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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 18, 2005 as doi:10.1096/fj.04-3278fje. |
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* Centre of Inflammation and Metabolism, Department of Infectious Diseases and The Copenhagen Muscle Research Centre, University Hospital of Copenhagen, Copenhagen, Denmark;
Department of Medical Anatomy, The Panum Institute, University of Copenhagen, Copenhagen, Denmark; and
Animal Physiology Unit, Faculty of Sciences, Autonomous University of Barcelona, Barcelona, Spain
1 Correspondence: Department of Infectious Diseases and the Copenhagen Muscle Research Centre, University Hospital Rigshospitalet, Tagensvej 20, Copenhagen 2200, Denmark. E-mail: pernillekeller{at}yahoo.com
SPECIFIC AIMS
Contracting muscle fibers produce and release IL-6. We recently showed that infusion of IL-6 to humans increases IL-6 mRNA levels in human skeletal muscle suggesting an autocrine regulation in vivo. We hypothesized that exercise or IL-6 alone could induce IL-6 receptor production in skeletal muscle. We therefore investigated IL-6 receptor regulation in skeletal muscle biopsies: in humans in response to bicycle exercise and rhIL-6 infusion, and in IL-6 KO mice in response to exercise.
PRINCIPAL FINDINGS
1. Human skeletal muscle expresses the IL-6 receptor
Expression of the IL-6 receptor was detected at both the mRNA and protein level in human skeletal muscle (Fig. 1
and Fig. 2
, respectively).
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2. Both IL-6 receptor mRNA levels and protein staining increase after exercise
In human skeletal muscle, IL-6 receptor mRNA increased (P<0.01) in response to 3 h bicycle exercise when compared with resting subjects. IL-6 receptor mRNA levels were elevated at 4.5 h and peaked at 9 h, still being slightly elevated at 24 h. All subjects showed the same pattern of IL-6 receptor mRNA response to exercise (Fig. 1)
. IL-6 receptor protein in human skeletal muscle fibers showed an increased staining of the cellular membrane of muscle fibers after exercise. The staining of IL-6 receptor protein was most prominent at 9 h, thus the kinetics of the IL-6 receptor protein staining followed the same pattern as the IL-6 receptor mRNA levels (Fig. 2)
. The ubiquitously expressed gp130 was neither regulated by exercise nor by IL-6.
3. IL-6 increases protein levels of the IL-6 receptor in skeletal muscle whereas mRNA levels are unaffected
After rhIL-6 infusion in humans, IL-6 receptor mRNA levels did not differ from levels in the control group. However, expression of IL-6 receptor protein increased in the rhIL-6 infused group when compared with controls. The staining of IL-6 receptor protein was localized to the cell membrane and displayed the strongest staining at the end of the 3 h infusion and at 6 h.
In both IL-6 knockout mice and wild-type mice, IL-6 receptor mRNA levels increased in skeletal muscle 3 h after one hour of swimming exercise (P<0.01). No differences in expression pattern were observed between wild-type and IL-6 KO mice.
CONCLUSIONS AND SIGNIFICANCE
IL-6 is produced and released by working skeletal muscle fibers. IL-6 transcription during exercise is further enhanced if glycogen levels within muscle are low, and ingestion of carbohydrate during exercise decreases the plasma IL-6 response, suggesting that IL-6 is regulated by energy availability. A role for IL-6 in metabolism is suggested as infusion of rhIL-6 to humans increase lipolysis and fat oxidation and as IL-6 deficient mice develop late-onset obesity and become glucose intolerant. Thus, IL-6 seems to serve as an energy sensor.
We have previously shown that IL-6 infusion to humans increases IL-6 mRNA in skeletal muscle, suggesting an autocrine regulation. In this study we show that the IL-6 receptor is produced in skeletal muscle and that IL-6 receptor mRNA and protein levels are responsive to exercise. Increased expression of the IL-6 receptor in muscle fibers after an exercise bout suggests that the muscle is sensitized by IL-6. The peak in IL-6 receptor production occurs several hours after the end of the exercise-bout, at the time where IL-6 plasma levels are decreasing, expression of the IL-6 receptor may therefore be a mechanism whereby muscle is sensitized to the exercise-induced effects of IL-6 when IL-6 levels are sparse. As several metabolic genes are increased in the recovery period from exercise, and with IL-6 as a possible energy sensor, this suggests that the increase in the IL-6 receptor may be involved in energy restoration within skeletal muscle.
