1 Copenhagen Muscle Research Centre and 2 Department of Infectious Diseases, Rigshospitalet, University of Copenhagen, DK-2100 Copenhagen, Denmark; and 3 Department of Physiology, The University of Melbourne, Parkville, Victoria xxxx, Australia
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ABSTRACT |
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The aim of the present study was to
examine whether IL-6 and TNF- are expressed in, and released from,
human skeletal muscle during exercise. We hypothesized that the
skeletal muscle will release IL-6, but not TNF-
, during exercise
because of previous observations that TNF-
negatively affects
glucose uptake in skeletal muscle. Six healthy, male subjects performed
180 min of two-legged knee-extensor exercise. Muscle samples were
obtained from the vastus lateralis of one limb. In addition,
blood samples were obtained from a femoral artery and vein. Plasma was
analyzed for IL-6 and TNF-
. We detected both IL-6 and TNF-
mRNA
in resting muscle samples, and whereas IL-6 increased
(P < 0.05) ~100-fold throughout exercise, no
significant increase in TNF-
mRNA was observed. Arterial plasma
TNF-
did not increase during exercise. Furthermore, there was no net
release of TNF-
either before or during exercise. In contrast, IL-6
increased throughout exercise in arterial plasma, and a net IL-6
release from the contracting limb was observed after 120 min of
exercise (P < 0.05).
cytokines; interleukins; exercise; metabolism; endocrine system
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INTRODUCTION |
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CYTOKINES ARE A
GROUP OF PROTEINS produced by many cells and/or tissues in
response to stress (1). Given that physical exercise can
markedly disrupt cellular homeostasis, it is not surprising that
exercise elevates plasma levels of several cytokines, including
interleukin (IL)-6 and tumor necrosis factor (TNF)- (22). Until recently, it was hypothesized that the
exercise-induced increase in these cytokines was a consequence of an
immune response due to local damage in the working muscles
(19). However, work from our research group has
demonstrated that the immune cells are not the source of the increase
in plasma TNF-
or IL-6 during exercise (32).
In addition to the large body of research examining the immunological
effects of elevations in TNF- and IL-6, recent studies have also
focused on the metabolic effects of these cytokines. Both have been
found to be expressed in human skeletal muscle (29, 31),
and both are associated with insulin resistance and type 2 diabetes
(15). Work from our laboratory has focused on the origin
of the exercise-induced increase in IL-6. We have demonstrated that
muscle contraction rapidly increases IL-6 gene expression in skeletal
muscles in both rats (12) and humans (21, 31,
33). In addition, we have shown that the intramuscular nuclear
transcriptional activity of IL-6 is rapidly increased with the onset of
exercise (14) and that IL-6 protein is released from
skeletal muscle during prolonged exercise (33, 34). The contraction-induced increased transcriptional activity of the IL-6 gene
and the IL-6 protein release is further elevated when intramuscular
glycogen stores are low (14, 33). We have also recently
shown that cultured human primary muscle cells are capable of
increasing IL-6 mRNA when incubated with the calcium ionophore ionomycin (13). Therefore, it is likely that myocytes
produce IL-6 in response to muscle contraction, an affect that is
exacerbated by low intramuscular glycogen stores, and production of
IL-6 by such tissue accounts for the exercise-induced increase in
plasma levels of this cytokine.
In contrast with the well described literature concerning IL-6, less is
known regarding the cells/organs responsible for the increased plasma
levels of TNF- observed during strenuous exercise (21,
32). TNF-
is expressed in human skeletal muscle
(29), and it is therefore possible that contracting
skeletal muscle is the source of this increase. However, TNF-
expression is augmented in the skeletal muscle of patients with type 2 diabetes (29), and it decreases insulin-stimulated rates
of glucose storage in cultured human muscle cells (11).
