Interleukin-6 modifies mRNA expression in mouse skeletal muscle

Acta Physiol 2011, 202, 165–173

Interleukin-6 modifies mRNA expression in mouse skeletal muscle H. Adser,1,2,3 J. F. P. Wojtaszewski,2,4 A. H. Jakobsen,1,2,3 K. Kiilerich,1,2,3 J. Hidalgo5 and H. Pilegaard1,2,3 1 2 3 4 5

Centre of Inflammation and Metabolism, Copenhagen, Denmark Copenhagen Muscle Research Centre, Copenhagen, Denmark Department of Biology, University of Copenhagen, Copenhagen, Denmark Department of Sport Sciences, University of Copenhagen, Copenhagen, Denmark Institute of Neurosciences and Department of Cellular Biology, Physiology and Immunology, Autonomous University of Barcelona, Barcelona, Spain

Received 29 October 2010, revision requested 16 December 2010, revision received 20 January 2011, accepted 17 February 2011 Correspondence: H. Pilegaard, August Krogh Building, Universitetsparken 13, 2100 Copenhagen Ø, Denmark. E-mail: [email protected]

Abstract Aim: The aim of this study was to test the hypothesis that interleukin (IL)-6 plays a role in exercise-induced peroxisome proliferator-activated receptor c co-activator (PGC)-1a and tumor necrosis factor (TNF)-a mRNA responses in skeletal muscle and to examine the potential IL-6-mediated AMP-activated protein kinase (AMPK) regulation in these responses. Methods: Whole body IL-6 knockout (KO) and wildtype (WT) male mice (4 months of age) performed 1 h treadmill exercise. White gastrocnemius (WG) and quadriceps (Quad) muscles were removed immediately (0¢) or 4 h after exercise and from mice not run acutely. Results: Acute exercise reduced only in WT muscle glycogen concentration to 55 and 35% (P < 0.05) of resting level in Quad and WG respectively. While AMPK and Acetyl CoA carboxylase (ACC) phosphorylation increased 1.3-fold (P < 0.05) in WG and twofold in Quad immediately after exercise in WT mice, no change was detected in WG in IL-6 KO mice. The PGC-1a mRNA content was in resting WG 1.8-fold higher (P < 0.05) in WT mice than in IL-6 KO mice. Exercise induced a delayed PGC-1a mRNA increase in Quad in IL-6 KO mice (12-fold at 4 h) relative to WT mice (fivefold at 0¢). The TNF-a mRNA content was in resting Quad twofold higher (P < 0.05) in IL-6 KO than in WT, and WG TNF-a mRNA increased twofold (P < 0.05) immediately after exercise only in IL-6 KO. Conclusion: In conclusion, IL-6 affects exercise-induced glycogen use, AMPK signalling and TNF-a mRNA responses in mouse skeletal muscle. Keywords AMP activated protein kinase, exercise, interleukin-6, mRNA, skeletal muscle, tumor necrosis factor a.

A physically inactive lifestyle is associated with increased risk of metabolically related diseases like type 2 diabetes (Chakravarthy & Booth 2004), but the mechanisms behind the beneficial effect of regular physical activity are still not fully elucidated. However, gene responses in skeletal muscle to each single exercise

bout are at least one likely contributing event (Chakravarthy & Booth 2004). Such exercise-induced gene responses vary in magnitude and timing of the responses in accordance with the function of the protein that the gene encodes (Pilegaard et al. 2000, 2002, Keller et al. 2001). Thus, the mRNA of genes

