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Inhibits Serum and Insulin-Like Growth Factor-I Stimulated Protein Synthesis1
Departments of Medicine (R.A.F, M.C.G) and Surgery (C.H.L.), State University of New York at Stony Brook, Stony Brook, New York 11794
Address all correspondence and requests for reprints to: Marie C. Gelato, Division of Endocrinology, Health Science Center T-15, Room 060, State University of New York at Stony Brook, Stony Brook, New York 11794-8154. E-mail: mgelato{at}epo.som.sunysb.edu
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Abstract |
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(TNF-) induces cachexia and is postulated
to be responsible for muscle wasting in several pathophysiological
conditions. The purpose of the present study was to investigate whether
exposure of human myoblasts to TNF- could directly inhibit the
ability of serum or insulin-like growth factor I (IGF-I) to stimulate
protein synthesis as assessed by the incorporation of
[3H]phenylalanine into protein. Serum and IGF-I
stimulated protein synthesis dose dependently. Half-maximal stimulation
of protein synthesis occurred at 05% serum and 8 ng/ml of IGF-I,
respectively. TNF- inhibited IGF-I-stimulated protein synthesis in a
dose-dependent manner. Additionally, as little as 2 ng/ml of TNF-
impaired the ability of IGF-I to stimulate protein synthesis by 33%
and, at a dose of 100 ng/ml, TNF- completely prevented the increase
in protein synthesis induced by either serum or a maximally stimulating
dose of IGF-I. Inhibition of protein synthesis was independent of
whether TNF- and growth factors were added to cells simultaneously
or if the cells were pretreated with growth factors. Exposure of
myoblasts to TNF- for 10 min completely inhibited serum-induced
stimulation of protein synthesis. TNF- inhibited protein synthesis
up to 48 h after addition of the cytokine. TNF- also inhibited
serum-stimulated protein synthesis in human myoblasts that were
differentiated into myotubes. In contrast, exposure of myoblasts to
TNF- had no effect on IGF-I binding and failed to alter the ability
of either IGF-I or serum to stimulate [3H]thymidine
uptake. These data indicate that transient exposure of myoblasts or
myotubes to TNF- inhibits protein synthesis. Thus, the anabolic
actions of IGF-I on muscle protein synthesis may be impaired during
catabolic conditions in which TNF- is over expressed. |
Introduction |
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Muscle mass and nitrogen balance are influenced by hormones functioning in an endocrine fashion (1, 9), as well as by the autocrine production of proinflammatory cytokines (4, 10). Insulin-like growth factor I (IGF-I) is a hormone that stimulates muscle protein synthesis (11, 12) and impairs protein degradation (12). The plasma concentration of IGF-I is reduced in sepsis (1, 9, 13), in critically ill patients (14), in individuals with AIDS (15), and in other catabolic conditions (16). It is postulated that muscle wasting results from both a decrease in the intramuscular concentration of IGF-I (17) and changes in the responsiveness of muscle to stimulation by IGF-I (8, 18, 19).
The plasma concentration of the proinflammatory cytokine tumor
necrosis factor- (TNF-) is elevated in sepsis (20, 21),
neoplastic disease (2, 10), and in AIDS (4, 22). During some catabolic
states, the increase in TNF- appears sustained, whereas in other
conditions, the elevation is relatively transient (i.e.
several hours). Furthermore, the administration of TNF- to normal
animals induces cachexia and rapidly alters the serum concentration of
IGF-I and IGF binding proteins (23). Yet, the ability of TNF- and
other cytokines to directly influence muscle protein metabolism remains
controversial. TNF- has been shown to have either no effect on
muscle protein metabolism (24, 25) or to decrease muscle protein
synthesis (26, 27). Indeed a single laboratory has shown TNF- to
have distinct effects under different experimental conditions (25, 26, 27).