Furthermore, TNF-
administration impairs both insulin-mediated
capillary recruitment and glucose uptake in anesthetized rats
(42), whereas TNF-
-null mice are protected from insulin
resistance (39). It is well known that muscle contraction
rapidly increases glucose uptake; therefore, an exercise-induced
increase in TNF-
would, on the one hand, appear paradoxical. On the
other hand, cytokines are produced in many cells and/or tissues in
response to stress, and given that muscle contraction can markedly
disrupt muscle cell homeostasis, it is possible that such cells may be
producing TNF-
during contraction. To our knowledge, no studies have
examined the TNF-
expression in contracting skeletal muscle or
whether TNF-
is released from muscle during contraction.
Although we have presented hypotheses as to the biological role of IL-6
expression in contracting muscle (23), we have yet to
determine the precise biological role. Of note, it has been demonstrated that IL-6 can impair TNF- expression in cardiac muscle
(35); therefore, one potential role of IL-6 expression in
contracting muscle may be to downregulate TNF-
expression. Because
the contracting muscle is the organ that undergoes the greatest
disruption to homeostasis during exercise, we aimed to determine
whether this organ is responsible for the observed increase not only in
IL-6 but also in TNF-
during exercise. We hypothesized that the
skeletal muscle will release IL-6, but not TNF-
, during exercise
because of the previous observations that TNF-
promotes impaired
glucose uptake in skeletal muscle. In addition, we aimed to determine
whether the TNF-
expression increased during contraction and whether
the kinetics of any increase differed from the expression of IL-6, so
that we could explore the possibility that a role for IL-6 expression
in skeletal muscle may be to downregulate any increase in TNF-
expression.
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MATERIALS AND METHODS |
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Subjects
Six healthy, physically active but not specifically trained, male subjects [mean age 26 yr (range 22-33), mean weight 78.1 kg (range 70-93), mean height 1.87 m (range 1.75-1.93 m)] were recruited to participate in the study. The subjects were given both oral and written information about the experimental procedures before giving their written informed consent. The study was approved by the Copenhagen and Frederiksberg Ethics Committees, Denmark.Experimental Design
Preexperimental protocol. Each subject underwent preliminary exercise tests on the two-legged knee-extensor apparatus (5). After they became familiar with the knee-extensor exercise model, they underwent a maximal exercise test to determine their individual knee-extensor peak power output (Wmax, ke). Thereafter, they performed ~2 h of two-legged knee-extensor exercise at ~55% Wmax, ke (mean workload = 93 ± 4 W) to become fully accustomed to performing the exercise for prolonged periods. At 1700 on the day before the experimental trial, subjects reported to the laboratory and performed 60 min of upright bicycling exercise, which subjects could tolerate for the duration of the exercise period. Subjects were then provided with a diet consisting of 300 ml of fruit juice, 500 ml of cola, 100 g of raisins, and 500 g of pasta (20% protein, 75% carbohydrate, 5% fat). This diet was consumed by 2100 on the evening before the experimental trial.
Experimental procedures.
On the day of the experiment, subjects reported to the laboratory at
0730, voided, changed into appropriate exercise attire, and rested in a
supine position. Catheters were then placed in the femoral vein and
femoral artery of one limb with subjects under local anesthesia
(Lidocaine, 20 mg/ml), as previously described (2). A
blood sample was then drawn into a precooled tube containing EDTA,
mixed, and immediately centrifuged at 2,200 g for 15 min at
4°C. The plasma was stored at 80°C until analysis. At this point,
the femoral arterial blood flow was measured with ultrasound Doppler as
previously validated (26). A muscle biopsy was then obtained from vastus lateralis by use of the percutaneous biopsy technique with suction and immediately frozen in liquid nitrogen. The
subjects then performed two-legged dynamic knee-extensor exercise at
~55% of their maximal workload for 180 min (mean workload = 93 ± 4 W). Blood samples and concomitant femoral arterial blood flow measures were obtained at 60, 120, and 180 min during exercise. In
addition, further muscle samples were obtained at 30, 90, and 180 min
during exercise. Subjects were permitted to drink water ad libitum
during exercise but were not permitted to consume any food or drink.
