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1 Copenhagen Muscle Research Centre, Denmark2 Department of Infectious Diseases, Rigshospitalet University Hospital of Copenhagen, Denmark3 Skeletal Muscle Research Group, School of Medical Sciences, Royal Melbourne Institute of Technology, Victoria, Australia4 Department of Medical Anatomy, The Panum Institute, University of Copenhagen, Denmark5 Clinical Nutrition Research, Department of Public Health and Caring Sciences/Geriatrics, University of Uppsala, Sweden6 Division of Clinical Chemistry, Karolinska University Hospital, Sweden7 Research and Quality Department, Semper Foods AB, Stockholm, Sweden
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Abstract |
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-tocopherol (400 i.u. day–1) (Treatment, n= 7), or placebo (Control, n= 7). After 28 days of supplementation, the subjects performed 3 h of dynamic two-legged knee-extensor exercise at 50% of their individual maximal power output. Muscle biopsies from vastus lateralis were obtained at rest (0 h), immediately post exercise (3 h) and after 3 h of recovery (6 h). Leg blood flow was measured using Doppler ultrasonography. Plasma IL-6 concentration was measured in blood sampled from the femoral artery and vein. The net release of IL-6 was calculated using Fick's principle. Plasma vitamin C and E concentrations were elevated in Treatment compared to Control. Plasma 8-iso-prostaglandin F2, a marker of lipid peroxidation, increased in response to exercise in Control, but not in Treatment. In both Control and Treatment, skeletal muscle IL-6 mRNA and protein levels increased between 0 and 3 h. In contrast, the net release of IL-6 from the leg, which increased during exercise with a peak at 3.5 h in Control, was completely blunted during exercise in Treatment. The arterial plasma IL-6 concentration from 3 to 4 h, when the arterial IL-6 levels peaked in both groups, was 
50% lower in the Treatment group compared to Control (Treatment versus Control: 7.9 pg ml–1, 95% confidence interval (CI) 6.0–10.7 pg ml–1, versus 19.7 pg ml–1, CI 13.8–29.4 pg ml–1, at 3.5 h, P < 0.05 between groups). Moreover, plasma interleukin-1 receptor antagonist (IL-1ra), C-reactive protein and cortisol levels all increased after the exercise in Control, but not in Treatment. In conclusion, our results show that supplementation with vitamins C and E attenuated the systemic IL-6 response to exercise primarily via inhibition of the IL-6 protein release from the contracting skeletal muscle per se.
(Received 20 April 2004;
accepted after revision 24 May 2004;
first published online 28 May 2004)
Corresponding author C. P. Fischer: Department of Infectious Diseases, Rigshospitalet University Hospital of Copenhagen, Denmark. Email: cfischer{at}rh.dk
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Introduction |
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Recently, a few studies have shown that oral supplementation with antioxidants can attenuate the exercise-induced increase of IL-6 in plasma (Thompson et al. 2001; Vassilakopoulos et al. 2003). Antioxidants, such as vitamin C (ascorbic acid) and vitamin E (tocopherol), have been shown to attenuate oxidative stress at rest (Huang et al. 2002) as well as in response to exercise (Kanter et al. 1993), the latter being a potent stimulus for increased formation of reactive oxygen species (ROS) in skeletal muscle (Davies et al. 1982).
Interestingly, murine myotubes have been shown to express IL-6 when exposed to oxidative stress (Kosmidou et al. 2002), possibly via activation of the redox-sensitive transcription factor nuclear factor kappa B (NF-
B) (Schreck et al. 1992). Thus, it is likely that supplementation with antioxidants attenuates the skeletal muscle IL-6 gene transcription during exercise. However, it has not been investigated whether antioxidants affect IL-6 synthesis within contracting human skeletal muscle per se. In the present study, we hypothesized that oral supplementation with vitamin C and vitamin E would inhibit the exercise-induced IL-6 response in human skeletal muscle. We further aimed to determine if the supplementation would inhibit the skeletal muscle IL-6 synthesis at the level of gene transcription, translation to protein or translocation from the tissue into the circulation.
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Methods |
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Fourteen young, healthy men participated as subjects in the study. All subjects were physically active non-athletes. Subject characteristics are listed in Table 1. A general medical examination in combination with blood test screening was performed before inclusion. Exclusion criteria were history of febrile illness within 3 weeks prior to the examination, indication of ongoing disease, recent vaccination, use of any medication, or use of antioxidants.