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encoding mitochondrial enzymes is first upregulated several hours into recovery (Leick et al. 2010). This is in line with the suggestion that the cumulative effects of transient gene responses to each single exercise session potentially can be the basic underlying mechanism behind increased mitochondrial biogenesis with exercise training (Williams & Neufer 1996; Pilegaard et al. 2000). The transcriptional co-activator peroxisome proliferator-activated receptor c co-activator (PGC)-1a has been identified as a master regulator of mitochondrial biogenesis (Lin et al. 2005). PGC-1a transcription and mRNA expression is upregulated in human and mouse skeletal muscle during the initial hours after exercise (Pilegaard et al. 2003, Jorgensen et al. 2005, Leick et al. 2008) potentially leading to a transient increased PGC-1a protein expression in response to exercise. Moreover, AMP-activated protein kinase (AMPK) activity is upregulated in human and mouse skeletal muscles in response to a single exercise bout (Wojtaszewski et al. 2002, Jorgensen et al. 2005), and repeated activation of AMPK by AICAR treatment leads to mitochondrial biogenesis in mouse skeletal muscle (Jorgensen et al. 2007). In addition, AMPK has been shown to phosphorylate and activate PGC-1a in vitro (Jager et al. 2007) and AICAR treatment has been shown to increase PGC-1a mRNA expression in mouse skeletal muscle (Jorgensen et al. 2005). Thus, an exercise-induced PGC-1a expression and/or activation during the initial hours after exercise may lead to the later activation of mitochondrial enzyme genes. The factors initiating exercise-induced PGC-1a gene regulation and AMPK activation in skeletal muscle have not been fully clarified. However, IL-6 may be a likely candidate. IL-6 transcription and mRNA expression is markedly upregulated in human skeletal muscle already during exercise (Keller et al. 2001), and seems to signal through both autocrine, paracrine and hormonal mechanisms (Pedersen et al. 2001, Pedersen & Febbraio 2005). Because a previous study (Kelly et al. 2004) suggests that IL-6 regulates AMPK phosphorylation at rest, IL-6 may influence basal expression as well as exercise-induced expression of genes encoding proteins in metabolic regulation. Type 2 diabetes is associated with chronic low-grade inflammation with elevated circulating levels of cytokines like tumor necrosis factor (TNF)-a. TNF-a has been shown to lower glucose uptake in obese rats (Hotamisligil et al. 1993) and induce insulin resistance when infused in humans (Plomgaard et al. 2005). Because IL-6 infusion in humans reduces the LPSinduced increase in plasma TNF-a (Starkie et al. 2003), IL-6 has been suggested to inhibit TNF-a expression (Pedersen & Febbraio 2005) and exercise-induced IL-6 expression may mediate an inhibitory effect on TNF-a

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expression in skeletal muscle. Thus, a potential beneficial effect of exercise may be an IL-6-mediated reduction in TNF-a expression and a concomitant avoidance of a low-grade inflammation state. The aim of this study was therefore to test the hypothesis that IL-6 regulates basal and exerciseinduced PGC-1a and TNF-a mRNA expression in skeletal muscle and to further explore the potential role of IL-6 in exercise-induced AMPK regulation.

Materials and methods Mice C57 black wildtype (WT) and whole body IL-6 knockout (KO) male mice were used. The IL-6 KO strain has previously been described (Wallenius et al. 2002, Di Gregorio et al. 2004), including the demonstration that the IL-6 KO mice do not produce functional IL-6 protein (Kopf et al. 1994). All mice were kept at 11 : 13 h light and dark cycle and received standard rodent chow (Altromin nr. 1324; Chr. Pedersen, Ringsted, Denmark) and water ad libitum. A total of 24 WT and 24 IL-6 KO mice were used at the age of 4 months.

Adaptation Exercise was performed on a treadmill (Model Exer-4 Treadmill, Columbus Instrument; Columbus, OH, USA) Before the experiment day, all mice were adapted to the treadmill by running 10 min on three consecutive days with a slope of 10% and starting at a velocity of 9.6 m min)1 on the first day and ending at 15 m min)1 on the third day.

Acute exercise After adaptation, the mice rested for 36 h and 16 WT and 16 IL-6 KO mice were then exposed to 1 h of treadmill running at a speed of 15.5 m min)1 and a slope of 10%. The mice were killed by cervical dislocation either immediately after exercise (0¢) or at 4 h of recovery from exercise. In addition, a group of WT and IL-6 KO mice (Rest) did not perform the acute exercise bout and were killed at the same time points as the exercise mice. White gastrocnemius (WG) and quadriceps (Quad) muscles as well as the liver were quickly removed and immediately frozen in liquid nitrogen and kept at )80 C. Quad muscles were crushed in liquid nitrogen to ensure homogeneity of the sample while pieces of WG were cut off. Quad and WG were used for glycogen, mRNA and protein determinations, whereas the liver was only used for glycogen.

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RNA isolation. RNA isolation was performed on approx. 20 mg of muscle tissue using a guanidinium thiocyanate-phenol-choloroform method modified from Chomczynski & Sacchi (1987), as described previously (Pilegaard et al. 2000). Reverse transcription (RT) was performed using the superscript II RNase H-system (Invitrogen, Carlsbad, CA, USA) as previously described (Pilegaard et al. 2000), and diluted in nuclease-free H2O.

5¢-GCTTAATTACACATGTTCTCTGGGAAA-3¢ 5¢-ATGGCCCAGACCCTCACA-3¢ 5¢-AGCCAAACCAACAACTTTATCTCTTC-3¢ IL-6 TNF-a PGC-1a

Reverse primer Forward primer Gene

Table 1 Primers and TaqMan probes for real-time PCR

SDS-Page and Western blotting. Pieces of WG and Quad were homogenized using a polytron (PT 1200 and PT 3100, Kinematica AG, Litauen) and rotated ‘endover-end’ for 1 h at 4 C. Afterwards, the samples were centrifuged at 17 500 g, 4 C, for 30 min and the supernatant (the lysate) was transferred to new tubes and stored at )80 C. The muscle lysate protein content was determined using the bicinchonimic acid method (Thermo Scientific, Rockford., IL, USA). Equal amount of muscle lysat protein was separated using a 7.5 or 15% polyacrylamide gel (Bio-Rad, Sundbyberg, Sweden) and transferred (semi-dry) to polyvinylidene fluoride (PVDF) membranes (Immobilion Transfer Membrane; Millipore Copenhagen, Denmark). After blocking in skin milk (2%) overnight, the membranes were incubated with primary antibody against either a-AMPK Thr172

