Fibre-type specificity of interleukin-6 gene transcription during muscle contraction in rat: association with calcineurin activity
1 Department of Human Factors, Centre de Recherches du Service de Sante des Armees, La Tronche, France
Abstract
In this study, we quantified the transcription of the interleukin-6 (IL-6) gene in individual fibres and the associated changes in calcineurin activity assessed at the cellular level during prolonged muscle contraction. Individual myofibres were isolated from plantaris and soleus muscles of rats at the end of an exhaustive running exercise test (n = 10), categorized according to their myosin heavy chain isoform content, and compared to those of resting rats (n = 10). Using real-time PCR analysis in individual fibres, a marked rise in IL-6 transcript levels occurred in type I and IIa fibres at the end of exercise (P < 0.05). Transcription of the gene encoding for the modulatory calcineurin-interacting protein-1 (MCIP-1), a sensitive indicator of calcineurin activity, also mainly increased in type I and IIa fibres (P < 0.05). Moreover, a slight increase in MCIP-1 mRNA levels was observed in type IIx (P < 0.05). Fibre types determined by immunohistochemistry were qualitatively examined for glycogen content using periodic acid–Shiff staining, and no direct relationship was found, at the cellular level, between glycogen content, fibre-type and IL-6 transcription. Our data clearly suggest that IL-6 gene transcription was mainly observed in early recruited myofibres and that contraction-induced IL-6 transcription could be associated with enhanced calcineurin activity.
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Introduction
It has been recently shown that interleukin-6 (IL-6) plasma levels increase dramatically during prolonged concentric exercise in man (for review see Febbraio & Pedersen, 2002). Increased IL-6 mRNA levels were reported in human muscle biopsies at the end of exercise, related to mechanisms other than muscle damage (Ostrowski et al. 1998). In a one-legged exercise test, high muscle IL-6 net release occurred only in the contracting limb (Steensberg et al. 2000). Together, these results strongly suggest that muscle is the main source of plasma IL-6 during exercise and that this production is directly associated with muscle contraction and does not result from an exercise-related systemic effect. Subjects exercising with low intramuscular glycogen levels showed a higher plasma IL-6 peak (Keller et al. 2001), independent of systemic influences (Steensberg et al. 2001). It has thus been hypothesized that muscle-derived IL-6 is linked to energy availability and could play an important role in carbohydrate homeostasis during exercise by contributing to contraction-mediated glucose uptake and by acting as an endocrine signal of muscle energy stores to favour hepatic glucose production and white adipose tissue lipolysis (for review see Febbraio & Pedersen, 2002).
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However, skeletal muscle contains several cell types that are known to be able to produce IL-6. Blood mononuclear cells do not account for the exercise-induced increase in IL-6 plasma levels (Ullum et al. 1994; Starkie et al. 2000; Moldoveanu et al. 2000). Human myoblasts (Bartoccioni et al. 1994), smooth muscle cells (Detmer et al. 2001) and endothelial cells (Sterpetti et al. 1993) can produce IL-6 when exposed to several stimuli such as inflammatory cytokines, endotoxins or mechanical stress. The cellular origin of IL-6 production in muscle has been examined in two recent studies. The immunohistochemical detection of IL-6 protein in skeletal muscle showed an increase in positive myofibres at the end of exercise, suggesting that myofibres per se could be a source of IL-6 production during contraction (Penkowa et al. 2003). Moreover, using in situ hybridization in human muscle, it has recently been shown that myofibres contain IL-6 mRNA at the end of prolonged exercise (Hiscock et al. 2004). These findings clearly show that muscle fibres are a source of IL-6, and because myofibres consume and need energy during muscle contraction, they reinforce the hypothesis of an energy-sensing function of IL-6.
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Adult rat skeletal muscles comprise at least four fibre types ranging from slow-twitch predominantly oxidative fibres (type I) to fast-twitch predominantly oxidative, intermediate oxidative and low oxidative fibres (types IIa, IIx and IIb, respectively). Muscle fibres are distributed among motor units and it is well accepted that during muscle contraction, motor units are recruited in an orderly manner. According to the ‘size principle’ of Henneman & Olson (1965), the smallest motor units comprising type I fibres are first recruited, while the largest, comprising type IIx and type IIb fibres, are recruited long after the beginning of muscle contraction, when local fatigue occurs in slow and oxidative motor units (Fallentin et al. 1993). Because type I and type IIa fibres have small glycogen stores, whereas type IIx and IIb fibre have large glycogen stores, and IL-6 may work as a sensor of carbohydrate availability (Febbraio & Pedersen, 2002), a fibre-type specificity of IL-6 gene expression could be expected at the end of prolonged exercise. This issue has been recently addressed and controversial findings were reported. No difference was detected between muscle fibre types at the protein level at the end of exercise (Penkowa et al. 2003), whereas Fischer et al. (2004) described preferential staining in type I fibres. More recent data suggest that type II fibres predominantly produce IL-6 during exercise in humans (Hiscock et al. 2004). Moreover, the results of this latter study suggested that a reduced level of glycogen was not directly involved in IL-6 gene expression during muscle contractile activity. Whether the expression of the IL-6 gene varies between fibre types during exercise therefore remains to be elucidated.
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Because the nuclear transcriptional rate of the IL-6 gene is remarkably rapid after the onset of exercise, it has been hypothesized that rapid changes in cytosolic Ca2+ concentration could be involved in IL-6 gene expression (Febbraio & Pedersen, 2002). Changes in intracellular free calcium concentration ([Ca2+]i) is a key event during myocyte contraction, which has recently been shown to induce a rapid increase in IL-6 mRNA and protein expression in rat isolated muscles (Holmes et al. 2004). Prolonged contractile activity is characterized by low-amplitude and sustained elevations in [Ca2+]i, leading to increased activity of calcineurin, a ubiquitous Ca2+–calmodulin-dependent protein phosphatase known to be a key mediator of Ca2+ signalling in muscle cells. Several downstream effectors of calcineurin have been identified in skeletal muscle, including the nuclear factor of the activated T cell (NFAT), and the calcineurin–NFAT pathway is involved in the acquisition and maintenance of the slow oxidative phenotype (for review see Bassel-Duby & Olson, 2003). Whether this transcription factor is involved in IL-6 gene transcription has recently been examined (Chan et al. 2004). No evidence was provided that IL-6 gene transcription is activated by NFAT, whereas the phosphorylation of nuclear p38 mitogen-activated protein kinase (MAPK) was considered as an activator for candidate transcription factors that may bind to the IL-6 promoter region in myofibres. However, whether the increased cytosolic Ca2+ accumulation expected within single specific fibres during prolonged muscle contraction is associated with activated calcineurin and enhanced IL-6 gene expression has not been examined to date.
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The aims of this study were to examine the fibre-type specificity of IL-6 gene transcription in individual myofibres at the end of prolonged exercise and the involvement of calcineurin activity in the control of IL-6 gene expression. In light of the recruitment pattern of muscle fibres during prolonged submaximal exercise and the differences in pre-exercise glycogen content, we hypothesized that (1) IL-6 production could be fibre-type specific and that IL-6 mRNA levels could be higher in type I and type IIa fibres than in type IIx and type IIb at the end of exercise, and (2) high IL-6 mRNA levels could be observed in fibres showing high calcineurin activity. To address these issues, we first quantified the level of IL-6 gene transcription within individual fibres taken from two skeletal muscles at the end of exercise in rat. Second, we examined whether IL-6 gene expression was associated with high levels of calcineurin activity. As previously suggested, transcription of the gene encoding for the modulatory calcineurin-interacting protein-1 (MCIP-1) was used as a sensitive indicator of calcineurin activity (Yang et al. 2000).
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Methods
Animals
Three-month-old male Wistar rats (n = 30) were purchased from Charles River (L'Arbresle, France). They were housed three per cage in a thermoneutral environment (22 ± 2°C), on a 12–12 h light–dark period, and were provided with food and water ad libitum. They were randomly assigned to three experimental groups: (1) control rats (n = 10); (2) active rats (n = 10); and (3) recovering rats (n = 10). Experiments received prior approval from the animal ethics committee of the Centre de Recherche du Service Sante des Armees (La Tronche, France).
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Exercise protocol
All animals were accustomed to running on a rodent treadmill for 15 min per day for 5 days at a moderate level (10–20 m min–1; 0 deg gradient). After 3 days at rest, active and recovering animals ran on the treadmill (25 m min–1, 8 deg gradient) until exhaustion, defined as the moment where animals were unable to keep in pace with the treadmill.
Tissue processing
Animals were removed and anaesthetized either immediately (active group), or 2 h after the end of exercise (recovering group), with intraperitoneal injection of pentobarbital (70 mg (kg body weight)–1). Plantaris and soleus muscles of both hindlimbs were excised and cleaned of adipose and connective tissue. Plantaris muscles were also separated in superficial and deep portions. While the left muscles were immediately frozen in liquid nitrogen and stored at –80°C, the right muscles were placed in 400 μl RNAlater solution (Ambion, Austin, TX, USA), kept at 4°C for 24 h and then frozen at –20°C. Animals were killed by removal of the heart.