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Supplementation
A single-blind placebo-controlled design with randomization was used in order to acquire two equally sized groups (Treatment and Control). Subjects in the Treatment group (n= 7) received oral supplementation with vitamin C (ascorbic acid, 500 mg day–1) and vitamin E (RRR--tocopherol, 400 i.u. day–1), while subjects in the Control group (n= 7) received placebo with similar appearance. The supplementation was ingested once a day together with breakfast. The length of the supplementation period was 29 days. The duration of the supplementation was chosen from a previous study (Meydani et al. 1997), which showed that the plasma concentration of tocopherol reaches steady state after at least 14 days of oral supplementation with vitamin E.
Pre-experimental protocol
Three weeks prior to the exercise experiment, the individual maximal power output of the subjects was determined during two-legged dynamic knee-extensor exercise. The test was carried out using a modified ergometer previously described (Andersen et al. 1985). In brief, the subjects performed a 5 min warm-up session consisting of 60 knee-extensions with passive repositions per minute at 60 W after becoming accustomed to the exercise model. This was followed by an incremental increase of the workload by 10 W every 2 min until volitional exhaustion. The highest workload that was maintained for at least 1 min was defined as the maximal power output, Pmax.
Experimental protocol
On the 28th day of the supplementation period the subjects reported to the laboratory at 08.00 h. Subjects had fasted overnight, and were asked to consume the supplement at 07.00 h. Furthermore, the subjects were instructed to refrain from exercise for at least 24 h prior to the experiment. Upon arrival, subjects changed into hospital attire and rested in a supine position. Under sterile conditions and with use of local anaesthesia (lidocaine (lignocaine), 20 mg ml–1; SAD, Copenhagen, Denmark), indwelling catheters (Arrow International, Reading, PA, USA) were placed in the femoral artery and vein of the right leg using the guidewire (Seldinger) technique. The femoral vein was cannulated 2 cm below the inguinal ligament, and an 18 gauge catheter was advanced 5 cm in the distal direction in order to minimize contamination with venous blood from the pelvic region and saphenous vein (Van Hall et al. 1999). The femoral artery was cannulated at the same level as the femoral vein, and a 20 gauge catheter was advanced 10 cm in the proximal direction.
Prior to the onset of exercise, subjects in both groups ingested two boiled eggs to ensure maximal uptake of the supplementation ingested earlier the same morning, while avoiding administration of carbohydrates, since intake of carbohydrates has been shown to attenuate the exercise-induced IL-6 response (Starkie et al. 2001).
The exercise bout consisted of 3 h of two-legged dynamic knee-extensor exercise at 60 extensions per minute and 50% of the individual Pmax.
After the exercise period, the subjects remained supine in the laboratory for a 3 h recovery period. Water was consumed ad libitum throughout the exercise and recovery period, but food was not permitted until the cessation of the recovery period. The following morning (the 29th day of supplementation) subjects reported at the laboratory for the final samples of the recovery period.
Blood flow
Blood flow of the femoral artery was measured using Doppler ultrasonography (model CFM 800, Vingmed Sound, Horten, Norway) as previously described (Radegran, 1997). The Doppler was equipped with an annular phased array transducer probe (diameter 11.5 mm, Vingmed Sound), which operated at an imaging frequency of 7.5 MHz and variable Doppler frequencies of 4.0–6.0 MHz (high-pulsed repetition frequency mode, 4–36 kHz). Both measurements of diameter and velocity were performed in triplicates at a level below the inguinal ligament, but above the bifurcation of the artery into the superficial and deep femoral branch. The femoral artery was visualized with a fixed perpendicular angle, and the diameter was determined along the central path of the ultrasound beam. The blood velocity was measured immediately prior to each blood sample.
Blood samples
Blood samples from the femoral vein and artery were drawn at rest (0 h), during exercise (0.5, 1, 2 and 3 h), and during recovery (3.5, 4, 5 and 6 h). The last blood sample (23 h) was obtained from an antecubital vein.