TaqMan probe

Real-time PCR. The mRNA content of selected genes was determined by real-time PCR (ABI prism 7900 Sequence Detection System; Applied Biosystems, Foster City, CA, USA). Primers and Taqman probes were designed from mouse-specific sequence databases (Entrez-NIH and Ensembl; Sanger Institute) using computer software (Primer Express; Applied Biosystems) and are given in Table 1. For each of the genes, a Blast Search revealed that sequence homology was obtained only for the target mRNA. All TaqMan probes were 5¢6-carboxyfluorescein (FAM) and 3¢6-carboxyN,N,N¢,N¢-tetramethylrhodamine (TAMRA) labelled. Primers and Taqman probes were optimized as previously described (Pilegaard et al. 2003). Samples were run in triplicates in a 10 ll reaction volume as previously described (Lundby et al. 2005). A serial dilution of a pooled RT sample was run together with the samples on each plate and used to construct a standard curve from which the cycle threshold (Ct) of each unknown sample was converted to a relative amount. The target mRNA content was normalized to the content of single-stranded cDNA measured by Oligreen Reagent (Molecular Probe, Leiden, the Netherlands) as previously described (Lundby et al. 2005).

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IL, interleukin; TNF, tumor necrosis factor; PGC, peroxisome proliferator-activated receptor c co-activator.

Analysis

5¢-ATCAGAATTGCCATTGCACAACTCTTTTCTCAT-3¢ 5¢-TCAGATCATCTTCTCAAAATTCGAGTGACAAGC-3¢ 5¢-AGAGTCACCAAATGACCCCAAGGGTTCC-3¢

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5¢-CAAGTGCATCATCGTTGTTCATAC-3¢ 5¢-TTGCTACGACGTGGGCTACA-3¢ 5¢-TTAAGGTTCGCTCAATAGTCTTGTTC-3¢

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phosphorylation (#2531; Cell Signaling Technology Boston, MA, USA) or Acetyl CoA carboxylase (ACC) Ser79 phosphorylation (Upstate Biotechnology, Waldham, MA) followed by incubation with horseradish peroxidase conjugated secondary antibodies (DakoCytomation, Glostrup, Denmark). Finally, the bands were visualized and quantified using a CCD-image sensor and 1D software (Kodak Image Station, 2000MM; Kodak, Denmark). Phosphorylation is expressed in arbitrary units relative to a standard sample run on each gel.

Glycogen WG, Quad and liver glycogen content was determined as glycosyl units after acid hydrolysis using an automatic spectrophotometer (Cobas FARA 2; Roche Diagnostic, Basel, Switzerland) as previously described (Lowry & Passonneau 1971).

WT mice. Four hours after exercise, the liver glycogen level had returned to resting level (Fig. 1c).

Content of mRNA The level of IL-6 mRNA in Quad was at both time points after exercise two- to threefold higher (P < 0.05) than at rest in both the WT and KO mice. In addition, at the time points after exercise, the IL-6 mRNA content was more than twofold higher (P < 0.05) in KO than WT mice (Fig. 2a). WG IL-6 mRNA content increased twofold immediately after exercise (P < 0.05) in IL-6 KO mice. The IL-6 mRNA content in WG was 2.5- to 6-fold higher (P < 0.05) in the IL-6 KO mice than in WT (Fig. 2b). There was no significant effect of exercise on TNF-a mRNA content in Quad in either WT or IL-6 KO mice.

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In WT mice, the muscle glycogen content was reduced (P < 0.05) immediately after exercise to approx. 55% and approx. 35% of the resting WG and Quad level in respectively. Four hours after exercise, the glycogen content had returned to the same level as before exercise in the WT mice. There was, however, no difference in the level of muscle glycogen before and immediately after exercise in the IL-6 KO mice (Fig. 1a,b). WG muscle glycogen content was 20% higher (P < 0.05) and Quad muscle glycogen was 35% higher (P < 0.05) in WT than IL-6 KO at rest and tended to be 35% higher (0.05 £ P < 0.1) in KO mice than WT immediately after exercise (Fig. 1a,b). The exercise bout reduced (P < 0.05) liver glycogen content similarly in the two genotypes to 15 and 30% of the resting level in WT and IL-6 KO mice respectively. However, the liver glycogen content immediately after exercise was fourfold higher (P < 0.05) in IL-6 KO than

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All values are presented as means  standard error. Two-way anova was used to test the effect of genotype and exercise on mRNA content, phosphorylation and glycogen content. In addition, one-way anova was used to test the effect of exercise separately within each genotype. A Student–Newman–Keuls post hoc test was used to locate differences. Student’s t-test was used for testing genotype differences in resting animals. Differences are considered statistically significant at P < 0.05 and a tendency for significance is reported for 0.05 £ P < 0.1.