In addition, the interpretation of data from in vivo studies
is confounded by the ability of TNF- to stimulate the production of
a cascade of other cytokines (21). Thus, it is difficult to conclude
whether TNF- has a direct effect on protein synthesis or whether the
observed changes are secondary to perturbations in other hormones.
Because IGF-I positively influences both whole body and muscle protein
synthesis (12), we have examined whether IGF-I can stimulate protein
synthesis in human myoblasts and whether its biological activity can be
influenced by TNF-. In addition, because TNF- expression is often
only transient but its effects long lasting, we examined whether
protein synthesis in human myoblasts is altered by a brief exposure to
TNF-.
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Methods |
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[3H]Thymidine uptake
Human myoblasts in 24-well dishes (1.5 x 105
cells/well) were treated with either serum-free MEM alone, recombinant
human IGF-I (Upstate Biotechnology, Lake Placid, NY), recombinant human
TNF- (PeproTech, Princeton, NJ), or a combination of both peptides
for 21 h. Cells were labeled with
[3H]methylthymidine (6.7 Ci/mmol, Dupont-NEN, Boston, MA)
at 0.5 µCi/well for the final 6 h. Cells were washed three times
in ice-cold HBSS, isolated in trypsin-EDTA, and precipitated at 4 C
overnight with 5% trichloroacetic acetic acid (TCA), as previously
described (29). After washing, TCA precipitable radioactivity was
solubilized in 1 N sodium hydroxide and liquid
scintillation cocktail (Scintverse II; Fisher Scientific, Springfield,
NJ) and counted in a liquid scintillation counter (Wallac,
Gaithersburg, MD).
Protein synthesis
Human myoblasts were treated as described above with either MEM
alone, IGF-I, TNF-, or a combination of both peptides for 5 h.
Cells were labeled with 2 µCi/well of [3H]phenylalanine
(132 Ci/mmol, Amersham, Arlington Heights, IL) for the entire period.
Cells were washed and isolated as described above and precipitated
overnight at 4 C with 10% TCA. In some experiments, the TCA
precipitate was base hydrolyzed in 0.3 N sodium hydroxide
for 0.5 h at 37 C to solubilize nucleic acid-bound phenylalanine.
Radioactively labeled protein was subsequently reprecipitated with TCA.
Human serum used in these experiments was collected from adult male
volunteers after an overnight fast (30). Serum was pooled and diluted
into culture media to a final concentration of 0.25%. Similar results
were observed when the serum pool was obtained from the same
individuals 4 h after being fed.
IGF-I binding assays
Human recombinant IGF-I was iodinated with
[125I]radionuclide (Amersham) and purified by gel
filtration chromatography, as previously described (31). Human
myoblasts were grown and subcultured into 24-well cluster plates as
described above. Forty minutes before the binding assays, half of the
wells were incubated with TNF- (100 ng/ml). Cells were subsequently
rinsed with ice-cold HBSS and incubated with 125I-labeled
IGF-I in HBSS with 0.1% gelatin at 4 C for 12 h in the presence
of increasing amounts of unlabeled IGF-I. Cells were rinsed with HBSS,
solubilized in 1 N sodium hydroxide, and bound
radioactivity measured in a 
-counter (Wallac). Results are expressed
as the percentage of 125I-labeled IGF-I bound to myoblasts
after subtraction of nonspecific binding determined in the presence of
1 µg/ml unlabeled IGF-I.
Statistical analysis
All experiments were repeated on at least three separate
occasions. Individual data points are expressed as the mean ±
SE for triplicate wells. Statistical comparisons were made
by ANOVA followed by Student-Newman-Keuls test to determine treatment
effect. Statistical significance was set at P <
0.05.
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Results |
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). Maximal
stimulation occurred with as little as 0.25% human serum. IGF-I also
stimulated protein synthesis dose-dependently (Fig. 1B). Half-maximal
stimulation occurred at 8 ng/ml. The response of myoblasts to IGF-I was
not altered by increasing concentrations of unlabeled phenylalanine
(0.2–0.8 mM). In addition, the incorporation of
[3H]phenyl-alanine into protein was confirmed by the
ability of the protein synthesis inhibitor cycloheximide to inhibit
incorporation (Fig. 2A) and by the
resistance of TCA precipitable material to base hydrolysis (Fig. 2B).