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Single-fibre isolation
The single-fibre study was adapted from the methods of Birot et al. (2003). Muscle conserved in RNAlater was thawed on ice and placed in a small dish containing RNAlater. A small bundle of muscle fibres was first isolated under the light microscope using sharp-ended tweezers. Single muscle fibres were then separated and cut into two equal parts. One half was placed in 40 μl Nanoprep lysis buffer supplemented with 0.7% 2-mercaptoethanol (Stratagene, La Jolla, CA, USA), kept for 2 h at 4°C and then frozen at –80°C before RNA extraction. The other half-fibre was placed in 20 μl myosin extraction solution containing (mM): NaCl 300, NaH2PO4 100, Na2HPO4 50, Na4P2O7 10, MgCl2.6H2O 1, EDTA 10 and 2-mercaptoethanol 1.4; pH 6.5, for further myosin heavy chain (MHC) content analysis.
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MHC isoform analysis
Single fibres were subjected to MHC isoform analysis using SDS-PAGE as previously described (Birot et al. 2003). After incubation for 24 h at 4°C, the half-fibre was digested and the 20 ml mixture was then diluted with 20 ml glycerol. Extracts were stored at –20°C until required for the separation process. Electrophoresis was performed using a Mini Protean II system (Biorad, Marnes-la-Coquette, France). The separating gel solution contained 30% glycerol, 8% acrylamide–bis (50: 1), 0.2 M Tris, 0.1 M glycine and 0.4% SDS. The stacking gel was composed of 30% glycerol, 4% acrylamide–bis (50: 1), 70 mM Tris, 4 mM EDTA and 0.4% SDS. Then 10 ml myofibril samples were denatured using 10 ml buffer containing 5% 2-mercaptoethanol, 100 mM Tris base, 5% glycerol, 4% SDS and bromophenol blue, for 3 min at 100°C. Myofibrillar homogenates were loaded onto vertical gels, whilst two lanes were loaded with protein extract from a control plantaris muscle known to contain the four adult MHC isoforms. Gels were run at constant voltage (72 V) for 31 h and then silver-stained (Agbulut et al. 1996). The MHC protein isoform bands were scanned using a densitometer system equipped with an integrator (GS-700, Biorad, Marnes-la-Coquette, France). The MHC isoforms expressed by single fibres were identified by comparing them with bands of myosin extracts from control plantaris muscle.
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RNA extraction
Muscle tissue mRNA was isolated using the MagNA Pure LC instrument (Roche Applied Science, Mannheim, Germany). We disrupted 10 mg frozen soleus and deep plantaris muscles using two tungsten carbide beads in 100 μl MagNA Pure LC mRNA isolation kit II (Roche Applied Science) lysis buffer with a Mixer Mill MM300 (Rescht, Haan, Germany) for 40 s (30 Hz). Lysate was centrifuged (12 000 g, 5 min, room temperature), the liquid phase was transferred in a fresh tube and lysis buffer added to a final volume of 300 μl. Extraction was then performed following the manufacturer's protocol with an 880 μl sample volume, a 50 μl elution volume and no dilution.
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The total RNA of the fibres was isolated using RNA Insta-pure reagent (Eurogentec, Saraing, Belgium) with a modified protocol. Samples were extracted using 200 μl RNA Insta-pure reagent and 20 μl chloroform (Sigma-Aldrich, Steinheim, Germany). An additional iso-volume chloroform extraction was performed. RNA isopropanol precipitation was carried out using 10 μg glycogen (Sigma-Aldrich) as carrier. The ethanol washing step was performed twice and the RNA pellet was dried in a vacuum for 3 min. RNA was then resuspended in 10 μl RNase-free water.
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Reverse transcription
Muscle tissue mRNA reverse transcription was carried out using the Reverse Transcriptase Core Kit (Eurogentec, Saraing, Belgium). The reaction was performed in a 10 μl final volume following the manufacturer's instructions. The reaction mix contained 1 μl buffer (10 x), 2 μl dNTP mix (2.5 mM each deoxyribronucleotide triphosphate (dNTP)), 0.5 μl oligo-dT primer (50 μM), 0.2 μl RNase inhibitor (20 units μl–1), 2 μl MgCl2 (25 mM), 0.25 μl Euroscript reverse transcriptase (50 U μl–1), 1.05 μl RNase-free water and 3 μl template mRNA.
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Fibre RNA reverse transcription was carried out using Sensiscript Reverse Transcriptase (Qiagen S.A, Courtaboeuf, France), specially designed for highly sensitive reverse transcription with small amounts of RNA. The reaction was performed in a 20 μl final volume following the manufacturer's instructions. The mix contained 2 μl buffer (10 x), 2 μl dNTP mix (5 mM each dNTP), 2 μl oligo-dT primer (10 μM), 1 μl RNase inhibitor (10 U μl–1), 1 μl Sensiscript reverse transcriptase, 4 μl RNase-free water and 8 μl template RNA.
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PCR primer design
All primers used in this study were designed with the MacVector software (Accelrys, Orsay, France) and synthesized at Eurogentec (Saraing, Belgium). Primer sequences and resulting amplified product characteristics are shown in Table 1.
Real-time PCR
The PCR reactions were carried out in a 20 μl final volume with the LC Fast Start DNA Master SYBR Green kit (Roche Applied Science) using 0.2 μl (whole muscle) or 0.5 μl (single-fibre) cDNA solution. Quantitative PCRs were performed using LightCycler (Roche Applied Science) for 45 amplification cycles using a 5 s annealing step and an 8 s 72°C elongation step (detailed conditions available in Table 1). Specificity was checked for each sample by melting-curve analysis. Transcription levels were normalized using an internal control gene with the comparative threshold cycle method (Livak & Schittgen, 2001) using RelQuant software (Roche Applied Science). A pool of 40 samples randomly chosen in the groups was used as a calibrator as previously described (Peinnequin et al. 2004). Cyclophyllin A (CycA) was used as reference gene because its expression is known to be unaffected at the end of an endurance exercise test in skeletal muscle (Mahoney et al. 2004). Furthermore, this was controlled in our experiment on whole muscle tissue.
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Purity of single fibres
The single-fibre approach we used, first described by Birot et al. (2003), makes it possible to remove fibroblasts, smooth muscle cells and blood mononuclear cells because conjunctive tissue and vessels are clearly visible under the microscope. In contrast, endothelial cells and infiltrating mononuclear cells cannot be seen, can adhere to the myofibres and are capable of producing great amounts of IL-6. Before including fibres in our study, we needed to check the purity of all half-fibres and confirm the absence of endothelial cells or monocytic cells. Every half-fibre of the active group was tested by PCR amplification for platelet endothelial cell adhesion molecule (PECAM) and cluster of differentiation 11b (CD11b). When there was a positive signal for PECAM and/or CD11b, the sample was excluded from the study.
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Immunohistochemistry and periodic acid–Schiff staining
Serial transverse sections (14 μm thick) were cut from the mid-belly portion of soleus and plantaris muscles in a cryostat maintained at –20°C. Plantaris sections were labelled with mouse monoclonal antibodies against myosin reacting with (1) slow type I (Novocastra, reference NCL-MHCS, Newcastle upon Tyne, UK), (2) all adult fast and developmentally regulated epitopes but not with slow myosin (MY-32, Sigma-Aldrich), or (3) fast type IIa (SC-71). Both plantaris and soleus sections were stained for glycogen content using the periodic acid–Shiff (PAS) staining system (Sigma-Aldrich) according to the manufacturer's instructions.
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Statistics
All data are presented as means ± S.E.M. Student's unpaired t test was used to determine differences between groups. Statistical significance was accepted at P < 0.05.
Results
Running time
Both active and recovering rats performed an exhaustive running exercise. The mean duration of this exercise was 101 ± 13 min.
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As expected, there was an increase in IL-6 mRNA levels in both plantaris and soleus muscles at the end of running exercise (P < 0.05). IL-6 mRNA returned to baseline values approximately 2 h after exercise stopped. There was no significant difference in IL-6 transcript levels between plantaris and soleus muscles in control, active and recovery groups (Fig. 1). Because IL-6 mRNA levels decreased 2 h after the end of exercise, gene expression within single fibres was only considered in control and active groups.
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Data are means ± S.E.M. *Significantly different from control values, P < 0.05; significantly different from active group, P < 0.05.
Fibre-type specificity of IL-6 mRNA expression
In soleus muscles, 91% of myofibres were pure type I fibres, while 9% expressed both MHC I and MHC IIa. In deep plantaris muscles, 49% of myofibres expressed only one MHC isoform. Therefore, only 171 of the 351 successfully typed fibres were included for further study. Before IL-6 mRNA quantification, each individual fibre was tested for PECAM, CD11b and CycA mRNA expression. A negative signal for both PECAM and CD11b mRNA proved the lack of contamination by endothelial or monocytic cell material, respectively, while a positive expression of CycA gene confirmed the presence of biological material. After all these validation analyses, 37% of single, successfully typed fibres taken from soleus and plantaris muscles were used for further assays.
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At the end of exercise, IL-6 mRNA levels increased significantly in myofibres of soleus muscle, which were mainly type I fibres (P < 0.05) (Fig. 2A). In myofibres taken from plantaris muscles, there was a marked and significant increase in IL-6 mRNA in only type I and type IIa fibres at the end of prolonged exercise (P < 0.05). In contrast, acute treadmill exercise did not significantly change the IL-6 mRNA levels in type IIx and IIb fibres (P < 0.05) (Fig. 2B).
, http://www.100md.com Plantaris fibres were categorized into four types: type I, IIa, IIx and IIb, according to their MHC isoform content. Data are means ± S.E.M. *Significantly different from control values, P < 0.05; significantly different from IIa in active group values, P < 0.05.