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Figure 1 Glycogen content (mmol kg)1) in quadriceps (a), white gastrocnemius (WG) (b) and liver (c) of wildtype (WT) mice and whole body interleukin (IL)-6 knockout (KO) mice at rest, immediately after 1 h of exercise (0¢) or at 4 h (4h) of recovery. Values are means  SE, n = 8. *Significantly different from Rest within given genotype, P < 0.05; #Significantly different from WT at given time point, P < 0.05; §Significant difference between genotypes when tested by t-test, P < 0.05.

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The TNF-a mRNA content was, however, twofold higher (P < 0.05) in IL-6 KO mice than WT at rest (Fig. 3a). In WT mice there was no change in WG TNF-a mRNA content in response to exercise, but in IL-6 KO mice WG TNF-a mRNA content increased (P < 0.05) approx. twofold immediately after exercise resulting in a genotype difference with twofold higher (P < 0.05) TNF-a mRNA content in IL-6 KO than in the WT mice in WG (Fig. 3b). Exercise increased (P < 0.05) Quad PGC-1a mRNA content in both WT and IL-6 KO mice. The PGC-1a mRNA level peaked in WT mice immediately after exercise with a fourfold induction (P < 0.05) and in KO mice 4 h after exercise with a 12-fold increase (P < 0.05) relative to at rest. While the PGC-1a mRNA level in Quad was higher (P < 0.05) in WT than in IL-6 KO immediately after exercise, the PGC-1a mRNA content was at 4 h of recovery higher (P < 0.05) in IL-6 KO than in WT (Fig. 4a). Exercise increased (P < 0.05) WG PGC-1a mRNA content twofold in WT and three fold in IL-6 KO mice at 4h of recovery relative to resting mice. The PGC-1a mRNA content was at rest approx. two fold higher (P < 0.05) in WT than IL-KO mice (Fig. 4b).

1.0

In WT mice, Quad AMPK and ACC phosphorylation increased (P < 0.05) two and 1.4-fold, respectively, immediately after exercise. The AMPK phosphorylation level was back at resting level at 4 h of recovery, while ACC was reduced (P < 0.05) to approx. 50% of the resting level at 4 h of recovery. Two- and one-way anova revealed no significant effect of exercise on AMPK and ACC phosphorylation in Quad in the IL-6 KO (Fig. 5a,b and 6). In WG, phosphorylation of AMPK and ACC increased (P < 0.05) approx. 1.5 times immediately after exercise only in WT mice. In WT, the AMPK phosphorylation was back to resting level 4 h after exercise (P < 0.05), while ACC phosphorylation was reduced to 50% of the resting level at 4 h of recovery. In IL-6 KO mice, exercise had no effect on AMPK and ACC phosphorylation in WG. WG AMPK phosphorylation was approx. 1.3-fold higher (P < 0.05) in KO than in WT at 4 h of recovery, while ACC phosphorylation in WG was approx.1.4-fold higher (P < 0.05) in WT than in IL-6 KO mice immediately after exercise (Fig. 5c,d and 6).

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Figure 2 Interleukin (IL)-6 mRNA content in quadriceps (a) and white gastrocnemius (WG) (b) of wildtype (WT) mice and whole body interleukin (IL)-6 knockout (KO) mice at Rest, immediately after 1 h of exercise (0¢) or at 4 h (4h) of recovery. The IL-6 mRNA content is normalized to the single standard DNA content in the samples. Values are means  SE, n = 8. *Significantly different from Rest within given genotype, P < 0.05; #Significantly different from WT at given time point, P < 0.05.

Figure 3 Tumor Necrosis factor (TNF)-a mRNA content in quadriceps (a) and white gastrocnemius (WG) (b) in wildtype (WT) mice and whole body interleukin (IL)-6 knockout (KO) mice at Rest, immediately after 1 h of exercise (0¢) or at 4 h of recovery (4h). The TNF-a mRNA content is normalized to the single standard DNA content in the samples. Values are means  SE, n = 8. *Significantly different from Rest within given genotype, P < 0.05; #Significantly different from WT at given time point, P < 0.05; §Significant difference between genotypes when tested by t-test, P < 0.05.

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Figure 4 Peroxisome proliferator-activated receptor c co-activator (PGC)-1a mRNA content in quadriceps (a) and white gastrocnemius (WG) (b) of wildtype (WT) mice and whole body interleukin (IL)-6 knockout (KO) mice at Rest, immediately after 1 h of exercise (0¢) or at 4 h (4h) of recovery. The PGC-1a mRNA content is normalized to the single standard DNA content in the samples. Values are means  SE, n = 8. #Significantly different from WT at given time point, P < 0.05; §Significant difference between genotypes when tested by t-test, P < 0.05.

