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1Copenhagen Muscle Research Centre, Department of Human Physiology, Institute of Exercise and Sport Sciences, University of Copenhagen, and 2Department of Infectious Diseases, Rigshospitalet, DK-1200 Copenhagen, Denmark
Submitted 10 March 2003 ; accepted in final form 30 July 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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0.8 pg/ml at rest to 4.5 pg/ml after 60 min of exercise. Activity of 
1-AMPK (160%) and 2-AMPK (145%) was increased at rest in the glycogen-depleted compared with the loaded situation. During exercise, 1-AMPK activity did not change from resting levels in both trials, whereas 2-AMPK activity increased only in the glycogen-depleted state. After 60 min of exercise in the glycogen-depleted state, individual values of 2-AMPK activity correlated significantly (r = 0.87, P < 0.006) with individual values of IL-6 release as well as with average IL-6 release over the entire 60 min (r = 0.86, P < 0.006). The present data are compatible with a role for AMPK in IL-6 release during exercise or a role for IL-6 in activating AMPK. Alternatively, both AMPK and IL-6 are independent sensors of a low muscle glycogen concentration during exercise. In addition, leg release of IL-6 cannot alone explain the increase in plasma IL-6 during exercise. interleukins; glycogen; 5'-AMP-activated protein kinase
The molecular signaling leading to IL-6 release from contracting muscle is not established. However, the fact that IL-6 release seems to depend on exercise intensity (6) and is stimulated by low muscle glycogen levels (14) may suggest that IL-6 release is related to AMPK activity. AMPK is a ubiquitously expressed sensor of cellular energy charge. The central concept is that the AMPK system protects cells by acting as a "low fuel warning" system. On activation, AMPK switches off ATP-consuming anabolic processes and turns on ATP-producing catabolic processes, via phosphorylation of several downstream metabolic enzymes and via effects on gene expression (4, 5). Two catalytic subunits of AMPK are known: The 1 isoform is widely distributed in different body tissues, and the 2 isoform is primarily expressed in skeletal muscle, heart, and liver (4). It has also been shown that 2-AMPK activity both at rest and during exercise is higher in muscles with low than in muscles with high glycogen content (19, 20). Thus the pattern of activation of AMPK during exercise fits with the pattern of IL-6 release from muscle.
In the present study, we tested the hypothesis that IL-6 release from muscle during exercise may be related to muscle AMPK activity. For this reason subjects were studied under conditions in which muscle glycogen content was either low or high in this way creating conditions in which both AMPK activity and IL-6 release would be expected to differ between the trials. It was hypothesized that covariation between AMPK activity and IL-6 release might be obtained. If so, this would support that AMPK activity and IL-6 release might be related.
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METHODS |
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O2 peak) was determined during an incremental cycling ergometer test (O2peak = 65 ± 1 ml · kg–1 · min–1). The results presented in this study are part of a larger study dealing with the effects of varying muscle glycogen levels on muscle glucose uptake and AMPK activity, which has partly been published previously (20).
Preexperimental treatment. The subjects randomly underwent two experimental trials separated by 2 wk. The subjects were instructed to eat a mixed diet and to avoid exercise training 2 days before each experiment. At noon the day before an experiment, the subjects underwent a glycogen-depleting cycle exercise protocol combining leg and arm exercise at varying power outputs. The glycogen-depleting exercise lasted for 5 h on average and was well tolerated by all the subjects. The subjects then left the laboratory and were instructed to eat a specified, controlled, isoenergetic diet for dinner and during the evening, consisting of 80 energy percent (E%) carbohydrate, 7 E% fat, and 13 E% protein (total energy intake: 17.4 MJ) in one of the trials and in the other trial 2 E% carbohydrate, 86 E% fat, and 12 E% protein (total energy intake: 16.3 MJ). The subjects were allowed to drink unlimited water and eat the specified diet until 11 PM.
Main experiment. The next morning the subjects ingested a light breakfast (75 E% carbohydrate, 8 E% fat, and 17 E% protein; total energy intake: 713 KJ) and arrived at the laboratory 2 h later using a minimum of physical activity. After 45 min of rest, Teflon catheters were inserted under the inguinal ligament in one femoral artery and one vein under local analgesia with lidocaine (1%). When 4 h had elapsed since breakfast, blood samples were drawn simultaneously from the arterial and venous catheters, and leg blood flow was measured by using the bolus-infusion thermodilution method as previously described (1). Then a needle biopsy was taken from the vastus lateralis under local analgesia with lidocaine, and resting pulmonary oxygen consumption was measured by using an on-line gas and airflow analyzer (Medgraphics). The subjects then performed exercise on a cycle ergometer for1hata relative workload of 70% O2 peak. Biopsies were taken again at 10 and 60 min of exercise. The biopsies were taken from one leg during the first trial and the contralateral leg during the second trial. The biopsies were taken through two incisions spaced 5–6 cm. Blood samples were drawn, and oxygen uptake was measured at 10, 20, 30, 45, and 60 min of exercise. During exercise, blood flow was measured by use of the constant-infusion thermodilution method (1).