Similar results were also obtained when [3H]tyrosine was
used as the labeled amino acid (data not shown). These data demonstrate
that protein synthesis can be quantitated in human myoblasts, and that
myoblasts are responsive to both human serum and IGF-I.
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inhibits IGF-I-stimulated protein synthesis is elevated in pathophysiological conditions characterized
by muscle wasting. Therefore, we examined whether TNF- could inhibit
basal and/or IGF-I stimulated protein synthesis in human myoblasts.
TNF- (100 ng/ml) decreased basal protein synthesis in human
myoblasts by 15% (Fig. 3A). IGF-I (40
ng/ml) stimulated protein synthesis by 18%. The effect of this
maximally stimulating dose of IGF-I was completely inhibited by the
addition of TNF- (100 ng/ml) to the cells (P <
0.05). TNF- inhibited the ability of IGF-I to stimulate protein
synthesis in a dose-dependent manner (Fig. 3B). Statistically
significant inhibition of this response occurred with as little as 2
ng/ml of TNF-.
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inhibits protein synthesis irrespective of sequence of
addition of growth factors could also inhibit the ability of whole serum to
stimulate protein synthesis. TNF- inhibited protein synthesis when
it was added simultaneously with serum, or if it was added 30 min after
myoblasts had been exposed to serum (Fig. 4A). TNF- also inhibited protein
synthesis when it was added 30 min before the addition of serum and
then removed for the remainder of the experiment (Fig. 4A). Similar
results were obtained when IGF-I was added to the cells instead of
serum (Fig. 4B).
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inhibits serum-stimulated protein
synthesis could inhibit the ability of serum to subsequently stimulate
protein synthesis, because the host is often only transiently exposed
to elevated plasma concentrations of cytokines. Exposure of human
myoblasts to TNF- for as little as 10 min inhibited the ability of
serum to stimulate protein synthesis over the next 6 h (Fig. 5A). Exposure of cells to TNF- for 20
min also inhibited the ability of serum to stimulate protein synthesis
when cells were challenged with serum 48 h later (Fig. 5B).
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does not inhibit IGF-I- or serum-stimulated thymidine
uptake has been shown to inhibit insulin-stimulated
glucose uptake in L6 myoblasts (34), we questioned whether TNF-
inhibited serum-stimulated events in general or whether it selectively
inhibited protein synthesis. Therefore, we examined whether TNF-
inhibited the ability of serum and IGF-I to stimulate thymidine uptake
in human myoblasts. IGF-I (40 ng/ml) and serum (0.25%) stimulated
thymidine uptake by 70% and 80%, respectively (Fig. 6, A and B). Although TNF- completely
inhibited the ability of serum and IGF-I to stimulate protein
synthesis, it failed to inhibit growth factor-stimulated thymidine
uptake (Fig. 6, A and B). There was a trend toward TNF- inhibiting
basal thymidine uptake in the absence of growth factors, but this
response did not reach statistical significance. Moreover, the affect
of TNF- was overcome by either serum or IGF-I (Fig. 6, A and B).
These data show that serum and IGF-I are biologically active in the
presence of TNF-, and that TNF- selectively inhibits only a
portion of growth factor stimulated events in human myoblasts.
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does not alter IGF-I binding to myoblasts may inhibit protein synthesis by altering IGF
receptor binding characteristics on myoblasts, we examined whether
TNF- affected the ability of 125I-labeled IGF-I to bind
to human myoblasts. Pretreatment of myoblasts with TNF- (100 ng/ml)
for 40 min had no affect on either the maximal binding of IGF-I to
myoblasts or the affinity of the IGF-I receptor for IGF-I (Fig. 7).