Plasma was obtained by drawing blood into pre-cooled EDTA-containing glass tubes, which were immediately centrifuged at 2200 g for 15 min at 4°C. Plasma for vitamin C analysis was stabilized with 5% metaphosphoric acid. The plasma was stored at –80°C until further analysis.
Haematocrit, haemoglobin concentration, plasma C-reactive protein concentration, and plasma creatine kinase activity
Systemic (arterial) blood samples were analysed for haematocrit, haemoglobin concentration, plasma C-reactive protein (CRP) concentration, as well as plasma creatine kinase (CK) activity using automated standard laboratory procedures.
Plasma ascorbic acid concentration
Ascorbic acid concentration was measured in plasma by a modified fluorometric method (Deutsch & Weeks, 1965; Vuilleumier & Keck, 1989). In brief, the assay included enzymatic oxidation of ascorbic acid with ascorbate oxidase to dehydroascorbic acid, which subsequently reacted with 1-ortho-phenylenediamine to the fluorescent compound quinoxaline, which was quantified using an excitation wavelength of 337 nm and an emission wavelength of 430 nm. The intra-assay coefficient of variation (CV) was 4% and inter-assay CV, 5%.
Plasma -tocopherol concentration
Plasma -tocopherol concentration was measured using HPLC with fluorescence detection (Ohrvall et al. 1993). In brief, 500 µl of plasma was extracted with 500 µl of ethanol containing 0.05 g l–1 of butylated hydroxytoluene and 2 ml of hexane. The supernatant (20 µl) was injected into an HPLC column (LiChrospher 100 NH2 250 mm x 4 mm) and fluorescence was measured with an excitation wavelength of 295 nm and an emission wavelength of 327 nm. Intra-assay CV was 4.5%.
Plasma 8-iso-prostaglandin F2 concentration
The concentration of the F2-isoprostane 8-iso-prostaglandin F2 (8-iso-PGF2) was determined by a radioimmunoassay (RIA) using an antibody raised in rabbits by immunization with 8-iso-PGF2 coupled to bovine serum albumin at the carboxylic acid by the 1,19-carbonyldiimmidazole method (Basu, 1998). Sensitivity was 23 pmol l–1 and intra-assay CV, 14.5%.
Plasma IL-6 and plasma IL-1ra concentrations
Plasma IL-6 concentration was measured using a high sensitivity ELISA kit (R & D, Minneapolis, MN, USA; code HS600), which detects total IL-6 independent of binding to soluble receptors, with a sensitivity of 0.094 pg ml–1, intra-assay CV, 11.1% and interassay CV, 16.5%.
The plasma concentration of IL-1ra was measured with an ELISA kit (R & D, code DRA00), with a sensitivity of 14 pg ml–1, intra-assay CV, 6.2% and inter-assay CV, 6.7%.
All samples were run in duplicates and the mean was calculated. On each ELISA plate, subjects from both groups, as well as all samples from each subject, were represented and analysed.
Plasma glucose
Systemic (arterial) plasma glucose concentration was measured using an automatic analyser (Cobas Fara, Roche, France).
Plasma cortisol concentration
Plasma cortisol concentration was measured using a RIA kit from Diagnostic Laboratories (Webster, TX, USA; code DSL-10-2000). Intra-assay CV was 5%.
Muscle biopsies
Muscle biopsies were obtained from the vastus lateralis using the percutaneous needle method (Bergström, Sweden) with suction (Bergstrom, 1962). Prior to each biopsy, local anaesthesia (lidocaine, 20 mg ml–1; SAD, Denmark) was applied to the skin and superficial fascia of the biopsy site. Time points for the biopsies were pre-exercise (0 h), immediately post exercise (3 h) and end of recovery (6 h). Visible connective tissue and blood contamination were carefully removed before the biopsies were frozen in liquid nitrogen and subsequently stored at –80°C until further analysis.
Skeletal muscle IL-6 mRNA expression
Total RNA was extracted from the muscle tissue using TRIzol according to the manufacturer's directions (Invitrogen, Grand Island, NY, USA). The resulting RNA pellet was dissolved in diethylpyrocarbonate (DEPC)-treated water. Reverse transcription reactions were performed on 1 µg of RNA using a RT kit (Applied Biosystems, Foster City, CA, USA) in a reaction volume of 50 µl.