Discussion The main findings of this study are that IL-6 seems to have an inhibitory effect on skeletal muscle TNF-a mRNA expression both at basal condition and after exercise, as well as influencing PGC-1a mRNA expression in skeletal muscle both at rest and after exercise. This study also supports that IL-6 is involved in eliciting exercise-induced AMPK signalling in skeletal muscle. Moreover, while liver glycogen is used similarly in WT and IL-6 KO mice in response to exercise, the IL-6 KO mice do not use muscle glycogen as WT mice during treadmill running. The present findings that TNF-a mRNA expression was elevated at basal state in quadriceps and increased by exercise in WG when functional IL-6 protein was absent indicate that IL-6 normally exerts an inhibitory effect on TNF-a expression in skeletal muscle. This suggestion is in accordance with a previous observation that IL-6 infusion blunted the LPS-induced increase in plasma TNF-a in humans (Starkie et al. 2003). As type 2 diabetes is associated with elevated circulating TNF-a levels (Andersen & Pedersen 2008), and TNF-a has been shown to reduce insulin-mediated glucose uptake (Plomgaard et al. 2005), mechanisms to reduce TNF-a

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expression are thought to be beneficial for the health of the individual (Petersen & Pedersen 2005). The present results thus suggest that IL-6 mediates such a favourable anti-inflammatory effect in skeletal muscle both at rest and during exercise. The different dependency of IL-6 on TNF-a mRNA expression in the various muscles in the present study indicates a muscle type specific effect, which may be related to fibre type specific expression of IL6, TNF-a and/or the IL6 receptor (Hiscock et al. 2004, Plomgaard et al. 2005). The observed exercise-induced upregulation of PGC1a mRNA in WT and IL-6 KO mice in the present study is in line with previous findings in mice subjected to a similar exercise protocol (Leick et al. 2008). But the delayed response in quadriceps in IL-6 KO mice relative to WT mice indicates that IL-6 influences the exerciseinduced regulation of the PGC-1a gene, although it should be noted that the role of IL-6 in PGC-1a mRNA regulation is not mandatory. In addition, the finding of lower basal PGC-1a mRNA levels in IL-6 KO WG further underlines that IL-6 appears to exert an impact on PGC1a mRNA expression and thereby potentially on PGC1a-mediated regulation. PGC-1a has been suggested to be important in regulating exercise-induced responses of genes encoding proteins in oxidative metabolism and training-induced expression of oxidative proteins when exercise is repeated (Pilegaard et al. 2003; Leick et al. 2010, Joseph et al. 2006). Thus, the present findings suggest that IL-6 may modify exercise-induced responses of metabolic genes and concomitantly influence traininginduced increases in oxidative capacity in skeletal muscle through effects on PGC-1a expression. AMP-activated protein kinase signalling is important in regulating metabolism in skeletal muscle during and after exercise (Winder & Hardie 1996, Park et al. 2002), and AMPK has been suggested to regulate gene expression in response to exercise (Jorgensen et al. 2006). In addition, AMPK has been shown to phosphorylate and activate PGC-1a in vitro (Jager et al. 2007). IL-6 mediated regulation of AMPK signalling in response to exercise therefore provides a potential link between IL-6 and a range of cellular and molecular events. In accordance, a previous study using IL-6 KO mice and isolated rat muscle (Kelly et al. 2004) provided evidence that IL-6 regulates AMPK phosphorylation. The present findings that exercise-induced AMPK and ACC phosphorylation was blunted in WG and quadriceps in IL-6 KO mice are thus in agreement with a role of IL-6 in AMPK regulation. The present results reveal no difference between genotypes in AMPK phosphorylation at rest, but differences in exercise inductions. However, Kelly et al. (2004) reported that AMPK phosphorylation was markedly reduced in KO muscles relative to WT both at rest and immediately after exercise and that the fold change in response to

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Figure 5 AMP-activated protein kinase (AMPK; a, c) and Acetyl CoA carboxylase (ACC; b, d) phosphorylation in quadriceps (a, b) and white gastrocnemius (WG; c, d) of wildtype (WT) mice and interleukin (IL)-6 knockout (KO) mice at Rest, immediately after 1 h of exercise (0¢) or at 4 h (4h) of recovery. Values are means  SE, n = 8. *Significantly different from Rest within given genotype, P < 0.05; #Significantly different from WT at given time point, P < 0.05.

exercise seemed higher in IL-6 KO than in WT mice. The reason for the observed differences in the two studies is not known, but may be attributable to the use of different muscles and/or exercise protocols, i.e. treadmill in the present and swimming in the previous study (Kelly et al. 2004). The observation that the effect of the IL-6 knockout on exercise-induced ACC phosphorylation was associated with an IL-6 effect on TNF-a mRNA content immediately after exercise may indicate that AMPK potentially could mediate this IL-6-regulated TNF-a effect. However, such Quadriceps 63 kDa