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inhibits serum-stimulated protein synthesis in myotubes). TNF- completely inhibited the
ability of serum to stimulate protein synthesis in myotubes (Fig. 8).
These responses are similar to those that we have observed for
myoblasts.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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. The current
data are consistent with previous work by Ewton et al. (32)
and Ballard et al. (33) showing that IGF-I stimulates amino
acid uptake and protein synthesis in the rat L6 muscle cell line. Our
work is also consistent with the finding by Fryburg (12), that IGF-I
stimulates protein synthesis in the forearm of healthy volunteers.
We demonstrate for the first time that TNF- can directly inhibit
both the basal level of protein synthesis, as well as the ability of
serum and IGF-I to stimulate protein synthesis in myoblasts. TNF-
expression may occur transiently in response to infectious-like
insults, but alterations in muscle protein balance are often manifested
over relatively longer periods of time. Many of the in vivo
effects of TNF- have previously been attributed to its ability to
stimulate the production of other cytokines (21). However, our in
vitro data shows that TNF- can also directly inhibit protein
synthesis. Exposure of myoblasts to TNF- for as little as 10 min
completely blocked the ability of serum and IGF-I to stimulate protein
synthesis. Moreover, the ability of IGF-I to stimulate protein
synthesis was impaired for at least 48 h after transient exposure
to the cytokine. These data suggest that a transient increase in the
plasma concentration of TNF- may impair protein synthesis long after
the cytokine has disappeared from the circulation.
The decrease in protein synthesis we observed in myoblasts and myotubes
treated with TNF- is similar to the inhibition of protein synthesis
observed in skeletal muscle from septic animals (11). In contrast to
our results obtained in cell culture, perfusion of the hindlimb with
IGF-I restores protein synthesis in the gastrocnemius of septic rats
(11). It is possible that the decrease in muscle protein synthesis
observed in this model results not from a direct effect of cytokines on
muscle, but from the dramatic decrease in the plasma concentration of
IGF-I that occurs with the insult (13). Perfusion of muscle with IGF-I
may restore protein synthesis by simply reexposing muscle to a high
concentration of IGF-I. It is also possible that the intramuscular
concentration of TNF- achieved in the animal model of sepsis (13)
may be below the concentration used in the present study.
Alternatively, myoblasts and myotubes may respond differently to IGF-I
and TNF- than does the hindlimb, which also contains nerve, adipose,
and connective tissue.
The concentration of serum chosen for this study was based on reports that have shown DNA synthesis to be maximally stimulated at very low concentrations of serum (i.e. < 1%) (35). Human serum, in the concentration range we have used, also contains the same amount of IGF-I that is necessary to maximally stimulate protein synthesis. This concentration of IGF-I is also comparable with that found in extracellular fluid to which muscle would be exposed, and the concentration of IGF-I that can be measured in rat muscle (17). High concentrations of IGF-I gave a suboptimal response in human myoblasts. This response may result from a down-regulation of the IGF-I receptor at high concentrations of IGF-I (36).
TNF- inhibited both IGF-I-stimulated protein synthesis and
serum-stimulated protein synthesis in myoblasts. This suggests that
TNF- may have a dominant negative effect not only on the ability of
IGF-I to stimulate protein synthesis, but also a negative effect on the
ability of other anabolic factors that are present in serum. We have
previously reported that TNF- decreases both the plasma and
intramuscular concentrations of IGF-I, and that there is a positive
correlation between the decreased content of IGF-I and the impaired
rate of protein synthesis in muscle of septic animals (17). Thus,
inflammatory stimuli appear to alter both the magnitude of the IGF-I
signal, and the ability of the IGF-I signal to be efficiently
transduced at the cellular level. These changes may, in part, be
responsible for the reduction in muscle protein synthesis during
infection and other conditions associated with the overexpression of
TNF-.