Tissue Analysis
Femoral arterial venous plasma was analyzed for IL-6 and TNF-Upon collection, muscle samples were divided into two portions. One
portion was analyzed for glycogen, as previously described (33). The second portion was analyzed for IL-6 and TNF-
mRNA. The muscle was extracted for total RNA with a modification of the
acid guanidinium thiocyanate-phenol-chloroform extraction of
Chomczynski and Sacchi (7), as previously described
(24). Final RNA pellets were dissolved in 0.1 mM EDTA (2 µl/mg original wet wt). Reverse transcription reactions were carried
out on 22 µl of sample with the superscript II RNAse H
Reverse Transcriptase (Invitrogen) in a reaction volume of 40 µl. All
samples were diluted in 160 µl of nuclear-free water.
Real-time PCR was employed to quantitate human IL-6 and TNF- gene
expression from the cDNA samples. Both human IL-6 and TNF-
were
designed (Primer Express version 1.0 Applied Biosystems) from the human
IL-6 and TNF-
gene sequences (GenBank/EBML accession nos. M54894 and
M38669 and X02910 and X02159 for IL-6 and TNF-
, respectively). An
81-base-length IL-6 fragment was amplified using the
forward primer 5'-GGTACATCCTCGACGGCATCT-3' and reverse primer
5'-GTGCCTCTTTGCTGCTTTCAC-3' (Sigma Geno-sys, Castle Hill, NSW,
Australia). A TaqMan fluorescent probe, 5'-FAM (6-carboxyfluorescein)TGTTACTCTTGTTACATGTCTCCTTTCTCAGGGCT-3' TAMRA (6-carboxy-tetramethylrhodamine) (Applied Biosystems) was included with
the primers in each reaction. For TNF-
, an 81-base-length fragment
was amplified using the forward primer
5'-CCCAGGCAGTCAGATCATCTTC-3' and reverse primer
5'-AGCTGCCCCTCAGCTTGA-3' (Sigma Geno-sys). A TaqMan fluorescent probe
5'-FAM (6-carboxyfluorescein)-TCTTCAAGGGCCAAGGCTGCCC-3' TAMRA
(6-carboxy-tetramethylrhodamine) (Applied Biosystems) was included with
the primers in each reaction. We also amplified 18S mRNA, and the
TaqMan probes and primers for this gene were supplied in a control
reagent kit (Applied Biosystems). We quantitated gene expression with a
multiplex comparative critical threshold (CT) method (ABI
PRISM 7700). A CT value reflects the cycle number at which
the cDNA amplification is first detected. This method detected our
reference genes (human 18S) and IL-6 or TNF-
in a single tube, where
the primers for 18S were limited to ensure that adequate amounts of
reagents were present for amplification of both genes.
It was possible to detect 18S in the same tube as our gene of interest
because the reporter dyes attached to the TaqMan probes fluoresce at
different emission wavelength maxima. In preliminary experiments, we
determined the relative efficiency of amplification of 18S vs. IL-6 or
TNF-. These experiments revealed approximately equal efficiencies of
18S and IL-6 or TNF-
amplifications over different starting template
concentrations. We also performed experiments to demonstrate that
multiplex vs. nonmultiplex experiments had no effect on CT
values, as well as primer-limited multiplex 18S vs. nonprimer-limited
nonmultiplex 18S reactions. Finally, we determined the linear dynamic
range for starting template concentrations.