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Figure 6 Representative Western blots of AMPK and AcetylCoA carboxylase (ACC) phosphorylation (pAMPK and pACC) in quadriceps and white gastrocnemius (WG) of wildtype (WT) and whole body interleukin (IL)-6 knockout (KO) mice at rest, immediately after 1 h of exercise (0¢) or at 4 h (4h) of recovery.

an association does not appear apparent for the IL-6mediated modification of the PGC-1a mRNA response suggesting that these changes may not be through AMPK. Measurements of muscle glycogen are often used to ensure similar recruitment of the implicated muscles during exercise making comparisons between groups reasonable. Therefore, the present finding that the acute exercise bout only reduced muscle glycogen in the muscles of WT mice could indicate that the muscles of the IL-6 KO mice were not recruited during the exercise bout. Although a previous study (Faldt et al. 2004) has reported reduced endurance capacity of IL-6 KO mice, the IL-6 KO mice in the present study did complete the exercise bout and despite this, the glycogen content was not reduced in WG or in quadriceps. In support of the notion that the IL-6 KO mice did indeed exercise as the WT mice is also the observation that liver glycogen was used similarly in the two genotypes. In addition, the clear inductions of PGC-1a mRNA in quadriceps and WG in the IL-6 KO mice in the present study support that the IL-6 KO muscles were indeed recruited during the exercise. Thus, the lack of muscle glycogen utilization in the IL-6 KO muscles seems instead to reflect that the IL-6 KO muscles used other substrates than muscle glycogen during the exercise, and that IL-6 therefore appears to influence substrate utilization in skeletal muscle during exercise. This possibility is supported by a recent study by Kelly et al. (2009), where incubation of rat EDL with IL-6 reduced the glycogen content in the muscle. Of note is that differences in muscle glycogen use may play a role in the IL-6–AMPK observed relationship.

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Hence, muscle glycogen levels have previously been shown to increase the activation of AMPK during exercise (Wojtaszewski et al. 2003). Moreover, the lack of muscle glycogen use in the IL-6 KO muscles was associated with no or less clear exercise-induced AMPK phosphorylation. Therefore, it is possible that the link between IL-6 and AMPK in the present study was through muscle glycogen levels. But whether IL-6 exerts a direct effect on AMPK phosphorylation or whether IL-6 may mediate this effect via modification of muscle glycogen utilization cannot be answered from the present data and remains to be determined. Because IL-6 is knocked out by inserting a neor cassette (Kopf et al. 1994), IL-6 mRNA is still made in the IL-6 KO mice and can be detected by the use of primers placed outside the inserted cassette. The markedly elevated IL-6 mRNA levels in both muscles at all time points in IL-6 KO mice show that the lack of functional IL-6 protein is sensed by the cell and a feedback mechanism elicited as previously described by Van Wagoner et al. (1999). Although IL-6 mRNA has been shown to be markedly increased in human skeletal muscle during prolonged exercise and to return quickly back to resting levels after exercise (Keller et al. 2001), IL-6 mRNA was in WT mice in the present study only increased immediately after exercise in quadriceps. These findings indicate that the effects of IL-6 on AMPK signalling and mRNA responses immediately after exercise may at least in WG be exerted by already existing IL-6 protein or by IL-6 derived from other muscles/tissues. In summary, this study provides insight into the function of IL-6 before and after exercise. First of all, the present findings show that TNF-a mRNA increases in skeletal muscle at basal conditions and after exercise when functional IL-6 protein is lacking, which supports that IL-6 is involved in keeping inflammation at a low level. The change in the exercise-induced PGC1a mRNA response, when IL-6 protein is absent, indicates that IL-6 plays a role in the regulation of PGC-1a expression and thus potentially regulation of oxidative enzymes with exercise training. In addition, the present findings support a role of IL-6 in AMPK regulation in skeletal muscle. Finally the observation that IL-6 KO mice did not use muscle glycogen during exercise suggests that IL-6 influences substrate utilization.

Conflicts of interest None. This work was supported by grants from The Novo Nordisk Foundation, Denmark, The Danish Medical Research Council, Denmark and from The European Commission FP6 Integrated

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Acta Physiol 2011, 202, 165–173 Project Exgenesis (ref. LSHM-CT-2004-005272). The Centre of Inflammation and Metabolism (CIM) is supported by a grant from the Danish National Research Foundation (02-51255). The Copenhagen Muscle Research Centre is supported by a grant from the Capital Region of Denmark. CIM and Molecular and Physiology group, Department of Sports Exercise and Sciences are part of the UNIK Project: Food, Fitness & Pharma for Health and Disease, supported by the Danish Ministry of Science, Technology and Innovation. The authors acknowledge D.G. Hardie University of Dundee, UK, for kindly donating the AMPKa1 antibody, and Horst Bluethmann for generously providing the breeding pairs.

