We tested the hypothesis that IL-6 release from muscle during exercise may be related to muscle activity of 5′-AMP-activated protein kinase (AMPK). Eight healthy, well-trained young men completed two 60-min trials on a bicycle ergometer at 70% of their peak oxygen uptake in either a glycogen-depleted or a glycogen-loaded state. IL-6 was released from the leg already after 10 min of exercise in the glycogen-depleted state, whereas no significant release was observed at any time in the loaded state. Nevertheless, plasma IL-6 increased similarly in the two trials from ∼0.8 pg/ml at rest to ∼4.5 pg/ml after 60 min of exercise. Activity of α1-AMPK (160%) and α2-AMPK (145%) was increased at rest in the glycogen-depleted compared with the loaded situation. During exercise, α1-AMPK activity did not change from resting levels in both trials, whereas α2-AMPK activity increased only in the glycogen-depleted state. After 60 min of exercise in the glycogen-depleted state, individual values of α2-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.
- 5′-AMP-activated protein kinase
increased circulating levels of IL-6 have been reported in response to exercise and the magnitude of the increase depends on the duration (for review, see Refs. 2, 13) and intensity (6) of exercise. It has been demonstrated that IL-6 is produced in and released from skeletal muscle during contractions (8, 14) and that release of IL-6 is higher during contractions when muscle glycogen level is low than when it is high (14). IL-6 has been proposed to play a signaling role between contracting muscle and hepatic glucose production as well as adipose tissue lipolysis during exercise (2, 12). A role for IL-6 in regulation of glucose homeostasis during exercise has also been suggested (2, 3).
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 α1 isoform is widely distributed in different body tissues, and the α2 isoform is primarily expressed in skeletal muscle, heart, and liver (4). It has also been shown that α2-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.
Subjects. Eight young (28 ± 1 yr), healthy, endurancetrained men gave their written, informed consent to participate in the study, which was approved by the Copenhagen Ethics Committee and conformed to the declaration of Helsinki. Body height, weight, and body mass index were 184 ± 2 cm, 81 ± 3 kg, and 24 ± 1 kg/m2, respectively. The subjects participated in physical exercise training 5–8 times per week (2–3 h per bout), for the most part performing cycle spinning but also performing some resistance training. One to two weeks before the experiments, peak pulmonary oxygen uptake (V̇o2 peak) was determined during an incremental cycling ergometer test (V̇o2peak = 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% V̇o2 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). α-Isoform-specific AMPK activity was measured in immunoprecipitates from 200 μg of muscle lysate protein using anti-α1- or anti-α2-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.
Muscle biopsies obtained before the experimental trial demonstrated that the depletion and diet protocol were successful in creating vastly different glycogen concentrations in the low-glycogen [LG; 163 ± 12 mmol/kg dry wt (dw)] and high-glycogen (HG; 909 ± 75 mmol/kg dw) conditions (Table 1). During the 60 min of cycle exercise, muscle glycogen content decreased progressively in the HG trial only, but at all time points it was higher than in the LG trial (Table 1).
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.
α2-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 α2-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). α1-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 α2-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 α2-AMPK activity at 60 min of exercise (r = 0.87, P < 0.006) (Fig. 2).
The main finding in the present study is that in endurance-trained subjects IL-6 release across the leg after 1 h of exercise at 70% of V̇o2 peak in the glycogen-depleted state correlates with α2-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, α2-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 α2-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 α2-AMPK activity at 60 min (r = 0.86, P = 0.006). Whereas correlation is not necessarily indicating a causative relationship between α2-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 V̇o2 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 α2-AMPK. In contrast, when preexercise muscle glycogen level is high, IL-6 release and α2-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.
The study was supported by grants from the Danish National Research Foundation (no. 504-14), by The Media and Grants Secretariat of the Danish Ministry of Culture, by the Danish Diabetes Association, by the Novo Nordisk Foundation, and by a Research & Technological Development Projects (QLG1-CT-2001-01488) funded by the European Commission. J. F. P. Wojtaszewski was supported by a Hallas Møller fellowship from the Novo Nordisk Foundation.
We thank Betina Bolmgren for skilled technical assistance.
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.
- Copyright © 2003 the American Physiological Society