PCR reactions were carried out in 25-µl reactions of TaqMan universal
PCR master mix (1×), 50 nM TaqMan 18S probe, 20 nM 18S forward primer,
80 nM 18S reverse primer, either 100 nM TaqMan IL-6 probe or 175 nM
TaqMan TNF- probe, 900 nM IL-6 forward primer or 900 nM TNF-
forward primer, 300 nM IL-6 reverse primer, or 900 nM TNF-
reverse
primer. Each reaction was made up to volume with RNase-free
H2O. Concentrations of the IL-6 and TNF-
probes and
primers were chosen on the basis of pilot analyses in which optimal
concentrations were determined. Fifty nanograms of cDNA and control
preparations were amplified using the following conditions: 50°C for
2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s
and 60°C for 1 min. For each sample, a change in (
)CT value was obtained by subtracting 18S CT values from IL-6
or TNF-
, with the resting value as the control. Resting values for
each subject were subtracted from the exercise samples for each subject to derive a
CT value. The expressions of human
IL-6 and TNF-
were then evaluated by
2
CT.
Statistical Analysis
All parameters were tested for normalcy in distribution. Accordingly, both IL-6 and TNF- ![]() |
RESULTS |
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Muscle glycogen averaged 398 ± 52 mmol/kg dry wt and
progressively decreased (P < 0.05) throughout exercise
such that values were 153 ± 50 mmol/kg dry wt after 180 min (Fig.
1). Plasma glucose did not change during
the exercise, whereas plasma free fatty acids (FFA) increased
(P < 0.05) (Table 1).
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We were able to detect both TNF- and IL-6 mRNA in the resting muscle
biopsy samples. Although there was a tendency (P = 0.08) for an increase in TNF-
mRNA when exercise values were
compared with those collected before exercise, results were not
significant (Fig. 2). In contrast, IL-6
mRNA increased (P < 0.05) after only 30 min of
exercise and peaked at the cessation of exercise, when values were
~100-fold higher compared with rest (Fig. 2).
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Arterial plasma TNF- concentration averaged 2.4 ± 0.2 pg/ml at
rest and did not increase during exercise. Furthermore, we could not
detect any arterial-femoral venous difference for TNF-
; therefore,
there was no net release of TNF-
either before or during exercise
(Fig. 3). In contrast, arterial
concentrations of IL-6 averaged 0.57 ± 0.18 pg/ml at rest but
increased (P < 0.05) progressively throughout
exercise. In addition, we observed an arterial-femoral venous
difference (P < 0.05) for IL-6 after 120 min of
exercise, and this remained until the cessation of exercise. As a
consequence, we observed net leg release (P < 0.05) of
IL-6 after 120 min of exercise, and this persisted until the cessation
of exercise (Fig. 3).
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Leg blood flow averaged 0.35 ± 0.07 l/min at rest and increased
(P < 0.05) to an average of 2.18 ± 0.09 l/min
during exercise (Fig. 4).
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DISCUSSION |
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The results from this study demonstrate that, despite 3 h of
strenuous exercise, contracting muscle increases gene expression and
protein release of IL-6, but not of TNF-. Therefore, our data
demonstrate a clear disassociation between IL-6 and TNF-
production
in skeletal muscle during exercise and are consistent with the
hypothesis that TNF-
, but not IL-6, impairs glucose uptake by
skeletal muscle.