Primers and probes were designed (Primer Express version 1.0, Applied Biosystems) from the gene sequences for human IL-6 (Starkie et al. 2001). An 81 base fragment was amplified using the forward primer 5'-GGTACATCCTCGACGGCATCT-3', the reverse primer 5'-GTGCCTCTTTGCTGCTTTCAC-3', and the TaqMan fluorescent probe, 5'-FAM-TGTTACTCTTGTTACATGTCTCCTTTCTCAGG-GCT-3' TAMRA (Applied Biosystems).
18S rRNA was used as reference. The TaqMan primers and probes for 18S rRNA were supplied in a control reagent kit (Applied Biosystems).
Gene expression of IL-6 was quantified using real-time PCR on the cDNA samples using a multiplex comparative critical threshold (CT) method (Starkie et al. 2001).
PCR reactions were carried out in 25 µl reactions of TaqMan universal PCR master mix (1 x), primer-limited 18S rRNA (1 x), 900 nM IL-6 forward primer, 300 nM IL-6 reverse primer, and 100 nM IL-6 TaqMan probe. Each reaction was made up to volume with RNase-free water. Fifty nanograms of cDNA and control preparations were amplified (ABI PRISM 7700 sequence detector, Applied Biosystems) following standard conditions using 50 cycles. All samples were performed in duplicates. Fold changes from resting conditions were calculated using the 2–
CT method (Starkie et al. 2001).
Skeletal muscle IL-6 protein expression and myofibrillar ATPase staining
Muscle tissue was cut in 6 µm consecutive sections on a cryostat, and the sections were immediately collected on glass slides, to be used for general histology and immunohistochemistry. Haematoxylin and eosin (HE) staining of the sections was performed according to standard procedures.
Myofibrillar ATPase staining with preincubations of pH 4.6 was used to identify muscle fibre types (Brooke & Kaiser, 1970).
For epitope retrieval, sections were preincubated overnight at 60°C in Tris-EGTA (TEG) buffer (1.211 g Tris, 0.95 g EGTA, 1 l distilled H2O) followed by incubation in 10% goat serum (In Vitro, Denmark; code 04009-1B) in TBS (TBS: 0.05 M Tris, pH 7.4, 0.15 M NaCl) with 0.01% Nonidet P-40 (TBS–Nonidet) for 30 min at room temperature. Sections were subsequently incubated overnight at 4° C with mouse anti-IL-6 diluted 1: 50 (Chemicon/Cymbus, USA; code CBL2117). The primary IL-6 antibody was detected using biotinylated goat anti-mouse IgG diluted 1: 200 (Sigma-Aldrich, USA; code B8774) for 30 min at room temperature followed by streptavidin–biotin–peroxidase complex (StreptABComplex/HRP, Dakopatts, Denmark; code K377) prepared at the manufacturer's recommended dilutions for 30 min at room temperature. Afterwards, sections were incubated with biotinylated tyramide and streptavidin–peroxidase complex (NEN, Life Science Products, USA; code NEL700A) prepared following the manufacturer's recommendations. The immunoreaction was visualized using 0.015% H2O2 in 3,3-di-aminobenzidine-tetrahydrochloride (DAB)–TBS for 10 min at room temperature.
In order to evaluate the extent of non-specific binding in the immunohistochemical experiments, control sections were incubated in the absence of primary antibody or in the blocking serum. Results were considered only if these controls were negative. For the simultaneous examination and recording of the stainings, a Zeiss Axioplan2 light microscope was used.
Calculations
All plasma concentrations were adjusted for possible changes in plasma volume according to the method described by Dill & Costill (1974). For each time point, the leg net IL-6 release was calculated as femoral venous [IL-6] minus arterial (systemic) [IL-6], multiplied by the femoral plasma flow (Fick's principle), since the amount of IL-6 bound to blood cells can be neglected (Castell et al. 1988). The plasma flow was calculated as blood flow x (1 – haematocrit). Finally, the cumulative net leg release of IL-6 was calculated as the area under the curve (AUC) of the net leg IL-6 release.
Statistics
All data were tested for normality of distribution before further statistical analysis. Plasma concentrations of ascorbic acid, -tocopherol, glucose and cortisol, as well as femoral blood flow, were normally distributed, while plasma concentrations of 8-iso-PGF2, IL-6 and CRP were normally distributed after log-transformation. Plasma IL-1ra and fold changes in skeletal muscle IL-6 mRNA expression were normally distributed after square root transformation. Leg net IL-6 release and cumulative IL-6 release were not normally distributed, and therefore presented as median ± interquartile range.
