IL-6 and TNF-alpha expression in, and release from, contracting human skeletal muscle

Adam Steensberg1,2, Charlotte Keller1,2, Rebecca L. Starkie3, Takuya Osada1, Mark A. Febbraio1,3, and Bente Klarlund Pedersen1,2

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The aim of the present study was to examine whether IL-6 and TNF-alpha are expressed in, and released from, human skeletal muscle during exercise. We hypothesized that the skeletal muscle will release IL-6, but not TNF-alpha , during exercise because of previous observations that TNF-alpha 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-alpha . We detected both IL-6 and TNF-alpha mRNA in resting muscle samples, and whereas IL-6 increased (P < 0.05) ~100-fold throughout exercise, no significant increase in TNF-alpha mRNA was observed. Arterial plasma TNF-alpha did not increase during exercise. Furthermore, there was no net release of TNF-alpha 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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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)-alpha (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-alpha or IL-6 during exercise (32).

In addition to the large body of research examining the immunological effects of elevations in TNF-alpha 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-alpha observed during strenuous exercise (21, 32). TNF-alpha is expressed in human skeletal muscle (29), and it is therefore possible that contracting skeletal muscle is the source of this increase. However, TNF-alpha 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-alpha administration impairs both insulin-mediated capillary recruitment and glucose uptake in anesthetized rats (42), whereas TNF-alpha -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-alpha 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-alpha during contraction. To our knowledge, no studies have examined the TNF-alpha expression in contracting skeletal muscle or whether TNF-alpha 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-alpha expression in cardiac muscle (35); therefore, one potential role of IL-6 expression in contracting muscle may be to downregulate TNF-alpha 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-alpha during exercise. We hypothesized that the skeletal muscle will release IL-6, but not TNF-alpha , during exercise because of the previous observations that TNF-alpha promotes impaired glucose uptake in skeletal muscle. In addition, we aimed to determine whether the TNF-alpha 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-alpha expression.


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

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-alpha by a commercially available enzyme-linked immunosorbent assay (ELISA; R&D Systems Europe, Oxon, UK). All measurements were performed in duplicate, and high-sensitivity kits were used. According to information provided by R&D Systems, the kits used for measuring IL-6 and TNF-alpha are insensitive to the addition of the recombinant forms of the soluble receptors (sIL-6R, sTNF-R1, and sTNF-R2, respectively); these measurements, therefore, correspond to both soluble and receptor-bound cytokines.

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-alpha 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-alpha gene expression from the cDNA samples. Both human IL-6 and TNF-alpha were designed (Primer Express version 1.0 Applied Biosystems) from the human IL-6 and TNF-alpha gene sequences (GenBank/EBML accession nos. M54894 and M38669 and X02910 and X02159 for IL-6 and TNF-alpha , 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-alpha , 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-alpha 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-alpha . These experiments revealed approximately equal efficiencies of 18S and IL-6 or TNF-alpha 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-alpha probe, 900 nM IL-6 forward primer or 900 nM TNF-alpha forward primer, 300 nM IL-6 reverse primer, or 900 nM TNF-alpha reverse primer. Each reaction was made up to volume with RNase-free H2O. Concentrations of the IL-6 and TNF-alpha 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 (Delta )CT value was obtained by subtracting 18S CT values from IL-6 or TNF-alpha , with the resting value as the control. Resting values for each subject were subtracted from the exercise samples for each subject to derive a Delta  -Delta CT value. The expressions of human IL-6 and TNF-alpha were then evaluated by 2-Delta -Delta CT.

Statistical Analysis

All parameters were tested for normalcy in distribution. Accordingly, both IL-6 and TNF-alpha mRNA values, as well as plasma IL-6, were not normally distributed. Hence, the data for these parameters were log transformed before analyses. A one-way analysis of variance (ANOVA) was performed on the data. After a significant F-test, pair-wise differences were identified using Newman-Keuls post hoc procedure. The significance level was set at P < 0.05. Data are presented as means ± SE unless otherwise stated.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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).