Myofibre glycogen content at the end of exercise
PAS staining and immunohistochemistical analyses showed that in soleus muscles (Fig. 3), type I fibres were markedly glycogen depleted, whereas glycogen only slightly decreased in type IIa fibres. Likewise, all type I fibres were glycogen-depleted at the end of exercise in plantaris muscles (Fig. 3); in contrast, type IIa fibres failed to be markedly glycogen depleted. As shown in Fig. 3, there was heterogeneity of glycogen depletion within the two other fast fibre types (type IIx and IIb fibres), with some fibres markedly depleted, while no detectable changes were reported in the others.
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Glycogen content in specific fibre types in soleus (A, B, E and F) and plantaris (C, D, G and H), before (A and C) and after exhaustive running exercise (B, D and E–H). Glycogen content was assessed by PAS staining (A–D) and fibre type by immunohistochemistry using antibodies directed against type I (F and H) and type IIa MHC isoforms (E and G). In soleus muscles, pure type IIa fibres () or hybrid fibres comprising both type I and type IIa MHC isoforms () (E and F) are shown in B with a lower glycogen depletion than pure type I fibres (). In plantaris muscles, type IIa fibres (G) are shown in D with slight or no glycogen depletion (); type I fibres (H) were markedly depleted (), while fibres negative for antibodies (i.e. fibres comprising either type IIx and/or type IIb MHC isoforms) (G and H) show a heterogeneity of glycogen depletion () (D). Scale bar, 100 μm.
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Fibre-type specificity of calcineurin activity at the end of exercise
The peculiar responsiveness of MCIP-1 gene expression to calcineurin activity was used to assess the activation state of calcineurin (Yang et al. 2000). A marked increase in MCIP-1 mRNA was shown in both plantaris and soleus muscles (P < 0.05 and P < 0.01, respectively) at the end of exercise (Fig. 4A). The exercise-induced increase in MCIP-1 mRNA was found at similar levels in slow-twitch (soleus) and fast-twitch muscles (plantaris).
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For n and categorization of fibres see Figs 1 (A) and 2B (B). Data are means ± S.E.M. *Significantly different from control values (P < 0.05); **significantly different from control values (P < 0.01); significantly different from IIx in the same group (P < 0.05); significantly different from IIb in the same group (P < 0.05).
As reported for IL-6 mRNA expression, there was a significant increase in MCIP-1 mRNA levels in both type I and IIa fibres at the end of prolonged exercise (P < 0.05) (Fig. 4B). Moreover, a slight but significant increase in MCIP-1 mRNA levels was shown in type IIx fibres of exercised rats (P < 0.05).
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Discussion
Despite emerging evidence for IL-6 production by myofibres during exercise, IL-6 mRNA levels were never quantified within individual fibres. In this study, we provide for the first time quantitative measures of IL-6 mRNA levels in muscle cells. The major and novel observations made in the current investigation were: (1) a fibre-type specificity of IL-6 gene expression during exercise, with a marked increase in IL-6 transcripts in type I and type IIa fibres; and (2) an increase in calcineurin activity in individual muscle cells in parallel with the exercise-induced enhancement in IL-6 gene transcription.
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Many recent studies showed increased levels of plasma IL-6 after exercise in humans, and demonstrated that the appearance of IL-6 in the circulation results from an increased transcription of the IL-6 gene in contracting muscles (Ostrowski et al. 1998; Steensberg et al. 2001; Keller et al. 2003). IL-6 is locally produced in skeletal muscle following both concentric and eccentric contractions in rats (Jonsdottir et al. 2000), but dynamic exercise such as running had never been used to examine the molecular events that control the IL-6 gene transcription in rodents. In the present study, IL-6 mRNA levels measured in whole muscle tissue before, at the end, and 2 h after running exercise in rats closely mimic the previously reported changes in human muscle biopsies after exercise. These results show that prolonged running exercise in the rat is a valid model to study contraction-induced IL-6 gene transcription in muscles.
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As previously and consistently shown, contracting skeletal muscle is the main source of IL-6 production during exercise (for review see Febbraio & Pedersen, 2002). However, the issue of the cell type responsible for IL-6 production within skeletal muscle has been only recently addressed using histochemical methods or a qualitative approach of IL-6 gene transcription (Penkowa et al. 2003; Hiscock et al. 2004; Fischer et al. 2004). In the present study, we provide the first quantitative analysis of IL-6 gene transcription rate by RT-PCR in single myofibres. The lack of myofibre contamination by endothelial and mononuclear cells was verified by a negative signal for PECAM and CD11b, and these controls ensured that samples selected for IL-6 mRNA determination were pure single myofibres. Consistent with a previous study, our results using RT-PCR analysis definitively show that muscle fibres are a source of IL-6 in contracting muscles (Hiscock et al. 2004).
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Because myofibres are recruited in an orderly manner and IL-6 production would be increased in glycogen-depleted fibres, it has been hypothesized that IL-6 mRNA expression could be fibre-type specific. This issue has been examined, and recent reports support the role played by the mode, intensity and duration of exercise on the fibre-type specificity of IL-6 gene transcription. Type I fibres are the main source of IL-6 during dynamic exercise at moderate intensity (Fischer et al. 2004), while bicycling exercise at higher intensity stimulated IL-6 synthesis in myofibres of all types (Penkowa et al. 2003). Surprisingly, IL-6 mRNA and protein were observed at a much greater level in type II than in type I fibres at the end of a bicycling exercise of lower intensity and lower duration, while these fibres also had greater glycogen content (Hiscock et al. 2004). One of the main results of the present study is that, using real-time RT-PCR analysis, a very accurate and reproducible method to quantify mRNA levels, IL-6 gene transcription was mainly enhanced in type I and type IIa fibres, two fibre types belonging to the earlier-recruited populations of motor units during prolonged and sustained muscle contraction. In contrast, no significant change in IL-6 gene transcription rate was observed in type IIx and type IIb fibres, which are fast glycolytic fibres, recruited later during prolonged exercise (Fallentin et al. 1993; Houtman et al. 2003). As previously discussed, the exercise mode, intensity and duration affect whether IL-6 mRNA is expressed in oxidative or glycolytic fibres. Our data lend support to the hypothesis that even if both type I and type II myofibres have the capacity to synthesize IL-6, this cytokine is produced by essentially oxidative myofibres during moderate prolonged exercise.
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Our histochemical analyses showed that IL-6 mRNA expression was markedly increased in type I fibres with low glycogen content, consistent with the hypothesis that this cytokine may be involved in the regulation of glucose homeostasis (Febbraio & Pedersen, 2002). However, only a limited glycogen depletion was reported in type IIa fibres, while IL-6 mRNA appeared predominantly in those fibres. Consistent with a previous study (Hiscock et al. 2004), our data failed to demonstrate that at the cellular level, IL-6 gene transcription was closely related to low glycogen content. This finding is not in disagreement with the possibility that IL-6 may act as an energy sensor at the whole muscle level. Some evidence clearly suggests that low pre-exercise skeletal muscle glycogen content exacerbates the IL-6 response to exercise (Keller et al. 2001; Steensberg et al. 2001; Chan et al. 2004). However, the lack of correlation between two biological factors at the myofibre level, does not reject the relationship at the whole muscle level, which could also result from local influences (Febbraio & Pedersen, 2002). Our findings suggest that in addition to factors related to low intramuscular glycogen stores, IL-6 gene transcription could be also enhanced by glycogen-independent mechanisms.
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Therefore, molecular signals or events generated by muscle contraction probably have a key role in IL-6 gene transcription during exercise. Tonic motor nerve activity has been shown to sustain [Ca2+]i at levels sufficient to activate calcineurin, a Ca2+-activated protein phosphatase involved in muscle development, adaptation and disease (Bassel-Duby & Olson, 2003). Alterations in [Ca2+]i have been implicated in several signalling cascades and as a potent signalling factor for IL-6 transcription (Febbraio & Pedersen, 2002; Keller et al. 2002), and recent data showed that ionomycin, a Ca2+ ionophore, increased IL-6 gene expression in isolated skeletal muscle (Holmes et al. 2004). These data support the hypothesis that increased [Ca2+]i is involved in the control of IL-6 gene expression during exercise. Several signalling molecules are involved in transducing the calcium signal, thereby including calcineurin, and it has been proposed that activation of this phosphatase could be involved in early IL-6 gene transcription during prolonged exercise (Febbraio & Pedersen, 2002). Our results show that MCIP-1 mRNA levels significantly increased in both plantaris and soleus muscles at the end of prolonged exercise. This increase in MCIP-1 mRNA, a marker for calcineurin activation in two skeletal muscles known to be recruited during running exercise in rats, supports the notion that calcineurin is activated at the end of prolonged exercise (Norrbom et al. 2004).
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A major finding of this study was that in parallel with IL-6 mRNA, MCIP-1 mRNA expression increased in both type I and type IIa fibres. This finding suggests that calcineurin is activated concomitant with the increase in IL-6 mRNA, mainly in the most active fibres, those expressing type I or type IIa MHC. Tonic motor nerve activity is characteristic of type I and to a lesser extent of type IIa fibres. Calcineurin activity is mainly sensitive to sustained, low-amplitude elevations of [Ca2+]i and the specific isoforms of some regulatory proteins and sarcoplasmic reticulum Ca2+-ATPase account for the sustained elevations in [Ca2+]i (Botinelli & Reggiani, 2000; Baylor & Hollingworth, 2003). In the present study, we cannot prove whether the association between IL-6 gene transcription and enhanced calcineurin activity was causal or related to a random association. However, these data provide a new argument to suggest that calcineurin activation could be an upstream signal for IL-6 gene transcription during exercise. Calcineurin activation facilitates alterations in gene expression through several transcriptional effectors, including NFAT transcription factors (Bassel-Duby & Olson, 2003). A recent study suggested that the exercise-induced increase in IL-6 mRNA occurred through an NFAT-independent mechanism (Chan et al. 2004). However, calcineurin has been shown to be involved in the regulation of skeletal muscle genes, but not through NFAT (Parsons et al. 2003; Giger et al. 2004). Collectively, these results suggest that the exercise-induced increase in IL-6 gene transcription could be associated with calcineurin activation, which could control gene expression through a mechanism independent of the NFAT nuclear accumulation. Future studies need to demonstrate the causative relationship between calcineurin activity and IL-6 gene expression and determine the downstream targets of calcineurin. While it has been previously shown that low pre-exercise muscle glycogen content enhanced IL-6 gene transcription during exercise through phosphorylation of p38 MAPK in the nucleus (Chan et al. 2004), our results suggest that increased calcineurin activity could also account for the exercise-induced increase in IL-6 mRNA.