For normally distributed data a one-way repeated measures ANOVA was used to detect the effect of time, followed by a post hoc paired t test with Bonferroni correction to identify differences from the pre-exercise values at specific time points. A two-way repeated measures ANOVA was used to detect differences between groups, followed by a post hoc two-sample t test with Bonferroni correction to identify time-point-specific differences between groups.
For data that were not normally distributed, a non-parametric Friedman ANOVA on ranks was used to test for the effect of time. If a time effect was detected, a Wilcoxon signed ranks test was used to identify time-point-specific differences from pre-exercise values. Time-point-specific differences between groups were detected using the non-parametric Kruskal-Wallis test. In general, P < 0.05 was considered as significant, except when using the Friedman test, where P < 0.01 was set as the level of significance.
The statistical analysis was performed using a statistical software package (Systat version 8.0 for Windows, SPSS, Chicago, IL, USA).
Results in the text are presented as mean ±S.E.M. or geometric mean with 95% confidence interval (CI), unless stated otherwise.
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Results |
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The maximal power output (Table 1) obtained during the pre-experimental test, thus workload employed during the exercise trial, was similar when comparing Treatment and Control groups. Exercise induced a rapid increase in the femoral blood flow in both groups (Fig. 1), without a significant difference between groups.
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0.07 l W–1 in both groups, which is in accordance with previous results using the same mode of exercise (Radegran, 1997). A moderate increase in heart rate during exercise was observed in both groups (mean during exercise was 113 ± 4 and 122 ± 4 beats min–1 in Treatment and Control groups, respectively, no difference between groups). There was no detectable increase of plasma CK levels in either group during the experimental day (not shown).
Plasma vitamin concentrations
On day 28 of the supplementation period the two groups differed when comparing plasma ascorbic acid concentration (Table 1) both at rest and after exercise (P < 0.05). The plasma ascorbic acid concentration was 100% higher in the Treatment group compared to Control. The large difference in plasma ascorbic acid concentration was maintained to the end of the exercise bout, although a significant decrease in plasma ascorbic acid was observed in the Treatment group in response to exercise. In contrast, although a high dose of -tocopherol was provided to the Treatment group, the plasma -tocopherol concentration in the Treatment group was only 35% higher than the level in the Control group (P < 0.05, end of exercise).
Plasma lipid peroxidation
Exercise induced a 2.4-fold increase of plasma 8-iso-PGF2 (Fig. 2) from 0 to 3 h in Control (P < 0.05), while levels remained unchanged in response to exercise in Treatment.
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The skeletal muscle IL-6 mRNA (Fig. 3) increased 16-fold (CI 7–31, P < 0.05) from 0 to 3 h in Treatment, while levels increased 22-fold (CI 12–37, P < 0.05) in Control. There was no difference between groups. At 6 h the skeletal muscle IL-6 mRNA level had returned to pre-exercise values.
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Muscle fibres from resting subjects in both groups showed very low IL-6 protein expression (Fig. 4A and B). Following exercise IL-6 protein expression increased significantly in the muscle tissue of both groups (Fig. 4C and D). In general, the IL-6 protein staining was at least as intense in Treatment as in Control. The exercise-induced increase in IL-6 protein expression was consistent, even though a few individual muscle fibres showed altering staining intensity. This variation in IL-6 protein expression after exercise was clearly related to differences in muscle fibre types, as judged by comparing neighbouring muscle tissue sections stained for myofibrillar ATPase (Fig. 4E and F) and IL-6, respectively. According to this, type 1 muscle fibres showed the highest increases in IL-6 expression after exercise, whereas exercise-induced IL-6 expression was more irregular in type 2 muscle fibres.