View larger version (9K):
[in this window]
[in a new window]
 
Fig. 1.   Muscle glycogen concentration before (Pre), during (30 and 90 min), and after (180 min) 3 h of two-legged knee-extensor exercise. Values are means ± SE (n = 6 subjects). * Significant difference (P < 0.05) compared with Pre.


                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Plasma glucose and FFA before, during, and after exercise

We were able to detect both TNF-alpha and IL-6 mRNA in the resting muscle biopsy samples. Although there was a tendency (P = 0.08) for an increase in TNF-alpha 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).


View larger version (8K):
[in this window]
[in a new window]
 
Fig. 2.   Muscle interleukin-6 (IL-6) and tumor necrosis factor (TNF)-alpha mRNA before (Pre), during (30 and 90 min), and after (180 min) 3 h of two-legged knee-extensor exercise. Values are means ± SE (n = 6). * Significant difference (P < 0.05) from Pre.

Arterial plasma TNF-alpha 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-alpha ; therefore, there was no net release of TNF-alpha 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).


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 3.   Plasma cytokine concentration (A), leg femoral venous-arterial (fv-a) cytokine difference (B), and net leg cytokine balance values (C) before (Pre), during (60 and 120 min), and after (180 min) 3 h of two-legged knee-extensor exercise. Values are means ± SE (n = 6). * Significant difference (P < 0.05) from Pre.

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).


View larger version (6K):
[in this window]
[in a new window]
 
Fig. 4.   Leg blood flow before (Pre), during (60 and 120 min), and after (180 min) 3 h of two-legged knee-extensor exercise. Values are means ± SE (n = 6). * Significant difference (P < 0.05) from Pre.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha . Therefore, our data demonstrate a clear disassociation between IL-6 and TNF-alpha production in skeletal muscle during exercise and are consistent with the hypothesis that TNF-alpha , but not IL-6, impairs glucose uptake by skeletal muscle.

Although TNF-alpha has been previously measured in human skeletal muscle (22), this is the first study to measure the effect of acute contraction on TNF-alpha in human muscle. These data support those of Greiwe et al. (10), who found that TNF-alpha 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-alpha mRNA was not significantly increased compared with rest. It could be argued that TNF-alpha mRNA and protein release did not increase because the glycogen levels were not reduced to very low levels and that TNF-alpha may be sensitive only to very low glycogen stores. Of note, however, there was an approximately fourfold increase (P = 0.08) in TNF-alpha mRNA after 30 min of exercise, after which time it did not increase any further. If TNF-alpha 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-alpha 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-alpha 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-alpha gene expression. However, it would appear from our cell culture experiments that any increase would be transient. In contrast to the pattern of TNF-alpha 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-alpha production. During contraction, muscle glucose uptake is markedly increased compared with rest; therefore, our hypothesis is consistent with the observation that TNF-alpha impairs glucose disposal in skeletal muscle (42). Indeed, there are data demonstrating that IL-6 can impair increases in TNF-alpha . Tanaka et al. (35) recently demonstrated that, during viral myocarditis, the serum TNF-alpha 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-alpha mRNA within skeletal muscle, we also observed that IL-6, but not TNF-alpha , 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-alpha protein was not expressed in contracting skeletal muscle. However, given the fact that TNF-alpha mRNA did not significantly increase within the muscle, this possibility appears remote. It is also important to note that arterial TNF-alpha concentration did not increase as a result of exercise, and therefore we cannot rule out the possibility that, in the studies in which TNF-alpha increased in the blood, the muscle was indeed the source of this increase. On careful examination of the literature, however, elevations in plasma TNF-alpha 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-alpha 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-alpha levels are not increased (38). These data lend support to the suggestion that TNF-alpha 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-alpha , these present data might also suggest that another role for muscle-derived IL-6 is to impair TNF-alpha 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-alpha . IL-6 inhibits LPS-induced TNF-alpha 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-alpha 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-alpha 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-alpha receptors, but not IL-1beta and TNF-alpha (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-alpha 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-alpha 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-alpha . Hence, our data are consistent with the hypothesis that TNF-alpha , but not IL-6, impairs glucose uptake by skeletal muscle.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abbas, AK, Lichtman AH, and Pober JS. Cellular and Molecular Immunology. Philadelphia, PA: Saunders, 1997.