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In summary, quantitative measures of IL-6 mRNA within muscle cells, demonstrate a fibre-type specificity of IL-6 gene expression during exercise, with a marked increase in IL-6 transcripts in type I and type IIa fibres during prolonged and sustained muscle contraction. Another major and novel finding of this study was that MCIP-1 mRNA expression also increased in both type I and type IIa fibres, suggesting that calcineurin activation is concomitant with the increase in IL-6 mRNA, mainly in the most active fibres. These data provide a new argument to suggest that calcineurin activation could be an upstream signal for IL-6 gene transcription during exercise. Our results suggest that in addition to factors related to the intracellular signalling of low intramuscular glycogen, molecular events related to contractile activity, such as increased calcineurin activity, would lead to IL-6 gene transcription.
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References
Agbulut O, Li Z, Mouly V & Butler-Browne GS (1996). Analysis of skeletal and cardiac muscle from desmin knock-out and normal mice by high resolution separation of myosin heavy-chain isoforms. Biol Cell 88, 131–135.
Bartoccioni E, Michaelis D & Hohlfeld R (1994). Constitutive and cytokine-induced production of interleukin-6 by human myoblasts. Immunol Lett 42, 135–138.
Bassel-Duby R & Olson EN (2003). Role of calcineurin in striated muscle: development, adaptation, and disease. Biochem Biophys Res Commun 311, 1133–1141.
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Baylor SM & Hollingworth S (2003). Sarcoplasmic reticulum calcium release compared in slow-twitch and fast-twitch fibres of mouse muscle. J Physiol 551, 125–138.
Birot OJ, Koulmann N, Peinnequin A & Bigard XA (2003). Exercise-induced expression of vascular endothelial growth factor mRNA in rat skeletal muscle is dependent on fibre type. J Physiol 552, 213–221.
Botinelli R & Reggiani C (2000). Human skeletal muscle fibres: molecular and functional diversity. Prog Biophys Mol Biol 73, 195–262.
, 百拇医药
Chan MH, McGee SL, Watt MJ, Hargreaves M & Febbraio MA (2004). Altering dietary nutrient intake that reduces glycogen content leads to phosphorylation of nuclear p38 MAP kinase in human skeletal muscle: association with IL-6 gene transcription during contraction. FASEB J 18, 1785–1787.
Detmer K, Wang Z, Warejcka D, Leeper-Woodford SK & Newman WH (2001). Endotoxin stimulated cytokine production in rat vascular smooth muscle cells. Am J Physiol Heart Circ Physiol 281, H661–H668.
, 百拇医药
Fallentin N, Jorgensen K & Simosen EB (1993). Motor unit recruitment during prolonged isometric contractions. Eur J Appl Physiol Occup Physiol 67, 335–341.
Febbraio MA & Pedersen BK (2002). Muscle-derived interleukin-6: mechanisms for activation and possible biological roles. FASEB J 16, 1335–1347.
Fischer CP, Hiscock N, Penkowa M, Basu S, Vessby B, Kallner A, Sjberg LB & Pedersen BK (2004). Supplementation with vitamins C and E inhibits the release of interleukin-6 from contracting human skeletal muscle. J Physiol 558, 633–645.
, 百拇医药
Giger JM, Haddad F, Qin AX & Baldwin KM (2004). Effect of cyclosporin A treatment on the in vivo regulation of type I MHC gene expression. J Appl Physiol 97, 475–483.
Henneman E & Olson CB (1965). Relations between structure and function in the design of skeletal muscles. J Neurophysiol 28, 581–598.
Hiscock N, Chan MH, Bisucci T, Darby IA & Febbraio MA (2004). Skeletal myocytes are a source of interleukin-6 mRNA expression and protein release during contraction: evidence of fiber type specificity. FASEB J 18, 992–994.
, http://www.100md.com
Holmes AG, Watt MJ, Carey AL & Febbraio MA (2004). Ionomycin, but not physiologic doses of epinephrine, stimulates skeletal muscle interleukin-6 mRNA expression and protein release. Metabolism 53, 1492–1495.
Houtman CJ, Stegeman DF, Van Dijk JP & Zwarts MJ (2003). Changes in muscle fiber conduction velocity indicate recruitment of distinct motor unit populations. J Appl Physiol 95, 1045–1054.
Jonsdottir IH, Schjerling P, Ostrowski K, Asp S, Richter EA & Pedersen BK (2000). Muscle contractions induce interleukin-6 mRNA production in rat skeletal muscles. J Physiol 528, 157–163.
, 百拇医药
Keller C, Hellsten Y, Pilegaard H, Febbraio MA & Pedersen BK (2002). Human muscle cells express IL-6 via a Ca2+ dependent pathway. J Physiol 539P, S096 (Abstract).
Keller P, Keller C, Carey AL, Jauffred S, Fischer CP, Steensberg A & Pedersen BK (2003). Interleukin-6 production by contracting human skeletal muscle: autocrine regulation by IL-6. Biochem Biophys Res Commun 310, 550–554.
Keller C, Steensberg A, Pilegaard H, Osada T, Saltin B, Pedersen BK & Neufer PD (2001). Transcriptional activation of the IL-6 gene in human contracting skeletal muscle: influence of muscle glycogen content. FASEB J 15, 2748–2750.
, 百拇医药
Livak KJ & Schittgen TD (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C (T)) Method. Methods 25, 402–408.
Mahoney DJ, Carey K, Fu MH, Snow R, Cameron-Smith D, Parise G & Tarnopolsky MA (2004). Real-time RT-PCR analysis of housekeeping genes in human skeletal muscle following acute exercise. Physiol Genomics 18, 226–231.
Moldoveanu AI, Shephard RJ & Shek PN (2000). Exercise elevates plasma levels but not gene expression of IL-1beta, IL-6, and TNF-alpha in blood mononuclear cells. J Appl Physiol 89, 1499–1504.
, 百拇医药
Norrbom J, Sunberg CJ, Ameln H, Kraus WE, Jansson E & Gustafsson T (2004). PGC-1alpha mRNA expression is influenced by metabolic perturbation in exercising human skeletal muscle. J Appl Physiol 96, 189–194.
Ostrowski K, Rohde T, Zacho M, Asp S & Pedersen BK (1998). Evidence that interleukin-6 is produced in human skeletal muscle during prolonged running. J Physiol 508, 949–953.
Parsons SA, Wilkins BJ, Bueno OF & Molkentin JD (2003). Altered skeletal muscle phenotypes in calcineurin Aalpha and Abeta gene-targeted mice. Mol Cell Biol 23, 4331–4343.
, http://www.100md.com
Peinnequin A, Mouret C, Birot O, Alonso A, Mathieu J, Clarencon D, Agay D, Chancerelle Y & Multon E (2004). Rat pro-inflammatory cytokine and cytokine related mRNA quantification by real-time polymerase chain reaction using SYBR green. BMC Immunol 5, 3.
Penkowa M, Keller C, Keller P, Jauffred S & Pedersen BK (2003). Immunohistochemical detection of interleukin-6 in human skeletal muscle fibers following exercise. FASEB J 17, 2166–2168.
, http://www.100md.com
Starkie RL, Angus DJ, Rolland J, Hargreaves M & Febbraio MA (2000). Effect of prolonged, submaximal exercise and carbohydrate ingestion on monocyte intracellular cytokine production in humans. J Physiol 528, 647–655.
Steensberg A, Febbraio MA, Osada T, Schjerling P, Van Hall G, Saltin B & Pedersen BK (2001). Interleukin-6 production in contracting human skeletal muscle is influenced by pre-exercise muscle glycogen content. J Physiol 537, 633–639.
, 百拇医药
Steensberg A, Van Hall G, Osada T, Sacchetti M, Saltin B & Pedersen BK (2000). 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.
Sterpetti AV, Cucina A, Morena AR, Di Donna S, D'Angelo LS, Cavalarro A & Tipa S (1993). Shear stress increases the release of interleukin-1 and interleukin-6 by aortic endothelial cells. Surgery 114, 911–914.
, http://www.100md.com
Ullum H, Haahr PM, Diamant M, Palmo J, Halkjaer-Kristensen J & Pedersen BK (1994). Bicycle exercise enhances plasma IL-6 but does not change IL-1 alpha, IL-1 beta, IL-6, or TNF-alpha pre-mRNA in BMNC. J Appl Physiol 77, 93–97.