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Plasma IL-6
The arterial (systemic) plasma [IL-6] (Fig. 5A) increased during exercise from 0 h in both Treatment and Control groups, with peak values at 4 h (Treatment: 12 pg ml–1, CI 8–22 pg ml–1, P < 0.05 versus 0 h; Control: 21 pg ml–1, CI 17–28 pg ml–1, P < 0.05 versus 0 h). However, the systemic plasma [IL-6] in Treatment was only 40% (P < 0.05 between groups) of the value in Control at 3.5 h, and 58% (not significant) of the value in Control at 4 h. The femoral venous plasma [IL-6] (Fig. 5B) showed a similar response to exercise, but with higher peak values at 4 h (Treatment: 20 pg ml–1, CI 12–37 pg ml–1, P < 0.05 versus 0 h; Control: 40 pg ml–1, CI 29–58 pg ml–1, P < 0.05 versus 0 h). The largest difference in femoral venous plasma [IL-6] between groups was observed at 3.5 h (P < 0.05 between groups). At rest, there was no net leg release of IL-6 (Fig. 5C) in either group. In Control, exercise induced a large net leg IL-6 release with peak at 3.5 h (9 ng min–1 leg–1, interquartile range 3–10 ng min–1 leg–1, P < 0.05 versus 0 h). A much lower net release of IL-6 was observed in Treatment at the same time point (1.8 ng min–1 leg–1, interquartile range 0.8–2.4 ng min–1 leg–1, P < 0.05 versus 0 h, P < 0.05 between groups). The cumulative net leg release of IL-6 (Fig. 5D) in Control at 6 h was approximately 6-fold higher than in the Treatment group (Treatment: 233 ng, interquartile range 157–581 ng; Control: 1420 ng, interquartile range 515–1585 ng, P < 0.05 between groups).
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There was no difference between groups in plasma [IL-1ra] at 0 h (Fig. 6). In Control only, there was an increase of plasma [IL-1ra] at 3 h and 6 h (P < 0.05 versus 0 h), while plasma [IL-1ra] remained unchanged from 0 h in Treatment (one-way ANOVA not significant).
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At rest (0 h), plasma [CRP] was below the detection limit (3 mg l–1) in all subjects in both groups. There was no increase from 0 h in plasma [CRP] at 3 h or 6 h in either Treatment or Control. However, although plasma [CRP] increased at 23 h in both groups, the plasma CRP level was markedly lower in Treatment compared to Control (4.1 mg l–1, CI 2.8–6.5 mg l–1, in Treatment, versus 12.5 mg l–1, CI 8.0–21.0 mg l–1, in Control, P < 0.05 between groups).
Plasma cortisol
At rest (0 h), there was no difference in plasma [cortisol] (Fig. 7) between groups. In Control, plasma [cortisol] increased during recovery (185 ± 17 ng ml–1 at 0 h versus 330 ± 35 ng ml–1 at 6 h, P < 0.05). In contrast, the plasma [cortisol] response to exercise was completely blunted in Treatment (177 ± 41 ng ml–1 at 0 h versus 197 ± 46 ng ml–1 at 6 h, one-way ANOVA not significant).
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There was no difference between groups in the arterial plasma glucose level (Table 1) at rest (0 h), immediately post exercise (3 h) or after 3 h of recovery (6 h). From 0 h the arterial blood glucose level decreased markedly at 3 h (P < 0.05) and 6 h (P < 0.05) in both groups.
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Discussion |
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The kinetics and magnitude of the circulating IL-6 response in the Control group is in accordance with other studies using the same mode of exercise (Steensberg et al. 2000; Keller et al. 2001). In addition, the supplementation with vitamins C and E reduced the systemic plasma IL-6 response by 50%, which is similar to the effect reported by Vassilakopoulos et al. (2003), who used an antioxidant cocktail consisting of vitamins A, C and E for 60 days, allopurinol for 15 days and N-acetylcysteine (NAC) for 3 days as well as a single dose of NAC immediately prior to 45 min of bicycling at 70% of the maximal oxygen consumption rate 
. Our results demonstrate that the attenuated systemic plasma IL-6 response to exercise is due to a remarkable decrease of the net leg IL-6 release from the contracting limb. Of note, five out of seven subjects in the Treatment group demonstrated a net leg IL-6 uptake during exercise instead of a release as expected a priori. On the other hand, exercise caused a marked increase of the skeletal muscle gene and protein expression of IL-6 in both groups. Of note, the staining for IL-6 protein in the skeletal muscle appeared at least just as intense or even more intense in muscle biopsy samples of the subjects in Treatment compared to Control. However, we could not determine whether the skeletal muscle IL-6 protein content was higher in Treatment than in Control, since the intensity of the stainings for IL-6 protein was not quantified. Nonetheless, our results indicate that translocation of IL-6 protein from the skeletal muscle tissue into the circulation is inhibited by supplementation with antioxidants, while IL-6 gene expression and subsequent translation into protein is much less affected. Thus, we find a different main effect of antioxidants in vivo compared to previous in vitro findings (Kosmidou et al. 2002). Interestingly, supplementation with carbohydrates (Starkie et al. 2001) also blunts the increase of plasma IL-6, but not the increase of skeletal muscle IL-6 mRNA, in response to exercise. We observed no differences in plasma glucose levels between groups at rest or in response to exercise. Thus, the effect of the antioxidants in the present study did not appear to be mediated indirectly by changes in plasma glucose availability.