In human hepatoma cells, the IL-6/IL-6 receptor complex is internalized and degraded. For sustained signaling through the IL-6 receptor to occur, de novo synthesis of the IL-6 receptor is required. Thus, the continuously elevated levels of IL-6 receptor mRNA and protein after a single exercise bout may affect the IL-6/IL-6 receptor system for more than 6 h after the end of exercise. The IL-6 receptor can be proteolytically cleaved, generating the soluble IL-6 receptor, thus any cell type that expresses the gp130 receptor can bind IL-6 in complex with its soluble receptor for initiation of signal transduction. As the plasma levels of the sIL-6 receptor were unaltered in response to exercise, this again suggests a local muscle-specific response to IL-6.
It seems that gp130 availability may limit IL-6 receptor signaling: however, as the gp130 is ubiquitously expressed and also serves as a receptor for many other cytokines, there is a surplus of the gp130 receptor. Thus, it is highly unlikely that it will limit IL-6 receptor signaling.
Despite IL-6 plasma levels being higher in the infusion trial compared with the exercise trial, IL-6 receptor mRNA levels were unaltered, suggesting that IL-6 is not the primary signal for the increase in IL-6 receptor mRNA observed in response to exercise. Thus, the exercise-induced increase in IL-6 receptor production most likely occurs via an IL-6 independent mechanism. This was further substantiated by the ability of the IL-6 KO mice to increase the IL-6 receptor mRNA levels to the same extent as control mice with exercise. Conversely, IL-6 increases both IL-6 receptor mRNA and protein levels in a human myeloma cell line. This discrepancy may be explained by variations in the IL-6 receptor response between cell types and dose of IL-6, and the latter data may not reflect the situation observed in skeletal muscle. However, rhIL-6 infusion in humans increased IL-6 receptor protein expression in skeletal muscle, possibly owing to post-transcriptional regulation of the IL-6 receptor in response to elevated IL-6 plasma levels. Although other receptor systems show decreased half-life of the receptors when exposed to saturating amounts of their ligands, our results showed increased IL-6 receptor protein levels after rhIL-6 infusion. This is consistent with results from canine kidney cells where IL-6 receptor half-life is independent of the presence of saturating amounts of IL-6.
We previously showed that IL-6 is produced in both type I and type II skeletal myofibers when subjects perform 3 h of intense ergometer bicycle exercise whereas another study reported specificity to fiber type II in response to 2 h of ergometer exercise. The studies indicate that the mode and intensity of exercise determines to which degree either fiber type expresses IL-6. In the present study, we studied 3 h of ergometer bicycle exercise and saw uniform expression of the IL-6 receptor in both type I and type II fibers, suggesting that both fiber types are rendered responsive to IL-6.
The kinetics of IL-6 and IL-6 receptor expression suggest that the pathways regulating IL-6 and the IL-6 receptor are linked, thus factors initiating IL-6 transcription may be inhibiting IL-6 receptor transcription. Activation of IL-6 transcription is regulated by energy availability, as low intra muscular glycogen levels further enhance transcription of the IL-6 gene. In response to exercise, IL-6 may act as a metabolic link, when energy levels within muscle become low, as rhIL-6 can increase lipolysis. Several metabolic genes are transcriptionally activated in the recovery phase from exercise, presumably to rebuild energy stores. It would seem reasonable if activation of transcription of the IL-6 receptor was also linked with factors involved in energy sensing, as energy stores are being rebuilt in the recovery phase from exercise, when IL-6 receptor levels peak. This would render the muscle fibers more responsive to IL-6 possibly initiating glycogen synthesis.
In conclusion, muscle contractions induce post-exercise expression of IL-6 receptor mRNA and protein levels in human skeletal muscle, possibly to sensitize muscle to the decreasing IL-6 plasma levels elicited by exercise. As exercise increases IL-6 receptor mRNA in both IL-6 KO mice and wild-type mice, exercise-induced transcription of IL-6 receptor in muscle fiber most likely occurs via an IL-6 independent mechanism. This is further supported by the human rhIL-6 infusion study. However, IL-6 may enhance IL-6 receptor production at a post-transcriptional level, as infusion of rhIL-6 increased IL-6 receptor protein expression.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3278fje; doi: 10.1096/fj.04-3278fje
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