References Andersen, K. & Pedersen, B.K. 2008. The role of inflammation in vascular insulin resistance with focus on IL-6. Horm Metab Res 40, 635–639. Chakravarthy, M.V. & Booth, F.W. 2004. Eating, exercise, and ‘‘thrifty’’ genotypes: connecting the dots toward an evolutionary understanding of modern chronic diseases. J Appl Physiol 96, 3–10. Chomczynski, P. & Sacchi, N. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Anal Biochem 162, 156–159. Di Gregorio, G.B., Hensley, L., Lu, T., Ranganathan, G. & Kern, P.A. 2004. Lipid and carbohydrate metabolism in mice with a targeted mutation in the IL-6 gene: absence of development of age-related obesity. Am J Physiol Endocrinol Metab 287, E182–E187. Faldt, J., Wernstedt, I., Fitzgerald, S.M., Wallenius, K., Bergstrom, G. & Jansson, J.O. 2004. Reduced exercise endurance in interleukin-6-deficient mice. Endocrinology 145, 2680– 2686. Hiscock, N., Chan, M.H., Bisucci, T., Darby, I.A. & Febbraio, M.A. 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. Hotamisligil, G.S., Shargill, N.S. & Spiegelman, B.M. 1993. Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance. Science 259, 87–91. Jager, S., Handschin, C., St-Pierre, J. & Spiegelman, B.M. 2007. AMP-activated protein kinase (AMPK) action in skeletal muscle via direct phosphorylation of PGC-1alpha. Proc Natl Acad Sci USA 104, 12017–12022. Jorgensen, S.B., Wojtaszewski, J.F., Viollet, B., Andreelli, F., Birk, J.B., Hellsten, Y., Schjerling, P., Vaulont, S., Neufer, P.D., Richter, E.A. & Pilegaard, H 2005. Effects of alphaAMPK knockout on exercise-induced gene activation in mouse skeletal muscle. FASEB J 19, 1146–1148. Jorgensen, S.B., Richter, E.A. & Wojtaszewski, J.F. 2006. Role of AMPK in skeletal muscle metabolic regulation and adaptation in relation to exercise. J Physiol 574, 17–31. Jorgensen, S.B., Treebak, J.T., Viollet, B., Schjerling, P., Vaulont, S., Wojtaszewski, J.F. & Richter, E.A. 2007. Role of AMPKalpha2 in basal, training-, and AICAR-induced GLUT4, hexokinase II, and mitochondrial protein expression in mouse muscle. Am J Physiol Endocrinol Metab 292, E331–E339.

 2011 The Authors Acta Physiologica  2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02269.x

Acta Physiol 2011, 202, 165–173 Joseph, A.M., Pilegaard, H., Litvintsev, A., Leick, L. & Hood, D.A. 2006. Control of gene expression and mitochondrial biogenesis in the muscular adaptation to endurance exercise. Essays Biochem 42, 13–29. Keller, C., Steensberg, A., Pilegaard, H., Osada, T., Saltin, B., Pedersen, B.K. & Neufer, P.D. 2001. Transcriptional activation of the IL-6 gene in human contracting skeletal muscle: influence of muscle glycogen content. FASEB J 15, 2748– 2750. Kelly, M., Keller, C., Avilucea, P.R., Keller, P., Luo, Z., Xiang, X., Giralt, M., Hidalgo, J., Saha, A.K., Pedersen, B.K. & Ruderman, N.B. 2004. AMPK activity is diminished in tissues of IL-6 knockout mice: the effect of exercise. Biochem Biophys Res Commun 320, 449–454. Kelly, M., Gauthier, M.S., Saha, A.K. & Ruderman, N.B. 2009. Activation of AMP-activated protein kinase by interleukin-6 in rat skeletal muscle: association with changes in cAMP, energy state, and endogenous fuel mobilization. Diabetes 58, 1953–1960. Kopf, M., Baumann, H., Freer, G., Freudenberg, M., Lamers, M., Kishimoto, T., Zinkernagel, R., Bluethmann, H. & Kohler, G. 1994. Impaired immune and acute-phase responses in interleukin-6-deficient mice. Nature 368, 339–342. Leick, L., Wojtaszewski, J.F., Johansen, S.T., Kiilerich, K., Comes, G., Hellsten, Y., Hidalgo, J. & Pilegaard, H. 2008. PGC-1alpha is not mandatory for exercise- and training-induced adaptive gene responses in mouse skeletal muscle. Am J Physiol Endocrinol Metab 294, E463–E474. Leick, L., Plomgaard, P., Grønløkke, L., Al-Abaiji, F., Wojtaszewski, J.F.P., Pilegaard, H. 2010. Endurance exercise induces mRNA of oxidative proteins in human skeletal muscle take in recovery. Scand J Med Sci Sports 20: 593–599. Lin, J., Handschin, C. & Spiegelman, B.M. 2005. Metabolic control through the PGC-1 family of transcription coactivators. Cell Metab 1, 361–370. Lowry, O.H. & Passonneau, J.V. 1971. Some recent refinements of quantitative histochemical analysis. Curr Probl Clin Biochem 3, 63–84. Lundby, C., Nordsborg, N., Kusuhara, K., Kristensen, K.M., Neufer, P.D. & Pilegaard, H. 2005. Gene expression in human skeletal muscle: alternative normalization method and effect of repeated biopsies. Eur J Appl Physiol 95, 351– 360. Park, H., Kaushik, V.K., Constant, S., Prentki, M., Przybytkowski, E., Ruderman, N.B. & Saha, A.K. 2002. Coordinate regulation of malonyl-CoA decarboxylase, sn-glycerol-3phosphate acyltransferase, and acetyl-CoA carboxylase by AMP-activated protein kinase in rat tissues in response to exercise. J Biol Chem 277, 32571–32577. Pedersen, B.K. & Febbraio, M. 2005. Muscle-derived interleukin-6 – a possible link between skeletal muscle, adipose tissue, liver, and brain. Brain Behav Immun 19, 371–376.