Although TNF- has been previously measured in human skeletal muscle
(22), this is the first study to measure the effect of
acute contraction on TNF-
in human muscle. These data support those
of Greiwe et al. (10), who found that TNF-
gene and
protein expression decreased in frail elderly patients after a
resistance training program. In the present study, despite 3 h of
continual contractile activity that resulted in a marked decrease in
intramuscular glycogen (Fig. 1), TNF-
mRNA was not significantly
increased compared with rest. It could be argued that TNF-
mRNA and
protein release did not increase because the glycogen levels were not reduced to very low levels and that TNF-
may be sensitive only to
very low glycogen stores. Of note, however, there was an approximately fourfold increase (P = 0.08) in TNF-
mRNA after 30 min of exercise, after which time it did not increase any further. If
TNF-
were sensitive to glycogen content, one would not have expected
the greatest degree of change, albeit not statistically significant, to
be observed after 30 min of exercise when glycogen stores were hardly
compromised. In contrast, the degree of increase in IL-6 mRNA peaked
after 180 min of exercise. We have been able to demonstrate, in
preliminary experiments, that TNF-
gene expression is inducible in
human skeletal muscle cells. We have shown that, when stimulated with
the calcium ionophore ionomycin, primary human muscle cells in culture
significantly increased TNF-
gene expression after 6 h of
incubation, after which time it fell to basal levels (Keller C,
Hellsten Y, Pilegaard H, Febbraio M, and Pedersen BK, unpublished data). Therefore, we cannot rule out the possibility that more intense exercise, which would increase the magnitude of calcium release
from the sarcoplasmic reticulum, would in turn increase TNF-
gene
expression. However, it would appear from our cell culture experiments
that any increase would be transient. In contrast to the pattern of
TNF-
expression, IL-6 peaked in the same stimulated culture
preparation after 24 h (13). Given these preliminary results and those from the present experiment, we propose that one
function of the marked increase in IL-6 gene expression in skeletal
muscle during muscle contraction may be to inhibit any increase in
TNF-
production. During contraction, muscle glucose uptake is
markedly increased compared with rest; therefore, our hypothesis is
consistent with the observation that TNF-
impairs glucose disposal
in skeletal muscle (42). Indeed, there are data
demonstrating that IL-6 can impair increases in TNF-
. Tanaka et al.
(35) recently demonstrated that, during viral myocarditis, the serum TNF-
concentration is markedly reduced in transgenic mice,
which overexpress IL-6 compared with wild-type mice.
Along with the differential expression of IL-6 and TNF- mRNA within
skeletal muscle, we also observed that IL-6, but not TNF-
, was
released during contraction. In the current study, we did not make
protein measures within the skeletal muscle, and therefore we cannot be
certain that TNF-
protein was not expressed in contracting skeletal
muscle. However, given the fact that TNF-
mRNA did not significantly
increase within the muscle, this possibility appears remote. It is also
important to note that arterial TNF-
concentration did not increase
as a result of exercise, and therefore we cannot rule out the
possibility that, in the studies in which TNF-
increased in the
blood, the muscle was indeed the source of this increase. On careful
examination of the literature, however, elevations in plasma TNF-
as
a result of exercise have been observed only after marathon running
(20, 21, 32, 37, 38) and not after other forms of
strenuous or prolonged exercise. During marathon running, a decrease in
blood flow to the splanchnic bed may occur that can induce an
ischemic state, resulting in gut wall bacterial translocation
(3). Indeed, endotoxemia has been observed after marathon
running (6), and because endotoxins are
lipopolysaccharides of gram-negative bacteria, it is probable that the
increased plasma TNF-
observed during marathon running is likely to
result from systemic endotoxemia rather than from contracting muscle
release. In addition, we have recently observed that, during eccentric
exercise that results in severe muscle membrane damage, TNF-
levels
are not increased (38). These data lend support to the
suggestion that TNF-
is enhanced in the plasma only after marathon
running and that this is due to endotoxemia.
Our observation of IL-6 gene expression within, and protein release
from, contracting skeletal muscle is consistent with our previous
observations (14, 31, 33, 34). Although we have not yet
determined the precise biological action of this phenomenon, we have
hypothesized that the role of IL-6 release from contracting muscle
during exercise is to act in a hormone-like manner to mobilize extramuscular substrates and/or augment substrate delivery during exercise (23). Of note, Wallenius et al. (40)
recently demonstrated that IL-6-deficient mice develop obesity and
glucose intolerance, whereas chronic treatment of these animals with
IL-6 partially attenuates these metabolic perturbations. Because the
kinetics of IL-6 gene expression differed markedly from those of
TNF-, these present data might also suggest that another role for
muscle-derived IL-6 is to impair TNF-
expression in skeletal muscle.