2.   Andersen, P, and Saltin B. Maximal perfusion of skeletal muscle in man. J Physiol 366: 233-249, 1985[Abstract].

3.   Bagby, GJ, Crouch LD, and Shepherd RE. Exercise and cytokines: spontaneous and elicited response. In: Exercise and Immune Function. New York: CRC, 1996, p. 55-78.

4.   Birkenkamp, KU, Esselink Mt, Kruijer W, and Wellenga E. An inhibitor of PI3-K differentially affects proliferation and IL-6 protein secretion in normal and leukemic myeloid cells depending on the stage of differentiation. Exp Hematol 28: 1239-1249, 2000[ISI][Medline].

5.   Blomstrand, E, Radegran G, and Saltin B. Maximum rate of oxygen uptake by human skeletal muscle in relation to maximal activities of enzymes in the Krebs cycle. J Physiol 501: 455-460, 1997[Abstract].

6.   Camus, G, Poortmans J, Nys M, Deby-Dupont G, Duchateau J, Deby C, and Lamy M. Mild endotoxaemia and the inflammatory response induced by a marathon race. Clin Sci (Colch) 92: 415-422, 1997[ISI][Medline].

7.   Chomczynski, P, and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156-159, 1987[ISI][Medline].

8.   Craig, R, Larkin A, Mingo AM, Thuerauf DJ, Andrews C, McDonough PM, and Glembotski CC. p38 MAPK and NF-kappa B collaborate to induce interleukin-6 gene expression and release. Evidence for a cytoprotective autocrine signaling pathway in a cardiac myocyte model system. J Biol Chem 275: 23814-23824, 2000[Abstract/Free Full Text].

9.   Del Aguila, LF, Claffey KP, and Kirwan JP. TNF-alpha impairs insulin signaling and insulin stimulation of glucose uptake in C2C12 muscle cells. Am J Physiol Endocrinol Metab 276: E849-E855, 1999[Abstract/Free Full Text].

10.   Greiwe, JS, Cheng B, Rubin DC, Yarasheski KE, and Semenkovich CF. Resistance exercise decreases skeletal muscle tumor necrosis factor alpha  in frail elderly humans. FASEB J 15: 475-482, 2001[Abstract/Free Full Text].

11.   Halse, R, Pearson SL, McCormack JG, Yeaman SJ, and Taylor R. Effects of tumor necrosis factor-alpha on insulin action in cultured human muscle cells. Diabetes 50: 1102-1109, 2001[Abstract/Free Full Text].

12.   Jonsdottir, IH, Schjerling P, Ostrowski K, Asp S, Richter EA, and Pedersen BK. Muscle contractions induce interleukin-6 mRNA production in rat skeletal muscles. J Physiol 528: 157-163, 2000[Abstract/Free Full Text].

13.  Keller C, Hellsten Y, Pilegaard H, Febbraio M, and Pedersen BK. Human muscle cells express IL-6 via a Ca2+ dependent pathway (Abstract). J Physiol 539P, 2002.

14.   Keller, C, Steensberg A, Pilegaard H, Osada T, Saltin B, Pedersen BK, and Neufer PD. Transcriptional activation of the IL-6 gene in human contracting skeletal muscle: influence of muscle glycogen content. FASEB J 15: 2748-2750, 2001[Abstract/Free Full Text].