Yang J, Rothermel B, Vega RB, Frey N, McKinsey TA, Olson EN, Bassel-Duby R & Williams RS (2000). Independent signals control expression of the calcineurin inhibitory proteins MCIP1 and MCIP2 in striated muscles. Circ Res 87, E61–E68., 百拇医药(Sebastien Banzet, Nathali)
Abstract
In this study, we quantified the transcription of the interleukin-6 (IL-6) gene in individual fibres and the associated changes in calcineurin activity assessed at the cellular level during prolonged muscle contraction. Individual myofibres were isolated from plantaris and soleus muscles of rats at the end of an exhaustive running exercise test (n = 10), categorized according to their myosin heavy chain isoform content, and compared to those of resting rats (n = 10). Using real-time PCR analysis in individual fibres, a marked rise in IL-6 transcript levels occurred in type I and IIa fibres at the end of exercise (P < 0.05). Transcription of the gene encoding for the modulatory calcineurin-interacting protein-1 (MCIP-1), a sensitive indicator of calcineurin activity, also mainly increased in type I and IIa fibres (P < 0.05). Moreover, a slight increase in MCIP-1 mRNA levels was observed in type IIx (P < 0.05). Fibre types determined by immunohistochemistry were qualitatively examined for glycogen content using periodic acid–Shiff staining, and no direct relationship was found, at the cellular level, between glycogen content, fibre-type and IL-6 transcription. Our data clearly suggest that IL-6 gene transcription was mainly observed in early recruited myofibres and that contraction-induced IL-6 transcription could be associated with enhanced calcineurin activity.
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Introduction
It has been recently shown that interleukin-6 (IL-6) plasma levels increase dramatically during prolonged concentric exercise in man (for review see Febbraio & Pedersen, 2002). Increased IL-6 mRNA levels were reported in human muscle biopsies at the end of exercise, related to mechanisms other than muscle damage (Ostrowski et al. 1998). In a one-legged exercise test, high muscle IL-6 net release occurred only in the contracting limb (Steensberg et al. 2000). Together, these results strongly suggest that muscle is the main source of plasma IL-6 during exercise and that this production is directly associated with muscle contraction and does not result from an exercise-related systemic effect. Subjects exercising with low intramuscular glycogen levels showed a higher plasma IL-6 peak (Keller et al. 2001), independent of systemic influences (Steensberg et al. 2001). It has thus been hypothesized that muscle-derived IL-6 is linked to energy availability and could play an important role in carbohydrate homeostasis during exercise by contributing to contraction-mediated glucose uptake and by acting as an endocrine signal of muscle energy stores to favour hepatic glucose production and white adipose tissue lipolysis (for review see Febbraio & Pedersen, 2002).
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However, skeletal muscle contains several cell types that are known to be able to produce IL-6. Blood mononuclear cells do not account for the exercise-induced increase in IL-6 plasma levels (Ullum et al. 1994; Starkie et al. 2000; Moldoveanu et al. 2000). Human myoblasts (Bartoccioni et al. 1994), smooth muscle cells (Detmer et al. 2001) and endothelial cells (Sterpetti et al. 1993) can produce IL-6 when exposed to several stimuli such as inflammatory cytokines, endotoxins or mechanical stress. The cellular origin of IL-6 production in muscle has been examined in two recent studies. The immunohistochemical detection of IL-6 protein in skeletal muscle showed an increase in positive myofibres at the end of exercise, suggesting that myofibres per se could be a source of IL-6 production during contraction (Penkowa et al. 2003). Moreover, using in situ hybridization in human muscle, it has recently been shown that myofibres contain IL-6 mRNA at the end of prolonged exercise (Hiscock et al. 2004). These findings clearly show that muscle fibres are a source of IL-6, and because myofibres consume and need energy during muscle contraction, they reinforce the hypothesis of an energy-sensing function of IL-6.
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Adult rat skeletal muscles comprise at least four fibre types ranging from slow-twitch predominantly oxidative fibres (type I) to fast-twitch predominantly oxidative, intermediate oxidative and low oxidative fibres (types IIa, IIx and IIb, respectively). Muscle fibres are distributed among motor units and it is well accepted that during muscle contraction, motor units are recruited in an orderly manner. According to the ‘size principle’ of Henneman & Olson (1965), the smallest motor units comprising type I fibres are first recruited, while the largest, comprising type IIx and type IIb fibres, are recruited long after the beginning of muscle contraction, when local fatigue occurs in slow and oxidative motor units (Fallentin et al. 1993). Because type I and type IIa fibres have small glycogen stores, whereas type IIx and IIb fibre have large glycogen stores, and IL-6 may work as a sensor of carbohydrate availability (Febbraio & Pedersen, 2002), a fibre-type specificity of IL-6 gene expression could be expected at the end of prolonged exercise. This issue has been recently addressed and controversial findings were reported. No difference was detected between muscle fibre types at the protein level at the end of exercise (Penkowa et al. 2003), whereas Fischer et al. (2004) described preferential staining in type I fibres. More recent data suggest that type II fibres predominantly produce IL-6 during exercise in humans (Hiscock et al. 2004). Moreover, the results of this latter study suggested that a reduced level of glycogen was not directly involved in IL-6 gene expression during muscle contractile activity. Whether the expression of the IL-6 gene varies between fibre types during exercise therefore remains to be elucidated.
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Because the nuclear transcriptional rate of the IL-6 gene is remarkably rapid after the onset of exercise, it has been hypothesized that rapid changes in cytosolic Ca2+ concentration could be involved in IL-6 gene expression (Febbraio & Pedersen, 2002). Changes in intracellular free calcium concentration ([Ca2+]i) is a key event during myocyte contraction, which has recently been shown to induce a rapid increase in IL-6 mRNA and protein expression in rat isolated muscles (Holmes et al. 2004). Prolonged contractile activity is characterized by low-amplitude and sustained elevations in [Ca2+]i, leading to increased activity of calcineurin, a ubiquitous Ca2+–calmodulin-dependent protein phosphatase known to be a key mediator of Ca2+ signalling in muscle cells. Several downstream effectors of calcineurin have been identified in skeletal muscle, including the nuclear factor of the activated T cell (NFAT), and the calcineurin–NFAT pathway is involved in the acquisition and maintenance of the slow oxidative phenotype (for review see Bassel-Duby & Olson, 2003). Whether this transcription factor is involved in IL-6 gene transcription has recently been examined (Chan et al. 2004). No evidence was provided that IL-6 gene transcription is activated by NFAT, whereas the phosphorylation of nuclear p38 mitogen-activated protein kinase (MAPK) was considered as an activator for candidate transcription factors that may bind to the IL-6 promoter region in myofibres. However, whether the increased cytosolic Ca2+ accumulation expected within single specific fibres during prolonged muscle contraction is associated with activated calcineurin and enhanced IL-6 gene expression has not been examined to date.
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The aims of this study were to examine the fibre-type specificity of IL-6 gene transcription in individual myofibres at the end of prolonged exercise and the involvement of calcineurin activity in the control of IL-6 gene expression. In light of the recruitment pattern of muscle fibres during prolonged submaximal exercise and the differences in pre-exercise glycogen content, we hypothesized that (1) IL-6 production could be fibre-type specific and that IL-6 mRNA levels could be higher in type I and type IIa fibres than in type IIx and type IIb at the end of exercise, and (2) high IL-6 mRNA levels could be observed in fibres showing high calcineurin activity. To address these issues, we first quantified the level of IL-6 gene transcription within individual fibres taken from two skeletal muscles at the end of exercise in rat. Second, we examined whether IL-6 gene expression was associated with high levels of calcineurin activity. As previously suggested, transcription of the gene encoding for the modulatory calcineurin-interacting protein-1 (MCIP-1) was used as a sensitive indicator of calcineurin activity (Yang et al. 2000).
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Methods
Animals
Three-month-old male Wistar rats (n = 30) were purchased from Charles River (L'Arbresle, France). They were housed three per cage in a thermoneutral environment (22 ± 2°C), on a 12–12 h light–dark period, and were provided with food and water ad libitum. They were randomly assigned to three experimental groups: (1) control rats (n = 10); (2) active rats (n = 10); and (3) recovering rats (n = 10). Experiments received prior approval from the animal ethics committee of the Centre de Recherche du Service Sante des Armees (La Tronche, France).
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Exercise protocol
All animals were accustomed to running on a rodent treadmill for 15 min per day for 5 days at a moderate level (10–20 m min–1; 0 deg gradient). After 3 days at rest, active and recovering animals ran on the treadmill (25 m min–1, 8 deg gradient) until exhaustion, defined as the moment where animals were unable to keep in pace with the treadmill.
Tissue processing
Animals were removed and anaesthetized either immediately (active group), or 2 h after the end of exercise (recovering group), with intraperitoneal injection of pentobarbital (70 mg (kg body weight)–1). Plantaris and soleus muscles of both hindlimbs were excised and cleaned of adipose and connective tissue. Plantaris muscles were also separated in superficial and deep portions. While the left muscles were immediately frozen in liquid nitrogen and stored at –80°C, the right muscles were placed in 400 μl RNAlater solution (Ambion, Austin, TX, USA), kept at 4°C for 24 h and then frozen at –20°C. Animals were killed by removal of the heart.
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Single-fibre isolation
The single-fibre study was adapted from the methods of Birot et al. (2003). Muscle conserved in RNAlater was thawed on ice and placed in a small dish containing RNAlater. A small bundle of muscle fibres was first isolated under the light microscope using sharp-ended tweezers. Single muscle fibres were then separated and cut into two equal parts. One half was placed in 40 μl Nanoprep lysis buffer supplemented with 0.7% 2-mercaptoethanol (Stratagene, La Jolla, CA, USA), kept for 2 h at 4°C and then frozen at –80°C before RNA extraction. The other half-fibre was placed in 20 μl myosin extraction solution containing (mM): NaCl 300, NaH2PO4 100, Na2HPO4 50, Na4P2O7 10, MgCl2.6H2O 1, EDTA 10 and 2-mercaptoethanol 1.4; pH 6.5, for further myosin heavy chain (MHC) content analysis.
