential for a successful response against invading pathogens (19, 41, 43). On the other hand, TNF-
is considered to play a major role in muscle catabolism. First, circulating levels of
TNF-
are markedly increased in catabolic states (3).
Second, enhanced protein degradation and muscle loss have been observed in TNF-
-transgenic animals (29) after chronic
administration of the cytokine (23, 54) or in animals
bearing TNF-
-producing tumors (12). Furthermore,
administration of anti-TNF-
antibodies or of TNF-
-soluble
receptor attenuates the catabolic reaction (28, 42).
Catabolic actions of TNF- in skeletal muscle can be exerted in
different ways. One possibility is that TNF-
acts directly on
skeletal muscle to induce muscle catabolism. Indeed, TNF-
has been
reported to inhibit protein synthesis and myogenesis in myoblasts
(22, 25, 34). Recent observations indicate that TNF-
can also stimulate the proteolysis of myosin heavy chains in
C2C12 myotubes by activating the
ubiquitin-proteasome pathway (36). Besides, TNF-
increases apoptotic cell death in skeletal muscle
(39). Alternatively, TNF-
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-
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-
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-
mRNA
and second to investigate the role of TNF-
in the decrease of muscle
IGF-I caused by LPS.
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MATERIALS AND METHODS |
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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-,
interleukin-1
, and interferon-
(rmTNF-
, rmIL-1
, rmIFN-
)
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 µg1 · 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.
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-B
(5'-TCGAGGGCTGGGGATTCCCCATCTC-3') were radiolabeled with
[
-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-
B subunits were incubated with the extract
samples for 5 min before the addition of the labeled probe. NF-
B p65
and p50 antibodies are, respectively, rabbit and goat affinity-purified polyclonal antibodies raised against a peptide mapping the carboxy terminus of NF-
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
End point PCR of TNF- 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.
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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.
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RESULTS |
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Regulation of muscle expression of IGF-I and TNF- by LPS.
In a first step, we investigated the changes produced by LPS injection
in muscle TNF-
mRNA and IGF-I mRNA.
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Regulation of myotube expression of IGF-I by TNF-.
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-.
Because in vivo results suggested a causal relationship between the
increase in TNF-
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-
has been reported to exert a catabolic
action leading to decrease in MHC content (37).
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Reversibility and relation to differentiation state of the
inhibition of IGF-I gene expression by TNF-.
The inhibition of IGF-I mRNA levels induced by 24-h exposure to TNF-
(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-
was removed (Fig.
6). This result indirectly indicates that
the TNF-
inhibitory effect on IGF-I mRNA is independent of a
cytotoxic effect of the cytokine.
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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-1, IL-6, and IFN-
(5 ng/ml) on
4-day-differentiated myotubes. Although, as expected, TNF-
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-1
, IL-6, or IFN-
) modified consistently the IGF-I mRNA levels in muscle cells (Fig. 8A). All together, these
observations indicate that, of all the cytokines tested, TNF-
is the
only one to significantly inhibit IGF-I mRNA.
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Effect of the combination of TNF- and IFN-
on IGF-I gene
expression.
Because IFN-
can potentiate some TNF-
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-
(3 ng/ml) and IFN-
(5 ng/ml) alone or in combination. As
expected, IFN-
did not significantly modify IGF-I mRNA (29%
decrease, P > 0.05), whereas TNF-
alone caused a
74% decrease of IGF-I mRNA levels (P < 0.01 vs.
control). When myotubes were challenged with the combination of TNF-
and IFN-
, 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-
alone), suggesting more an
additive action of both cytokines than a potentiation (Fig.
9).
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Expression of TNF- receptors on C2C12
myotubes.
To assess the nature of TNF-
receptor isoforms expressed in
C2C12 myotubes, total cDNA was prepared and
amplified for transcripts encoding the different TNF-
receptor
isoforms p60 (TNFR1) and p80 (TNFR2). Both types of TNF-
receptors
(TNFR1 and TNFR2) were found to be expressed at the mRNA level in this
cell line (Fig. 10).
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Stimulation of NO production by TNF- and IFN-
.
Inducible nitric oxide synthase (iNOS) plays an important role in the
transduction of TNF-
signal in catabolic states (7). Hence, we examined the possibility that TNF-
inhibition of the IGF-I
gene would be mediated through NO production. Myotubes were stimulated
for 24 h with TNF-
(3 ng/ml), IFN-
(5 ng/ml), or their
combination. Neither TNF-
nor IFN-
exposure increased nitrite
production in C2C12 myotubes. However, marked
nitrite production occurred only when myotubes were stimulated with the combination of TNF-
and IFN-
(47-fold the control values;
P < 0.001; Table 2).
This result clearly shows a synergy of action between TNF-
and
IFN-
on NO production in our model.