It could be argued that an even longer duration of antioxidant supplementation prior to the exercise experiment would affect the exercise-induced skeletal muscle IL-6 gene expression, since the incorporation of tocopherol into the lipid bilayer is slow and varies from tissue to tissue (Bourre & Clement, 1996). On the other hand, a shorter period of supplementation would probably have been insufficient to exert an effect on the exercise-induced response, although we cannot exclude some acute effect of the vitamin C dose provided a few hours prior to the exercise. However, short-term supplementation with vitamin C alone causes no or only a minor effect on the exercise-induced IL-6 response (Nieman et al. 2002; Thompson et al. 2003). In the present study the supplementation was sufficient with regard to producing differences in plasma vitamin and F2-isoprostane levels between groups. The latter, of which 8-iso-PGF2 is a major isoform (Morrow et al. 1994), is formed by a non-enzymatic interaction between reactive oxygen species and arachidonic acid (Morrow et al. 1992), and is therefore suitable as a marker of lipid peroxidation. In contrast to our results, no effect on plasma IL-6 was detected in a study by Nieman et al. (2000), where a single dose of vitamin C was employed prior to an ultramarathon race. Also, in a previous study from our group (Petersen et al. 2001), a combination of vitamins C and E ingested for 14 days prior to 90 min of 5% downhill running at 70% of 
did not affect the systemic plasma IL-6 response, even though the peak plasma IL-6 levels were at least as high as those observed in the study by Vassilakopoulos et al. Thus, a high exercise-induced plasma IL-6 level itself does not appear to be crucial for the observation of an effect of antioxidant supplementation. The shorter duration of the supplementation with antioxidants employed in both these studies compared to the present study can explain some of the difference regarding the effect of antioxidants on the exercise-induced IL-6 response. However, the mode of exercise may also explain the observed differences, since the increase of plasma-IL-6 was attenuated in the two studies using a pure concentric mode of exercise, but not in the studies using exercise including a considerable eccentric component associated with muscle damage and subsequent inflammation. This point of view is supported by previous studies demonstrating no effect of antioxidant supplementation on muscle damage (Beaton et al. 2002) nor IL-6 associated with inflammation (Bruunsgaard et al. 2003).
When comparing the immunohistochemical staining for IL-6 protein and the staining for myofibrillar ATPase activity we observed that IL-6 protein was accumulated predominantly, though not exclusively, in type 1 muscle fibres in both groups after 3 h of dynamic knee-extensor exercise. In contrast, Hiscock et al. (2004) recently observed that type 2 fibres are the predominant source of skeletal muscle IL-6 protein in response to 2 h of bicycling at 55% of 
. On the other hand, Penkowa et al. (2003) demonstrated a marked IL-6 protein expression in all fibres independent of fibre type using bicycling for 3 h at 60% of 
as the mode of exercise. Also, the IL-6 protein expression peaked 3 h after the cessation of the exercise and remained elevated from pre-exercise even the day after the exercise in the study by Penkowa et al. Taken together, these results indicate that both type 1 and type 2 muscle fibres have the capacity to synthesize IL-6, depending on the mode, intensity and duration of exercise. Thus, the fixed low cadence and moderate intensity used in the present study appear to stimulate IL-6 synthesis mainly in type 1 fibres, while bicycling at moderate intensity as used in the study by Hiscock et al. stimulates IL-6 synthesis predominantly in type 2 fibres, despite the fact that primarily type 1 fibres were glycogen depleted. Furthermore, the findings by Penkowa et al. suggest that following bicycling with higher intensity and longer duration, all fibre types eventually synthesize IL-6. While it has previously been demonstrated that exercise intensity affects the increase in circulating IL-6 (Ostrowski et al. 2000), our results together with the results reported by Penkowa et al. and Hiscock et al. indicate that the exercise mode and intensity also affect whether IL-6 protein is expressed in type 2 fibres or in type 1 fibres, as well as the duration of elevated IL-6 protein levels within the skeletal muscle. However, muscle biopsies obtained during exercise of different modes and intensities would add useful information to the observed discrepancies in skeletal muscle IL-6 protein expression found in the different studies at this point.