Assays. The concentration of IL-6 in plasma was measured by using a commercially available ELISA kit (R&D Systems, Minneapolis, MN). The concentrations of epinephrine and norepinephrine in arterial plasma were determined by radioimmunoassay (KatCombi, Immuno-Biological Laboratories).
Muscle biopsies were quickly frozen in liquid nitrogen while still situated in the biopsy needles. After termination of exercise, <30 s elapsed before the "exercise" biopsy was frozen. The frozen biopsies were freeze-dried and dissected free of visible blood, fat, and connective tissue before any analysis was performed.
Glycogen content was determined as glycosyl units after acid hydrolysis (10). Muscle AMPK activities were measured in muscle lysates prepared as described previously (21). -Isoform-specific AMPK activity was measured in immunoprecipitates from 200 µg of muscle lysate protein using anti-1- or anti-2-antibodies kindly provided by D. G. Hardie. A p81-filter paper assay using SAMS-peptide (HMRSAMS-GLHLVKRR) (200 µmol/l) as substrate was used to measure AMPK activity as previously described (21).
Calculations and statistics. Control samples of human muscle were added to all activity assays, and assay-to-assay variation was accounted for by expressing the data relative to these samples. Net IL-6 release across the limb was calculated by multiplying arteriovenous differences with plasma flow. Data are expressed as means ± SE. Statistical evaluation was performed by two-way ANOVA with repeated measures. When ANOVA revealed significant differences, a post hoc test was used to correct for multiple comparisons (Student-Newman-Keuls test). Pearson's correlation analysis was performed to establish the presence of correlations. Differences between groups were considered statistically significant when P < 0.05.
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RESULTS |
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At rest, the respiratory exchange ratio was lower in the LG compared with the HG condition (0.81 ± 0.003 vs. 0.84 ± 0.007, respectively; P < 0.05). During exercise, the average respiratory exchange ratio value was significantly lower in the LG condition (0.73 ± 0.01) compared with the HG condition (0.93 ± 0.01) (P < 0.001). Pulmonary oxygen uptake during exercise was on average found to be significantly higher in the LG (47 ± 0.3 ml · kg–1 · min–1) compared with the HG trial (45 ± 0.3 ml · kg–1 · min–1) (P < 0.001) even though the average work load was exactly the same in the two trials (256 ± 9 W).
Leg blood flow at rest averaged 0.4 ± 0.03 l/min in both trials. During exercise, leg blood flow increased rapidly in both trials, albeit to a slightly higher level in LG (8.2 ± 0.2 l/min) compared with HG (7.5 ± 0.2 l/min), corresponding to a 9% higher flow (P < 0.02).
The catecholamine concentrations increased during exercise and the increase was higher in the LG compared with the HG trial (P < 0.05) (Table 2). Arterial concentrations of IL-6 were similar at rest in both trials and increased during exercise in similar fashion in both trials (Fig. 1). This was surprising in light of the very different leg release during exercise in the two conditions. In the HG trial there was no significant release of IL-6 at any time points during exercise. In contrast to HG, there was a significant release of IL-6 from the exercising leg already after 10 min in LG, and this release remained significantly elevated above resting values throughout the entire exercise period (P < 0.01) (Fig. 1). No measurable release of IL-6 from the leg was present in the resting condition in either trial.
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2-AMPK activity in the vastus lateralis muscle increased from a resting value of 1.6 ± 0.2 to 2.4 ± 0.4 at 10 min to 2.6 ± 0.3 pmol · mg–1 · min–1 at 60 min of exercise in LG (Table 3). In HG, the resting 2-AMPK activity was found to be significantly lower (1.1 ± 0.2 pmol · mg–1 · min–1) and there was no significant increase in kinase activity during exercise (Table 3). 1-AMPK activity was unchanged during exercise in both trials but was significantly higher in the LG than in the HG condition (Table 3). A strong positive correlation was found between the average IL-6 release (average of 10-, 20-, 30-, 45-, and 60-min values) in LG and 2-AMPK activity at 60 min (r = 0.86, P < 0.006) (Fig. 2). An equally strong correlation was found when the individual values for IL-6 release in LG at 60 min of exercise were correlated against the individual values of 2-AMPK activity at 60 min of exercise (r = 0.87, P < 0.006) (Fig. 2).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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O2 peak in the glycogen-depleted state correlates with 2-AMPK activity in the exercising muscle. Furthermore, despite differences in leg IL-6 release, arterial IL-6 concentrations rose similarly in the two trials. Finally, a novel observation is that IL-6 is released from the leg already after 10 min of exercise when preexercise muscle glycogen stores are low. Thus, in this respect the present findings extend and confirm previous findings indicating that IL-6 release from an exercising limb depends on the preexercise glycogen store (14). In that study, IL-6 release was measured after 1 h of exercise in the glycogen-depleted leg, whereas 2 h elapsed before release was detectable in the leg with normal preexercise glycogen levels.