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MHC isoform analysis
Single fibres were subjected to MHC isoform analysis using SDS-PAGE as previously described (Birot et al. 2003). After incubation for 24 h at 4°C, the half-fibre was digested and the 20 ml mixture was then diluted with 20 ml glycerol. Extracts were stored at –20°C until required for the separation process. Electrophoresis was performed using a Mini Protean II system (Biorad, Marnes-la-Coquette, France). The separating gel solution contained 30% glycerol, 8% acrylamide–bis (50: 1), 0.2 M Tris, 0.1 M glycine and 0.4% SDS. The stacking gel was composed of 30% glycerol, 4% acrylamide–bis (50: 1), 70 mM Tris, 4 mM EDTA and 0.4% SDS. Then 10 ml myofibril samples were denatured using 10 ml buffer containing 5% 2-mercaptoethanol, 100 mM Tris base, 5% glycerol, 4% SDS and bromophenol blue, for 3 min at 100°C. Myofibrillar homogenates were loaded onto vertical gels, whilst two lanes were loaded with protein extract from a control plantaris muscle known to contain the four adult MHC isoforms. Gels were run at constant voltage (72 V) for 31 h and then silver-stained (Agbulut et al. 1996). The MHC protein isoform bands were scanned using a densitometer system equipped with an integrator (GS-700, Biorad, Marnes-la-Coquette, France). The MHC isoforms expressed by single fibres were identified by comparing them with bands of myosin extracts from control plantaris muscle.
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RNA extraction
Muscle tissue mRNA was isolated using the MagNA Pure LC instrument (Roche Applied Science, Mannheim, Germany). We disrupted 10 mg frozen soleus and deep plantaris muscles using two tungsten carbide beads in 100 μl MagNA Pure LC mRNA isolation kit II (Roche Applied Science) lysis buffer with a Mixer Mill MM300 (Rescht, Haan, Germany) for 40 s (30 Hz). Lysate was centrifuged (12 000 g, 5 min, room temperature), the liquid phase was transferred in a fresh tube and lysis buffer added to a final volume of 300 μl. Extraction was then performed following the manufacturer's protocol with an 880 μl sample volume, a 50 μl elution volume and no dilution.
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The total RNA of the fibres was isolated using RNA Insta-pure reagent (Eurogentec, Saraing, Belgium) with a modified protocol. Samples were extracted using 200 μl RNA Insta-pure reagent and 20 μl chloroform (Sigma-Aldrich, Steinheim, Germany). An additional iso-volume chloroform extraction was performed. RNA isopropanol precipitation was carried out using 10 μg glycogen (Sigma-Aldrich) as carrier. The ethanol washing step was performed twice and the RNA pellet was dried in a vacuum for 3 min. RNA was then resuspended in 10 μl RNase-free water.
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Reverse transcription
Muscle tissue mRNA reverse transcription was carried out using the Reverse Transcriptase Core Kit (Eurogentec, Saraing, Belgium). The reaction was performed in a 10 μl final volume following the manufacturer's instructions. The reaction mix contained 1 μl buffer (10 x), 2 μl dNTP mix (2.5 mM each deoxyribronucleotide triphosphate (dNTP)), 0.5 μl oligo-dT primer (50 μM), 0.2 μl RNase inhibitor (20 units μl–1), 2 μl MgCl2 (25 mM), 0.25 μl Euroscript reverse transcriptase (50 U μl–1), 1.05 μl RNase-free water and 3 μl template mRNA.
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Fibre RNA reverse transcription was carried out using Sensiscript Reverse Transcriptase (Qiagen S.A, Courtaboeuf, France), specially designed for highly sensitive reverse transcription with small amounts of RNA. The reaction was performed in a 20 μl final volume following the manufacturer's instructions. The mix contained 2 μl buffer (10 x), 2 μl dNTP mix (5 mM each dNTP), 2 μl oligo-dT primer (10 μM), 1 μl RNase inhibitor (10 U μl–1), 1 μl Sensiscript reverse transcriptase, 4 μl RNase-free water and 8 μl template RNA.
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PCR primer design
All primers used in this study were designed with the MacVector software (Accelrys, Orsay, France) and synthesized at Eurogentec (Saraing, Belgium). Primer sequences and resulting amplified product characteristics are shown in Table 1.
Real-time PCR
The PCR reactions were carried out in a 20 μl final volume with the LC Fast Start DNA Master SYBR Green kit (Roche Applied Science) using 0.2 μl (whole muscle) or 0.5 μl (single-fibre) cDNA solution. Quantitative PCRs were performed using LightCycler (Roche Applied Science) for 45 amplification cycles using a 5 s annealing step and an 8 s 72°C elongation step (detailed conditions available in Table 1). Specificity was checked for each sample by melting-curve analysis. Transcription levels were normalized using an internal control gene with the comparative threshold cycle method (Livak & Schittgen, 2001) using RelQuant software (Roche Applied Science). A pool of 40 samples randomly chosen in the groups was used as a calibrator as previously described (Peinnequin et al. 2004). Cyclophyllin A (CycA) was used as reference gene because its expression is known to be unaffected at the end of an endurance exercise test in skeletal muscle (Mahoney et al. 2004). Furthermore, this was controlled in our experiment on whole muscle tissue.
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Purity of single fibres
The single-fibre approach we used, first described by Birot et al. (2003), makes it possible to remove fibroblasts, smooth muscle cells and blood mononuclear cells because conjunctive tissue and vessels are clearly visible under the microscope. In contrast, endothelial cells and infiltrating mononuclear cells cannot be seen, can adhere to the myofibres and are capable of producing great amounts of IL-6. Before including fibres in our study, we needed to check the purity of all half-fibres and confirm the absence of endothelial cells or monocytic cells. Every half-fibre of the active group was tested by PCR amplification for platelet endothelial cell adhesion molecule (PECAM) and cluster of differentiation 11b (CD11b). When there was a positive signal for PECAM and/or CD11b, the sample was excluded from the study.
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Immunohistochemistry and periodic acid–Schiff staining
Serial transverse sections (14 μm thick) were cut from the mid-belly portion of soleus and plantaris muscles in a cryostat maintained at –20°C. Plantaris sections were labelled with mouse monoclonal antibodies against myosin reacting with (1) slow type I (Novocastra, reference NCL-MHCS, Newcastle upon Tyne, UK), (2) all adult fast and developmentally regulated epitopes but not with slow myosin (MY-32, Sigma-Aldrich), or (3) fast type IIa (SC-71). Both plantaris and soleus sections were stained for glycogen content using the periodic acid–Shiff (PAS) staining system (Sigma-Aldrich) according to the manufacturer's instructions.
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Statistics
All data are presented as means ± S.E.M. Student's unpaired t test was used to determine differences between groups. Statistical significance was accepted at P < 0.05.
Results
Running time
Both active and recovering rats performed an exhaustive running exercise. The mean duration of this exercise was 101 ± 13 min.
, 百拇医药 Exercise-induced increase in IL-6 in whole muscle tissue
As expected, there was an increase in IL-6 mRNA levels in both plantaris and soleus muscles at the end of running exercise (P < 0.05). IL-6 mRNA returned to baseline values approximately 2 h after exercise stopped. There was no significant difference in IL-6 transcript levels between plantaris and soleus muscles in control, active and recovery groups (Fig. 1). Because IL-6 mRNA levels decreased 2 h after the end of exercise, gene expression within single fibres was only considered in control and active groups.
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Data are means ± S.E.M. *Significantly different from control values, P < 0.05; significantly different from active group, P < 0.05.
Fibre-type specificity of IL-6 mRNA expression
In soleus muscles, 91% of myofibres were pure type I fibres, while 9% expressed both MHC I and MHC IIa. In deep plantaris muscles, 49% of myofibres expressed only one MHC isoform. Therefore, only 171 of the 351 successfully typed fibres were included for further study. Before IL-6 mRNA quantification, each individual fibre was tested for PECAM, CD11b and CycA mRNA expression. A negative signal for both PECAM and CD11b mRNA proved the lack of contamination by endothelial or monocytic cell material, respectively, while a positive expression of CycA gene confirmed the presence of biological material. After all these validation analyses, 37% of single, successfully typed fibres taken from soleus and plantaris muscles were used for further assays.
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At the end of exercise, IL-6 mRNA levels increased significantly in myofibres of soleus muscle, which were mainly type I fibres (P < 0.05) (Fig. 2A). In myofibres taken from plantaris muscles, there was a marked and significant increase in IL-6 mRNA in only type I and type IIa fibres at the end of prolonged exercise (P < 0.05). In contrast, acute treadmill exercise did not significantly change the IL-6 mRNA levels in type IIx and IIb fibres (P < 0.05) (Fig. 2B).
, http://www.100md.com Plantaris fibres were categorized into four types: type I, IIa, IIx and IIb, according to their MHC isoform content. Data are means ± S.E.M. *Significantly different from control values, P < 0.05; significantly different from IIa in active group values, P < 0.05.