Of note, a minor systemic increase of IL-6 was observed during exercise in Treatment, even though the release of IL-6 from the contracting limb was completely blunted. While peripheral blood mononuclear cells do not contribute to the circulating IL-6 in response to exercise (Ostrowski et al. 1998), both the central nervous system (Nybo et al. 2002) and adipose tissue (Lyngso et al. 2002) has been demonstrated to release IL-6, the latter in particular during recovery from exercise. Also, in response to long-distance running, large peritendinous concentrations of IL-6 along the Achilles' tendon have been observed using the microdialysis technique (Langberg et al. 2002). However, a major contribution from tendons to circulating IL-6 in the present study is not likely, partly because the tendons are quantitatively much smaller compared to the contracting muscle, partly because of the limited load on tendons during the exercise employed in the present study. Of note, the immunohistochemical stainings in both the present study, as well in the studies by Penkowa et al. (2003) and Hiscock et al. (2004) demonstrate IL-6 protein expression predominantly in the muscle fibres, but not in other cell types within the skeletal muscle tissue. However, non-muscle cells within the skeletal muscle may contribute more significantly to circulating IL-6 levels in response to exercise associated with pronounced muscle damage and subsequent inflammation. Nonetheless, although it is evident that tissues other than skeletal muscle contribute to IL-6 in the circulation in response to exercise, our results clearly show that the contribution of IL-6 from skeletal muscle plays an important role, since the peak systemic plasma IL-6 level was reduced by 50%, while the exercise-induced increase of plasma IL-1ra and cortisol levels was totally blunted in the antioxidant-supplemented group. In addition, the increase of plasma CRP at 23 h was markedly lower in the Treatment group. The latter observations are in accordance with IL-6 being a potent direct (Bethin et al. 2000) or indirect (Naitoh et al. 1988) inducer of cortisol, IL-1ra and CRP (Steensberg et al. 2003). Although we cannot exclude an additional direct effect of the antioxidants on the cortisol, IL-1ra as well as the CRP response to exercise, it is likely that IL-6 released from the contracting muscle mediates a significant part of the observed effects.
Interestingly, it has been suggested that IL-6 possesses important hormone-like properties (Febbraio & Pedersen, 2002). Thus, the IL-6 response to exercise has been shown to be dependent on pre-exercise skeletal muscle glycogen content (Steensberg et al. 2001) and carbohydrate supplementation (Starkie et al. 2001), while infusion with recombinant human IL-6 increases lipolysis and fat oxidation in humans (Van Hall et al. 2003). In addition, IL-6 appears to be involved in glucose homeostasis (Stouthard et al. 1995; Gleeson, 2000). Our findings support the view that IL-6 derived from skeletal muscle is involved in the systemic immunological and metabolic response to exercise. Thus, we speculate that since antioxidants apparently attenuate the normal physiological response to concentric exercise, the use of antioxidant supplementation may be less desirable from a long-term health perspective. This point of view is partly supported by large studies showing no or even a detrimental effect of antioxidant supplementation on morbidity and mortality (Heart Protection Study Collaborative Group, 2002; Vivekananthan et al. 2003).
In conclusion, the present study shows that oral supplementation with vitamins C and E inhibits the release of IL-6 from contracting human skeletal muscle, thus causing a marked attenuation of IL-6 in the circulation, as well as blunting the increase of IL-1ra and cortisol in response to exercise.
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, Treatment group; •, Control group. 
Significant difference (P < 0.05) versus pre-exercise (0 h) in the Treatment group. 
Significant difference (P < 0.05) versus pre-exercise in the Control group. Mean ±S.E.M.