H Adser et al.

Æ IL-6-induced AMPK and mRNA responses

Pedersen, B.K., Steensberg, A. & Schjerling, P. 2001. Musclederived interleukin-6: possible biological effects. J Physiol 536, 329–337. Petersen, A.M. & Pedersen, B.K. 2005. The anti-inflammatory effect of exercise. J Appl Physiol 98, 1154–1162. Pilegaard, H., Ordway, G.A., Saltin, B. & Neufer, P.D. 2000. Transcriptional regulation of gene expression in human skeletal muscle during recovery from exercise. Am J Physiol Endocrinol Metab 279, E806–E814. Pilegaard, H., Keller, C., Steensberg, A., Helge, J.W., Pedersen, B.K., Saltin, B. & Neufer, P.D. 2002. Influence of preexercise muscle glycogen content on exercise-induced transcriptional regulation of metabolic genes. J Physiol 541, 261–271. Pilegaard, H., Saltin, B. & Neufer, P.D. 2003. Exercise induces transient transcriptional activation of the PGC-1alpha gene in human skeletal muscle. J Physiol 546, 851–858. Plomgaard, P., Bouzakri, K., Krogh-Madsen, R., Mittendorfer, B., Zierath, J.R. & Pedersen, B.K. 2005. Tumor necrosis factor-alpha induces skeletal muscle insulin resistance in healthy human subjects via inhibition of Akt substrate 160 phosphorylation. Diabetes 54, 2939–2945. Starkie, R., Ostrowski, S.R., Jauffred, S., Febbraio, M. & Pedersen, B.K. 2003. Exercise and IL-6 infusion inhibit endotoxin-induced TNF-alpha production in humans. FASEB J 17, 884–886. Van Wagoner, N.J., Oh, J.W., Repovic, P. & Benveniste, E.N. 1999. Interleukin-6 (IL-6) production by astrocytes: autocrine regulation by IL-6 and the soluble IL-6 receptor. J Neurosci 19, 5236–5244. Wallenius, V., Wallenius, K., Ahren, B., Rudling, M., Carlsten, H., Dickson, S.L., Ohlsson, C. & Jansson, J.O. 2002. Interleukin-6-deficient mice develop mature-onset obesity. Nat Med 8, 75–79. Williams, R.S., Neufer, P.D. 1996. Regulation of gene expression in skeletal muscle by contractile activity. In: Handbook of Physiology. Exercise: Regulation and Integration of multiple systems. Bethesda, MD: Am. Physiol. Soc. sect 12, chapt 25: 1124–1146. Winder, W.W. & Hardie, D.G. 1996. Inactivation of acetylCoA carboxylase and activation of AMP-activated protein kinase in muscle during exercise. Am J Physiol 270, E299– E304. Wojtaszewski, J.F., Mourtzakis, M., Hillig, T., Saltin, B. & Pilegaard, H. 2002. Dissociation of AMPK activity and ACCbeta phosphorylation in human muscle during prolonged exercise. Biochem Biophys Res Commun 298, 309–316. Wojtaszewski, J.F., MacDonald, C., Nielsen, J.N., Hellsten, Y., Hardie, D.G., Kemp, B.E., Kiens, B. & Richter, E.A. 2003. Regulation of 5¢AMP-activated protein kinase activity and substrate utilization in exercising human skeletal muscle. Am J Physiol Endocrinol Metab 284, E813–E822.

 2011 The Authors Acta Physiologica  2011 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2011.02269.x

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