15.   Kern, PA, Ranganathan S, Li C, Wood L, and Ranganathan G. Adipose tissue tumor necrosis factor and interleukin-6 expression in human obesity and insulin resistance. Am J Physiol Endocrinol Metab 280: E745-E751, 2001[Abstract/Free Full Text].

16.   Kirwan, JP, Del Aguila LF, Hernandez JM, Williamson DL, O'Gorman DJ, Lewis R, and Kirshnan RK. Regular exercise enhances insulin activation of IRS-1-associated PI3-kinase in human skeletal muscle. J Appl Physiol 88: 797-803, 2000[Abstract/Free Full Text].

17.   Matthys, P, Mitera T, Heremans H, Van Damme J, and Billiau A. Anti-gamma interferon and anti-interleukin-6 antibodies affect staphylococcal enterotoxin B-induced weight loss, hypoglycemia, and cytokine release in D-galactosamine-sensitized and unsensitized mice. Infect Immun 63: 1158-1164, 1995[Abstract].

18.   Mizuhara, H, O'Neill E, Seki N, Ogawa T, Kusunoki C, Otsuka K, Satoh S, Niwa M, Senoh H, and Fujiwara H. T cell activation-associated hepatic injury: mediation by tumor necrosis factors and protection by interleukin 6. J Exp Med 179: 1529-1537, 1994[Abstract].

19.   Nieman, DC, Nehlsen-Cannarella SL, Fagoaga OR, Henson DA, Utter A, Davis JM, Williams F, and Butterworth DE. Influence of mode and carbohydrate on the cytokine response to heavy exertion. Med Sci Sports Exerc 30: 671-678, 1998[ISI][Medline].

20.   Ostrowski, K, Rohde T, Asp S, Schjerling P, and Pedersen BK. Pro- and anti-inflammatory cytokine balance in strenuous exercise in humans. J Physiol 515: 287-291, 1999[Abstract/Free Full Text].

21.   Ostrowski, K, Rohde T, Zacho M, Asp S, and Pedersen BK. Evidence that interleukin-6 is produced in human skeletal muscle during prolonged running. J Physiol 508: 949-953, 1998[Abstract/Free Full Text].

22.   Pedersen, BK, and Hoffman-Goetz L. Exercise and the immune system: regulation, integration, and adaptation. Physiol Rev 80: 1055-1081, 2000[Abstract/Free Full Text].

23.   Pedersen, BK, Steensberg A, and Schjerling P. Muscle-derived interleukin-6: possible biological effects. J Physiol 536: 329-337, 2001[Abstract/Free Full Text].

24.   Pilegaard, H, Ordway GA, Saltin B, and Neufer PD. Transcriptional regulation of gene expression in human skeletal muscle during recovery from exercise. Am J Physiol Endocrinol Metab 279: E806-E814, 2000[Abstract/Free Full Text].

25.   Pue, CA, Mortensen RF, Marsh CB, Pope HA, and Wewers MD. Acute phase levels of C-reactive protein enhance IL-1 beta and IL-1ra production by human blood monocytes but inhibit IL-1 beta and IL-1ra production by alveolar macrophages. J Immunol 156: 1594-1600, 1996[Abstract].

26.   Radegran, G. Ultrasound Doppler estimates of femoral artery blood flow during dynamic knee extensor exercise in humans. J Appl Physiol 83: 1383-1388, 1997[Abstract/Free Full Text].

27.   Richter, EA, Derave W, and Wojtaszewski JFP Glucose, exercise and insulin: emerging concepts. J Physiol 535: 313-322, 2001[Abstract/Free Full Text].

28.   Ryder, JW, Chibalin AV, and Zierath JR. Intracellular mechanisms underlying increases in glucose uptake in response to insulin or exercise in skeletal muscle. Acta Physiol Scand 171: 249-257, 2001[ISI][Medline].