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Activation of NF-B by TNF-
and IL-1
.
Because NF-
B activation is essential for the inflammatory response
(51), we assessed whether this transcription factor plays
a role in the inhibitory effect of TNF-
on IGF-I gene expression. Our data show that exposure of 4-day-differentiated myotubes for 30 min
to TNF-
(3 ng/ml) activates NF-
B (Fig.
11). The incubation of nuclear extracts
with specific antibodies demonstrated that the signal was indeed
NF-
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-
B oligonucleotide complex formed (Fig.
11).
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DISCUSSION |
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Our results show that LPS injection induces a rapid increase of
TNF- 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-
inhibits IGF-I gene and protein expression.
In our in vivo model, TNF- mRNA levels are induced rapidly and
transiently in tibialis anterior after LPS administration. This
indicates that the stimulation of TNF-
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-
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-
in skeletal muscle. Although the
possibility exists that TNF-
is produced, at least partially, by the
myocytes (15, 44), in our model of
C2C12 myotubes, LPS failed to induce
significant TNF-
expression (data not shown). In contrast, TNF-
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-
production in muscles exposed to LPS.
The mechanisms for increased TNF- 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-
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-
expression. On
the other hand, the second possibility is suggested by the fact that
TNF-
induces its own expression in vitro, both in immune cells and
in myocytes (1, 15, 53, 57). However, the concomitant
induction of TNF-
in liver and muscle after LPS injection favors a
major direct role of LPS in the induction of muscle TNF-
.
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- 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-
and decreases IGF-I
production (14) and by the fact that TNF-
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-
induction by LPS.
To demonstrate this hypothesis, we assessed the effects of TNF- on
IGF-I gene and protein expression in muscle cells in vitro. We show
here that TNF-
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-, 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- 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-
was similar to that of
controls. Second, the decrease in IGF-I expression caused by TNF-
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-
, even after 72 h of incubation
(37). Although induction of TNF-soluble receptors can
block TNF-
action, this seems not to be the case in our model, as
the TNF-
inhibitory effect on IGF-I was sustained for
72 h.
The inhibition of IGF-I mRNA by TNF- could result from
dedifferentiation of myotubes caused by the cytokine (34).
Indeed, TNF-
clearly impairs the differentiation of myoblasts into
myotubes by inhibiting several myogenic markers (25). In
the present study, TNF-
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-
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-
on IGF-I gene expression is probably independent of myotube dedifferentiation, although this phenomenon could be involved at later stages.
The TNF- 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-
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-
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-
,
produced by LPS, on IGF-I in skeletal muscle.
Whereas TNF- is not the sole cytokine capable of inducing a
catabolic state (38) and whereas some actions of TNF-
have been shown to be mediated by other cytokines (2), we
studied whether IL-1
, IL-6, or IFN-
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-1
and IFN-
, as the
former activated NF-
B and the latter potentiated the TNF-
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-
effect on IGF-I expression is not mediated by IL-1, IL-6, or IFN-
.
Nevertheless, the mechanism by which TNF-
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- expression in C2C12 myotubes.
Because most of the TNF- catabolic actions are mediated through
TNFR1, we searched for the presence of this receptor in
C2C12 myotubes. This cell line actually
expresses both TNF-
receptor isoforms, TNFR1 and TNFR2. We cannot,
therefore, affirm the nature of the TNF-
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-, the role of the NO pathway in the IGF-I gene inhibition by
TNF-
can be excluded, as TNF-
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-
appears to be essential
(55). This seems to be also the case in our model, where
only the combination of TNF-
and IFN-
resulted in a dramatic NO
production, as assessed by nitrite concentration in the medium. The
essential role of IFN-
in inducing NO production might result from
its ability to upregulate TNF-
receptor expression (9)
or from a synergy operated at the transcriptional level (10). A synergistic action between TNF-
and IFN-
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-B is a crucial step for many TNF-
actions
(51). Our data show that TNF-
indeed activates NF-
B
in myotubes, confirming the results reported by Li et al.
(37). Nevertheless, our observations extend their findings
by showing that NF-
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-
B by TNF-
might suggest that this transcription factor plays a role in the
inhibition of IGF-I expression caused by TNF-
. However, IL-1
also
activates NF-
B in C2C12 myotubes, similarly
to TNF-
but without inhibiting IGF-I expression. Activation of
NF-
B is therefore not sufficient to inhibit IGF-I gene expression.
Taken together, our data indicate that TNF- induced by LPS in
skeletal muscle may exert its catabolic effects in an
autocrine/paracrine manner by reducing IGF-I gene and protein expression.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Josiane Verniers, Pascale Lause, Ivo Vanderheyden, and Prof. Marianne Philippe for expert technical assistance.
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FOOTNOTES |
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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.
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