We have found the effect of TNF- on protein synthesis to persist
well past the initial exposure of myoblasts to the cytokine. Our data
suggest that TNF- rapidly binds to myoblasts, and initiates a series
of events that inhibit the ability of serum and IGF-I to stimulate
protein synthesis. These cellular changes do not require continuous
stimulation by TNF-, and suggest that a key component of the IGF-I
signal transduction pathway is altered by exposure to TNF.
Exposure of human myoblasts to TNF- did not alter IGF-I receptor
binding characteristics. Therefore, it appears that TNF- interferes
with the IGF-I signal transduction pathway at a point distal to
receptor binding and autophosphorylation. Our data suggest that the
IGF-I signaling pathway bifurcates, and that TNF- acts selectively
on a component of the pathway that is necessary for stimulation of
protein synthesis but not thymidine uptake. One possibility is that
TNF- interferes with kinases that are responsible for stimulating
components of the protein synthetic machinery. IGF-I and insulin have
been shown to stimulate the kinase that is responsible for
phosphorylating ribosomal protein S6 (p70/S6 kinase) (37). The p70/S6
kinase phosphorylates the eukaryotic initiation factor 4E (eIF4E)
binding protein, PHAS-1, and thus stimulates protein synthesis (38).
p70/S6 kinase activity is also correlated with the translation of
messenger RNAs for elongation factors and ribosomal proteins (39). It
is possible that TNF- may inhibit this process and/or other
components of the IGF-I signal transduction pathway that normally
result in enhanced protein synthesis.
TNF- did not significantly impair the stimulation of thymidine
uptake by either serum or IGF-I. This suggests that the proteins
necessary for thymidine uptake are present in the cell before addition
of TNF-, or that TNF- affects only the synthesis of a subset of
proteins that are not required for thymidine uptake. TNF- inhibited
basal protein synthesis by only 20%, leaving up to 80% of the maximum
protein synthetic capacity of the myoblast intact. This moderate change
in protein synthesis may explain why DNA synthesis is able to proceed
in cells treated with both TNF- and IGF-I. The changes in DNA and
protein synthesis we have observed are strikingly similar to the effect
that the p70/S6 kinase inhibitor rapamycin has on cycling cells
(40).
TNF- is cytostatic in many tumor cells, but is also a mitogen for
fibroblasts (41, 42). We find that there is a trend toward TNF-
inhibiting thymidine uptake in myoblasts, but that this effect does not
reach statistical significance. This slight inhibition can be overcome
with either serum or IGF-I. This confirms both the viability of the
myoblasts and the selectivity of TNF action.
In conclusion, the results of the present study indicate that TNF-
directly inhibits both the basal level of protein synthesis and growth
factor-stimulated protein synthesis in human myoblasts. TNF- also
inhibits the ability of serum to stimulate protein synthesis in
myotubes. TNF- acts rapidly, such that even a transient exposure to
TNF- is inhibitory. TNF- does not inhibit IGF-I or
serum-stimulated thymidine uptake, suggesting that TNF- acts
specifically on a component of the IGF-I signal transduction pathway
involved in stimulating protein synthesis. Furthermore, transient
exposure of myoblasts to TNF- inhibits protein synthesis for 48
h without the need for other cytokines. Hence, the human myoblast
culture system can be used to identify factors that affect muscle
protein synthesis. This system will be useful in determining the
mechanism(s) responsible for muscle wasting in various
pathophysiological conditions.
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Footnotes |
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2 Current address: Department of Cellular and Molecular Physiology,
College of Medicine, Pennsylvania State University, Hershey,
Pennsylvania 17033.
Received February 26, 1997.
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, P < 0.01,
different from IGF-I alone. B, Dose-dependent inhibition of
IGF-I-stimulated protein synthesis. Values are mean ±
SE (n = 3); *, P < 0.05 different
from control; 
) for 40 min. Cells were washed with
ice-cold HBSS and incubated with 125I-labeled IGF-I and an
increasing concentration of unlabeled IGF-I at 4 C for 12 h.
TNF-