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

30.   Schindler, R, Mancilla J, Endres S, Ghorbani R, Clark SC, and Dinarello CA. Correlations and interactions in the production of interleukin-6 (IL- 6), IL-1, and tumor necrosis factor (TNF) in human blood mononuclear cells: IL-6 suppresses IL-1 and TNF. Blood 75: 40-47, 1990[Abstract].

31.   Starkie, RL, Arkinstall MJ, Koukoulas I, Hawley JA, and Febbraio MA. Carbohydrate ingestion attenuates the increase in plasma interleukin-6, but not skeletal muscle interleukin-6 mRNA, during exercise in humans. J Physiol 533: 585-591, 2001[Abstract/Free Full Text].

32.   Starkie, RL, Rolland J, Angus DJ, Anderson MJ, and Febbraio MA. Circulating monocytes are not the source of elevations in plasma IL-6 and TNF-alpha levels after prolonged running. Am J Physiol Cell Physiol 280: C769-C774, 2001[Abstract/Free Full Text].

33.   Steensberg, A, Febbraio MA, Osada T, Schjerling P, van Hall G, Saltin B, and Pedersen BK. Interleukin-6 production in contracting human skeletal muscle is influenced by pre-exercise muscle glycogen content. J Physiol 537: 633-639, 2001[Abstract/Free Full Text].

34.   Steensberg, A, van Hall G, Osada T, Sacchetti M, Saltin B, and Klarlund PB. Production of interleukin-6 in contracting human skeletal muscles can account for the exercise-induced increase in plasma interleukin-6. J Physiol 529: 237-242, 2000[Abstract/Free Full Text].

35.   Tanaka, T, Kanda T, McManus BM, Kanai H, Akiyama H, Sekiguchi K, Yokoyama T, and Kurabayashi M. Overexpression of interleukin-6 aggravates viral myocarditis: impaired increase in tumor necrosis factor-alpha. J Mol Cell Cardiol 33: 1627-1635, 2001[ISI][Medline].

36.   Tilg, H, Dinarello CA, and Mier JW. IL-6 and APPs: anti-inflammatory and immunosuppressive mediators. Immunol Today 18: 428-432, 1997[ISI][Medline].

37.   Toft, AD, Jensen LB, Bruunsgaard H, Ibfelt T, Halkjaer-Kristensen J, Febbraio MA, and Pedersen BK. Cytokine response to eccentric exercise in young and elderly humans. Am J Physiol Cell Physiol 283: C289-C295, 2002[Abstract/Free Full Text].

38.   Toft, AD, Thorn M, Ostrowski K, Asp S, Moller K, Iversen S, Hermann C, Sondergaard SR, and Pedersen BK. N-3 polyunsaturated fatty acids do not affect cytokine response to strenuous exercise. J Appl Physiol 89: 2401-2406, 2000[Abstract/Free Full Text].

39.   Uysal, KT, Wiesbrock SM, Marino MW, and Hotamisligil GS. Protection from obesity-induced insulin resistance in mice lacking TNF-alpha function. Nature 389: 610-614, 1997[ISI][Medline].

40.   Wallenius, V, Wallenius K, Ahren B, Rudling M, Carlsten H, Dickson SL, Ohlsson C, and Jansson JO. Interleukin-6-deficient mice develop mature-onset obesity. Nature Med 8: 75-79, 2002[ISI][Medline].

41.   Yamauchi-Takihara, K, and Kishimoto T. Cytokines and their receptors in cardiovascular diseases---role of gp130 signalling pathway in cardiac myocyte growth and maintenance. J Exp Pathol 81: 1-16, 2000.

42.   Youd, JM, Rattigan S, and Clark MG. Acute impairment of insulin-mediated capillary recruitment and glucose uptake in rat skeletal muscle in vivo by TNF-alpha. Diabetes 49: 1904-1909, 2000[Abstract].


Am J Physiol Endocrinol Metab 283(6):E1272-E1278
0193-1849/02 $5.00 Copyright © 2002 the American Physiological Society