Myofibre glycogen content at the end of exercise
PAS staining and immunohistochemistical analyses showed that in soleus muscles (Fig. 3), type I fibres were markedly glycogen depleted, whereas glycogen only slightly decreased in type IIa fibres. Likewise, all type I fibres were glycogen-depleted at the end of exercise in plantaris muscles (Fig. 3); in contrast, type IIa fibres failed to be markedly glycogen depleted. As shown in Fig. 3, there was heterogeneity of glycogen depletion within the two other fast fibre types (type IIx and IIb fibres), with some fibres markedly depleted, while no detectable changes were reported in the others.
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Glycogen content in specific fibre types in soleus (A, B, E and F) and plantaris (C, D, G and H), before (A and C) and after exhaustive running exercise (B, D and E–H). Glycogen content was assessed by PAS staining (A–D) and fibre type by immunohistochemistry using antibodies directed against type I (F and H) and type IIa MHC isoforms (E and G). In soleus muscles, pure type IIa fibres () or hybrid fibres comprising both type I and type IIa MHC isoforms () (E and F) are shown in B with a lower glycogen depletion than pure type I fibres (). In plantaris muscles, type IIa fibres (G) are shown in D with slight or no glycogen depletion (); type I fibres (H) were markedly depleted (), while fibres negative for antibodies (i.e. fibres comprising either type IIx and/or type IIb MHC isoforms) (G and H) show a heterogeneity of glycogen depletion () (D). Scale bar, 100 μm.
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Fibre-type specificity of calcineurin activity at the end of exercise
The peculiar responsiveness of MCIP-1 gene expression to calcineurin activity was used to assess the activation state of calcineurin (Yang et al. 2000). A marked increase in MCIP-1 mRNA was shown in both plantaris and soleus muscles (P < 0.05 and P < 0.01, respectively) at the end of exercise (Fig. 4A). The exercise-induced increase in MCIP-1 mRNA was found at similar levels in slow-twitch (soleus) and fast-twitch muscles (plantaris).
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For n and categorization of fibres see Figs 1 (A) and 2B (B). Data are means ± S.E.M. *Significantly different from control values (P < 0.05); **significantly different from control values (P < 0.01); significantly different from IIx in the same group (P < 0.05); significantly different from IIb in the same group (P < 0.05).
As reported for IL-6 mRNA expression, there was a significant increase in MCIP-1 mRNA levels in both type I and IIa fibres at the end of prolonged exercise (P < 0.05) (Fig. 4B). Moreover, a slight but significant increase in MCIP-1 mRNA levels was shown in type IIx fibres of exercised rats (P < 0.05).
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Discussion
Despite emerging evidence for IL-6 production by myofibres during exercise, IL-6 mRNA levels were never quantified within individual fibres. In this study, we provide for the first time quantitative measures of IL-6 mRNA levels in muscle cells. The major and novel observations made in the current investigation were: (1) a fibre-type specificity of IL-6 gene expression during exercise, with a marked increase in IL-6 transcripts in type I and type IIa fibres; and (2) an increase in calcineurin activity in individual muscle cells in parallel with the exercise-induced enhancement in IL-6 gene transcription.
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Many recent studies showed increased levels of plasma IL-6 after exercise in humans, and demonstrated that the appearance of IL-6 in the circulation results from an increased transcription of the IL-6 gene in contracting muscles (Ostrowski et al. 1998; Steensberg et al. 2001; Keller et al. 2003). IL-6 is locally produced in skeletal muscle following both concentric and eccentric contractions in rats (Jonsdottir et al. 2000), but dynamic exercise such as running had never been used to examine the molecular events that control the IL-6 gene transcription in rodents. In the present study, IL-6 mRNA levels measured in whole muscle tissue before, at the end, and 2 h after running exercise in rats closely mimic the previously reported changes in human muscle biopsies after exercise. These results show that prolonged running exercise in the rat is a valid model to study contraction-induced IL-6 gene transcription in muscles.
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As previously and consistently shown, contracting skeletal muscle is the main source of IL-6 production during exercise (for review see Febbraio & Pedersen, 2002). However, the issue of the cell type responsible for IL-6 production within skeletal muscle has been only recently addressed using histochemical methods or a qualitative approach of IL-6 gene transcription (Penkowa et al. 2003; Hiscock et al. 2004; Fischer et al. 2004). In the present study, we provide the first quantitative analysis of IL-6 gene transcription rate by RT-PCR in single myofibres. The lack of myofibre contamination by endothelial and mononuclear cells was verified by a negative signal for PECAM and CD11b, and these controls ensured that samples selected for IL-6 mRNA determination were pure single myofibres. Consistent with a previous study, our results using RT-PCR analysis definitively show that muscle fibres are a source of IL-6 in contracting muscles (Hiscock et al. 2004).
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Because myofibres are recruited in an orderly manner and IL-6 production would be increased in glycogen-depleted fibres, it has been hypothesized that IL-6 mRNA expression could be fibre-type specific. This issue has been examined, and recent reports support the role played by the mode, intensity and duration of exercise on the fibre-type specificity of IL-6 gene transcription. Type I fibres are the main source of IL-6 during dynamic exercise at moderate intensity (Fischer et al. 2004), while bicycling exercise at higher intensity stimulated IL-6 synthesis in myofibres of all types (Penkowa et al. 2003). Surprisingly, IL-6 mRNA and protein were observed at a much greater level in type II than in type I fibres at the end of a bicycling exercise of lower intensity and lower duration, while these fibres also had greater glycogen content (Hiscock et al. 2004). One of the main results of the present study is that, using real-time RT-PCR analysis, a very accurate and reproducible method to quantify mRNA levels, IL-6 gene transcription was mainly enhanced in type I and type IIa fibres, two fibre types belonging to the earlier-recruited populations of motor units during prolonged and sustained muscle contraction. In contrast, no significant change in IL-6 gene transcription rate was observed in type IIx and type IIb fibres, which are fast glycolytic fibres, recruited later during prolonged exercise (Fallentin et al. 1993; Houtman et al. 2003). As previously discussed, the exercise mode, intensity and duration affect whether IL-6 mRNA is expressed in oxidative or glycolytic fibres. Our data lend support to the hypothesis that even if both type I and type II myofibres have the capacity to synthesize IL-6, this cytokine is produced by essentially oxidative myofibres during moderate prolonged exercise.
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Our histochemical analyses showed that IL-6 mRNA expression was markedly increased in type I fibres with low glycogen content, consistent with the hypothesis that this cytokine may be involved in the regulation of glucose homeostasis (Febbraio & Pedersen, 2002). However, only a limited glycogen depletion was reported in type IIa fibres, while IL-6 mRNA appeared predominantly in those fibres. Consistent with a previous study (Hiscock et al. 2004), our data failed to demonstrate that at the cellular level, IL-6 gene transcription was closely related to low glycogen content. This finding is not in disagreement with the possibility that IL-6 may act as an energy sensor at the whole muscle level. Some evidence clearly suggests that low pre-exercise skeletal muscle glycogen content exacerbates the IL-6 response to exercise (Keller et al. 2001; Steensberg et al. 2001; Chan et al. 2004). However, the lack of correlation between two biological factors at the myofibre level, does not reject the relationship at the whole muscle level, which could also result from local influences (Febbraio & Pedersen, 2002). Our findings suggest that in addition to factors related to low intramuscular glycogen stores, IL-6 gene transcription could be also enhanced by glycogen-independent mechanisms.
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Therefore, molecular signals or events generated by muscle contraction probably have a key role in IL-6 gene transcription during exercise. Tonic motor nerve activity has been shown to sustain [Ca2+]i at levels sufficient to activate calcineurin, a Ca2+-activated protein phosphatase involved in muscle development, adaptation and disease (Bassel-Duby & Olson, 2003). Alterations in [Ca2+]i have been implicated in several signalling cascades and as a potent signalling factor for IL-6 transcription (Febbraio & Pedersen, 2002; Keller et al. 2002), and recent data showed that ionomycin, a Ca2+ ionophore, increased IL-6 gene expression in isolated skeletal muscle (Holmes et al. 2004). These data support the hypothesis that increased [Ca2+]i is involved in the control of IL-6 gene expression during exercise. Several signalling molecules are involved in transducing the calcium signal, thereby including calcineurin, and it has been proposed that activation of this phosphatase could be involved in early IL-6 gene transcription during prolonged exercise (Febbraio & Pedersen, 2002). Our results show that MCIP-1 mRNA levels significantly increased in both plantaris and soleus muscles at the end of prolonged exercise. This increase in MCIP-1 mRNA, a marker for calcineurin activation in two skeletal muscles known to be recruited during running exercise in rats, supports the notion that calcineurin is activated at the end of prolonged exercise (Norrbom et al. 2004).
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A major finding of this study was that in parallel with IL-6 mRNA, MCIP-1 mRNA expression increased in both type I and type IIa fibres. This finding suggests that calcineurin is activated concomitant with the increase in IL-6 mRNA, mainly in the most active fibres, those expressing type I or type IIa MHC. Tonic motor nerve activity is characteristic of type I and to a lesser extent of type IIa fibres. Calcineurin activity is mainly sensitive to sustained, low-amplitude elevations of [Ca2+]i and the specific isoforms of some regulatory proteins and sarcoplasmic reticulum Ca2+-ATPase account for the sustained elevations in [Ca2+]i (Botinelli & Reggiani, 2000; Baylor & Hollingworth, 2003). In the present study, we cannot prove whether the association between IL-6 gene transcription and enhanced calcineurin activity was causal or related to a random association. However, these data provide a new argument to suggest that calcineurin activation could be an upstream signal for IL-6 gene transcription during exercise. Calcineurin activation facilitates alterations in gene expression through several transcriptional effectors, including NFAT transcription factors (Bassel-Duby & Olson, 2003). A recent study suggested that the exercise-induced increase in IL-6 mRNA occurred through an NFAT-independent mechanism (Chan et al. 2004). However, calcineurin has been shown to be involved in the regulation of skeletal muscle genes, but not through NFAT (Parsons et al. 2003; Giger et al. 2004). Collectively, these results suggest that the exercise-induced increase in IL-6 gene transcription could be associated with calcineurin activation, which could control gene expression through a mechanism independent of the NFAT nuclear accumulation. Future studies need to demonstrate the causative relationship between calcineurin activity and IL-6 gene expression and determine the downstream targets of calcineurin. While it has been previously shown that low pre-exercise muscle glycogen content enhanced IL-6 gene transcription during exercise through phosphorylation of p38 MAPK in the nucleus (Chan et al. 2004), our results suggest that increased calcineurin activity could also account for the exercise-induced increase in IL-6 mRNA.