Indeed, previous studies provide evidence for the notion that a primary
role of IL-6 may be to inhibit the production of TNF-. IL-6 inhibits
LPS-induced TNF-
production in both cultured human monocytes and the
human monocytic line U937 (30). The suppressive effect
occurs at the level of transcription in human peripheral blood
mononuclear cells. In in vivo endotoxin models, levels of TNF-
were
elevated in anti-IL-6-treated mice (17) and in
IL-6-deficient knockout mice compared with control mice, suggesting
that circulating IL-6 regulates TNF-
levels (18). IL-6
has strong anti-inflammatory effects. Thus IL-6 administration in
humans induces interleukin-1 receptor antagonist (IL-1ra) and soluble
TNF-
receptors, but not IL-1
and TNF-
(36).
Furthermore, IL-6 induces the production of C-reactive protein, which
has a role both in the induction of anti-inflammatory cytokines in
circulating monocytes and in the suppression of the synthesis of
proinflammatory cytokines in tissue macrophages (25).
Taking these observations together, we propose that muscle contractions
result in the generation of a strong anti-inflammatory response.
Both exercise and insulin stimulation increase glucose uptake in
skeletal muscles (27, 28). Two key proteins involved in,
respectively, insulin-stimulated and contraction-induced increased glucose uptake are phosphatidylinositol 3 (PI 3)-kinase and
mitogen-activated protein kinase (MAPK) (28). IL-6 can
bind to either a soluble IL-6 receptor or a membrane-bound receptor and
activate the gp130-signaling pathway (41). Stimulation of
the gp130 receptor in cardiac muscle increases the activity of both PI
3-kinase and MAPK (41). Importantly, insulin resistance is
associated with a decreased activity of PI 3-kinase in skeletal muscle
(28). Therefore, one possible function of the upregulated
IL-6 during exercise could be to increase glucose uptake in the
contracting skeletal muscle by activating MAPK. Second,
exercise-derived IL-6 could account for the increased insulin
sensitivity found in response to regular exercise through the
activation of PI 3-kinase (16). In contrast, TNF- has
been found to downregulate both PI 3-kinase and MAPK in skeletal muscle cells (9). However, in principle, PI 3-kinase and MAPK may also regulate IL-6; therefore, a possible relationship could be inverse. Thus MAPK, in particular p38, can activate IL-6
(8), whereas PI 3-kinase has an inhibitory effect on IL-6
expression, since pharmacological blockade of PI 3-kinase can enhance
monocyte IL-6 production severalfold (4). This implies
that increased MAPK activity during exercise would stimulate the IL-6
production. Furthermore, because of the reduced activity of PI 3-kinase
in insulin-resistant muscles, IL-6 expression would be increased in
these muscles.
In conclusion, we have demonstrated for the first time a clear
disassociation between IL-6 and TNF- production in skeletal muscle
during exercise. We have shown that, although intramuscular IL-6 gene
expression and protein release are remarkable during continuous
contractile activity, no such response is evident for TNF-
. Hence,
our data are consistent with the hypothesis that TNF-
, but not IL-6,
impairs glucose uptake by skeletal muscle.
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ACKNOWLEDGEMENTS |
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The technical assistance of Ruth Rousing and Hanne Willumsen is gratefully acknowledged. We also thank Dr. Irene Koukoulas for helpful advice with respect to real-time PCR technology.
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FOOTNOTES |
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This study was supported by a grant from the Danish National Research Foundation (504-14). T. Osada is a Visiting Fellow from the Department of Preventive Medicine and Public Health, Tokyo Medical University, Tokyo, Japan.
Address for reprint requests and other correspondence: A. Steensberg, The Copenhagen Muscle Research Centre, Rigshospitalet 7641, Blegdamsvej 9, DK-2100 Copenhagen, Denmark (E-mail: SBERG{at}rh.dk).
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.
August 20, 2002;10.1152/ajpendo.00255.2002
Received 10 June 2002; accepted in final form 13 August 2002.
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