Low glycogen levels have been shown to induce IL-6 gene transcription in skeletal muscle during exercise (8). The fact that IL-6 was released already after 10 min of exercise in the LG condition indicates that release must have been due to IL-6 stored in the muscle rather than to acute exercise-induced increase in IL-6 gene transcription. Supposedly gene transcription was activated during the glycogen-depletion trial the day before and was probably maintained during the low-carbohydrate diet consumed after the glycogen-depletion trial, whereas the glycogen supercompensation resulting from the carbohydrate-rich diet presumably inhibited IL-6 gene transcription. Nevertheless, because IL-6 release was identical at rest in the two trials, the rapid rise in IL-6 release in the LG condition indicates the existence of specific mechanisms for IL-6 release activated by muscle contractions when glycogen stores are low. So far, the underlying signaling mechanisms behind exercise-induced IL-6 release have not been clarified. Nevertheless, in the present experiment, 2-AMPK activity and IL-6 release only increased above resting values in the LG trial and, furthermore, a fairly strong positive correlation was found when the individual values for IL-6 release in LG at 60 min of exercise were correlated against the individual values of 2-AMPK activity at the same time (r = 0.87, P = 0.006). A similar strong correlation was obtained when average IL-6 release over the entire 60 min was correlated to 2-AMPK activity at 60 min (r = 0.86, P = 0.006). Whereas correlation is not necessarily indicating a causative relationship between 2-AMPK activity and IL-6 release, the present findings may implicate such a relationship.
It has previously been estimated that the increase in plasma IL-6 concentration during 5 h of knee extensor exercise could be fully accounted for by release from the working muscle (17), and it was hypothesized that leg release might signal to increase hepatic glucose output during exercise (2, 3). In the present study the increase in arterial IL-6 during 60 min of exercise was identical in the two conditions despite the fact that the leg only released IL-6 in the LG condition. This indicates that other sources of IL-6 than exercising muscle contribute to the increase in plasma IL-6 during 60 min of exercise at 70% of O2 peak. These may include the adipose tissue (7), the peritendinous tissue (9), and the brain (11). The possibility also exists that clearance of IL-6 was lower in the HG trial than in the LG trial. The similar increase in plasma IL-6 in the two trials indicates that the liver is exposed to similar IL-6 concentrations during the two trials despite different leg release of IL-6. Therefore, leg release of IL-6 cannot play an important signaling role to liver glucose output at least during exercise of up to 60 min. This is in agreement with recent findings from one of our laboratories that infusion of recombinant IL-6 into healthy resting volunteers does not increase hepatic glucose production (15). However, IL-6 may have other signaling properties because infusion of IL-6 has been shown to increase lipolysis and fat oxidation (18).
It was recently found that IL-6 release during knee extensor exercise correlated with the arterial plasma epinephrine concentration (6). Also, infusion of epinephrine at rest, at a rate resulting in arterial plasma epinephrine concentrations observed during strenuous whole body exercise, has been shown to increase the arterial IL-6 concentration. However, the increase was markedly smaller than the increase seen during strenuous exercise (16). In addition, in a study of one-legged exercise, IL-6 release was found in the exercising but not in the resting leg (17), pointing toward local factors as being more important than adrenaline in release of IL-6 during exercise. In the present study, IL-6 release during the LG trial did not correlate significantly with plasma epinephrine concentration, supporting the importance of local mechanisms in IL-6 release during exercise.
In conclusion, the present study has shown that, when preexercise muscle glycogen concentration is low, IL-6 release across the leg is measurable already after 10 min of dynamic exercise of moderate intensity and that after 60 min of exercise release of IL-6 correlated significantly with muscle activity of 2-AMPK. In contrast, when preexercise muscle glycogen level is high, IL-6 release and 2-AMPK activity do not increase for 1 h at this exercise intensity. Despite the difference in leg release of IL-6, arterial IL-6 concentrations increased similarly in the two trials, suggesting either that IL-6 is released mainly from other organs than muscle during exercise of this duration or that clearance of IL-6 is affected differently in the two trials. The present data are compatible with a role for AMPK in IL-6 release during exercise or a role for IL-6 in activating AMPK. Alternatively, both AMPK and IL-6 are independent sensors of a low muscle glycogen concentration during exercise.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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) and high-glycogen (HG; 
) trials. Values are means ± SE; n = 8. A: * indicates that arterial values were significantly (P < 0.05) higher than values at rest. B: * indicates that release was significantly (P < 0.05) different from zero during exercise only in the LG condition and it was also significantly higher in the LG vs. the HG condition.