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In summary, quantitative measures of IL-6 mRNA within muscle cells, demonstrate a fibre-type specificity of IL-6 gene expression during exercise, with a marked increase in IL-6 transcripts in type I and type IIa fibres during prolonged and sustained muscle contraction. Another major and novel finding of this study was that MCIP-1 mRNA expression also increased in both type I and type IIa fibres, suggesting that calcineurin activation is concomitant with the increase in IL-6 mRNA, mainly in the most active fibres. These data provide a new argument to suggest that calcineurin activation could be an upstream signal for IL-6 gene transcription during exercise. Our results suggest that in addition to factors related to the intracellular signalling of low intramuscular glycogen, molecular events related to contractile activity, such as increased calcineurin activity, would lead to IL-6 gene transcription.
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References
Agbulut O, Li Z, Mouly V & Butler-Browne GS (1996). Analysis of skeletal and cardiac muscle from desmin knock-out and normal mice by high resolution separation of myosin heavy-chain isoforms. Biol Cell 88, 131–135.
Bartoccioni E, Michaelis D & Hohlfeld R (1994). Constitutive and cytokine-induced production of interleukin-6 by human myoblasts. Immunol Lett 42, 135–138.
Bassel-Duby R & Olson EN (2003). Role of calcineurin in striated muscle: development, adaptation, and disease. Biochem Biophys Res Commun 311, 1133–1141.
, 百拇医药
Baylor SM & Hollingworth S (2003). Sarcoplasmic reticulum calcium release compared in slow-twitch and fast-twitch fibres of mouse muscle. J Physiol 551, 125–138.
Birot OJ, Koulmann N, Peinnequin A & Bigard XA (2003). Exercise-induced expression of vascular endothelial growth factor mRNA in rat skeletal muscle is dependent on fibre type. J Physiol 552, 213–221.
Botinelli R & Reggiani C (2000). Human skeletal muscle fibres: molecular and functional diversity. Prog Biophys Mol Biol 73, 195–262.
, 百拇医药
Chan MH, McGee SL, Watt MJ, Hargreaves M & Febbraio MA (2004). Altering dietary nutrient intake that reduces glycogen content leads to phosphorylation of nuclear p38 MAP kinase in human skeletal muscle: association with IL-6 gene transcription during contraction. FASEB J 18, 1785–1787.
Detmer K, Wang Z, Warejcka D, Leeper-Woodford SK & Newman WH (2001). Endotoxin stimulated cytokine production in rat vascular smooth muscle cells. Am J Physiol Heart Circ Physiol 281, H661–H668.
, 百拇医药
Fallentin N, Jorgensen K & Simosen EB (1993). Motor unit recruitment during prolonged isometric contractions. Eur J Appl Physiol Occup Physiol 67, 335–341.
Febbraio MA & Pedersen BK (2002). Muscle-derived interleukin-6: mechanisms for activation and possible biological roles. FASEB J 16, 1335–1347.
Fischer CP, Hiscock N, Penkowa M, Basu S, Vessby B, Kallner A, Sjberg LB & Pedersen BK (2004). Supplementation with vitamins C and E inhibits the release of interleukin-6 from contracting human skeletal muscle. J Physiol 558, 633–645.
, 百拇医药
Giger JM, Haddad F, Qin AX & Baldwin KM (2004). Effect of cyclosporin A treatment on the in vivo regulation of type I MHC gene expression. J Appl Physiol 97, 475–483.
Henneman E & Olson CB (1965). Relations between structure and function in the design of skeletal muscles. J Neurophysiol 28, 581–598.
Hiscock N, Chan MH, Bisucci T, Darby IA & Febbraio MA (2004). Skeletal myocytes are a source of interleukin-6 mRNA expression and protein release during contraction: evidence of fiber type specificity. FASEB J 18, 992–994.
, http://www.100md.com
Holmes AG, Watt MJ, Carey AL & Febbraio MA (2004). Ionomycin, but not physiologic doses of epinephrine, stimulates skeletal muscle interleukin-6 mRNA expression and protein release. Metabolism 53, 1492–1495.
Houtman CJ, Stegeman DF, Van Dijk JP & Zwarts MJ (2003). Changes in muscle fiber conduction velocity indicate recruitment of distinct motor unit populations. J Appl Physiol 95, 1045–1054.
Jonsdottir IH, Schjerling P, Ostrowski K, Asp S, Richter EA & Pedersen BK (2000). Muscle contractions induce interleukin-6 mRNA production in rat skeletal muscles. J Physiol 528, 157–163.
, 百拇医药
Keller C, Hellsten Y, Pilegaard H, Febbraio MA & Pedersen BK (2002). Human muscle cells express IL-6 via a Ca2+ dependent pathway. J Physiol 539P, S096 (Abstract).
Keller P, Keller C, Carey AL, Jauffred S, Fischer CP, Steensberg A & Pedersen BK (2003). Interleukin-6 production by contracting human skeletal muscle: autocrine regulation by IL-6. Biochem Biophys Res Commun 310, 550–554.
Keller C, Steensberg A, Pilegaard H, Osada T, Saltin B, Pedersen BK & Neufer PD (2001). Transcriptional activation of the IL-6 gene in human contracting skeletal muscle: influence of muscle glycogen content. FASEB J 15, 2748–2750.
, 百拇医药
Livak KJ & Schittgen TD (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C (T)) Method. Methods 25, 402–408.
Mahoney DJ, Carey K, Fu MH, Snow R, Cameron-Smith D, Parise G & Tarnopolsky MA (2004). Real-time RT-PCR analysis of housekeeping genes in human skeletal muscle following acute exercise. Physiol Genomics 18, 226–231.
Moldoveanu AI, Shephard RJ & Shek PN (2000). Exercise elevates plasma levels but not gene expression of IL-1beta, IL-6, and TNF-alpha in blood mononuclear cells. J Appl Physiol 89, 1499–1504.
, 百拇医药
Norrbom J, Sunberg CJ, Ameln H, Kraus WE, Jansson E & Gustafsson T (2004). PGC-1alpha mRNA expression is influenced by metabolic perturbation in exercising human skeletal muscle. J Appl Physiol 96, 189–194.
Ostrowski K, Rohde T, Zacho M, Asp S & Pedersen BK (1998). Evidence that interleukin-6 is produced in human skeletal muscle during prolonged running. J Physiol 508, 949–953.
Parsons SA, Wilkins BJ, Bueno OF & Molkentin JD (2003). Altered skeletal muscle phenotypes in calcineurin Aalpha and Abeta gene-targeted mice. Mol Cell Biol 23, 4331–4343.
, http://www.100md.com
Peinnequin A, Mouret C, Birot O, Alonso A, Mathieu J, Clarencon D, Agay D, Chancerelle Y & Multon E (2004). Rat pro-inflammatory cytokine and cytokine related mRNA quantification by real-time polymerase chain reaction using SYBR green. BMC Immunol 5, 3.
Penkowa M, Keller C, Keller P, Jauffred S & Pedersen BK (2003). Immunohistochemical detection of interleukin-6 in human skeletal muscle fibers following exercise. FASEB J 17, 2166–2168.
, http://www.100md.com
Starkie RL, Angus DJ, Rolland J, Hargreaves M & Febbraio MA (2000). Effect of prolonged, submaximal exercise and carbohydrate ingestion on monocyte intracellular cytokine production in humans. J Physiol 528, 647–655.
Steensberg A, Febbraio MA, Osada T, Schjerling P, Van Hall G, Saltin B & Pedersen BK (2001). Interleukin-6 production in contracting human skeletal muscle is influenced by pre-exercise muscle glycogen content. J Physiol 537, 633–639.
, 百拇医药
Steensberg A, Van Hall G, Osada T, Sacchetti M, Saltin B & Pedersen BK (2000). 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.
Sterpetti AV, Cucina A, Morena AR, Di Donna S, D'Angelo LS, Cavalarro A & Tipa S (1993). Shear stress increases the release of interleukin-1 and interleukin-6 by aortic endothelial cells. Surgery 114, 911–914.
, http://www.100md.com
Ullum H, Haahr PM, Diamant M, Palmo J, Halkjaer-Kristensen J & Pedersen BK (1994). Bicycle exercise enhances plasma IL-6 but does not change IL-1 alpha, IL-1 beta, IL-6, or TNF-alpha pre-mRNA in BMNC. J Appl Physiol 77, 93–97.
Yang J, Rothermel B, Vega RB, Frey N, McKinsey TA, Olson EN, Bassel-Duby R & Williams RS (2000). Independent signals control expression of the calcineurin inhibitory proteins MCIP1 and MCIP2 in striated muscles. Circ Res 87, E61–E68., 百拇医药(Sebastien Banzet, Nathali)