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Journal of Physiology (2002), 541.3, pp. 979-989
© Copyright 2002 The Physiological Society
DOI: 10.1113/jphysiol.2002.018044

Role of 5'AMP-activated protein kinase in glycogen synthase activity and glucose utilization: insights from patients with McArdle's disease

Jakob N. Nielsen*, Jørgen F. P. Wojtaszewski*, Ronald G. Haller†, D. Grahame Hardie‡, Bruce E. Kemp§, Erik A. Richter* and John Vissing¶

	ABSTRACT

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It has been suggested that 5'AMP-activated protein kinase (AMPK) is involved in the regulation of glucose and glycogen metabolism in skeletal muscle. We used patients with chronic high muscle glycogen stores and deficient glycogenolysis (McArdle's disease) as a model to address this issue. Six McArdle patients were compared with control subjects during exercise. Muscle 2AMPK activity increased in McArdle patients (from 1.3 ± 0.2 to 1.9 ± 0.2 pmol min^-1 mg^-1, P = 0.05) but not in control subjects (from 1.0 ± 0.1 to 1.3 ± 0.3 pmol min^-1 mg^-1). Exercise-induced phosphorylation of the in vivo AMPK substrate acetyl CoA carboxylase (ACC; Ser²²¹) was higher (P < 0.01) in McArdle patients than in control subjects (18 ± 3 vs. 10 ± 1 arbitrary units). Exercise-induced whole-body glucose utilization was also higher in McArdle patients than in control subjects (P < 0.05). No correlation between individual AMPK or ACC values and glucose utilization was observed. Glycogen synthase (GS) activity was decreased in McArdle patients from 11 ± 1.3 to 5 ± 1.2 % (P < 0.05) and increased in control subjects from 19 ± 1.6 to 23 ± 2.3 % (P < 0.05) in response to exercise. This was not associated with activity changes of GS kinase 3 or protein phosphatase 1, but the changes in GS activity could be due to changes in activity of AMPK or protein kinase A (PKA) as a negative correlation between either ACC phosphorylation (Ser²²¹) or plasma adrenaline and GS activity was observed. These findings suggest that GS activity is increased by glycogen breakdown and decreased by AMPK and possibly PKA activation and that the resultant GS activity depends on the relative strengths of the various stimuli. Furthermore, AMPK may be involved in the regulation of glucose utilization during exercise in humans, although the lack of correlation between individual AMPK activity or ACC phosphorylation (Ser²²¹) values and individual glucose utilization during exercise implies that AMPK may not be an essential regulator.

(Received 2 February 2002; accepted after revision 8 April 2002)
Corresponding author J. N. Nielsen: Copenhagen Muscle Research Centre, Department of Human Physiology, University of Copenhagen, 13, Universitetsparken, DK-2100 Copenhagen, Denmark. Email: jnnielsen{at}aki.ku.dk

	INTRODUCTION

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Glycogen synthase is the key enzyme in the regulation of glycogen synthesis in skeletal muscle. The activity of glycogen synthase is under a complex control of kinases and phosphatases, leading to deactivation and activation, respectively. It has been observed that glycogen synthase activity may be decreased during exercise, possibly due to activation of protein kinase A (PKA) (Yan et al. 1992) and/or inactivation of protein phosphatase 1 (PP1) (Kida et al. 1989; Katz & Raz, 1995) but apparently not due to activation of glycogen synthase kinase 3 (GSK3) (Wojtazsewski et al. 2001). 5'AMP-activated protein kinase (AMPK) has been shown to phosphorylate (Carling & Hardie, 1989) and co-immunoprecipitate (Chen et al. 1999) with glycogen synthase in vitro. Pharmacological activation of AMPK also leads to glycogen synthase deactivation in the rat (Wojtaszewski et al. 2002), but the effect of AMPK on glycogen synthase during and after exercise in human and rat skeletal muscle remains unclear. After exercise, glycogen synthase activity is rapidly increased by an insulin-independent mechanism that is poorly understood (Danforth, 1965; Kochan et al. 1979; Richter et al. 1984; Bak & Pedersen, 1990; Yan et al. 1992; Yan et al. 1993), but the glycogen synthase activity under these and other conditions has been shown to be negatively related to the muscle glycogen level (Danforth, 1965; Bogardus et al. 1983; Zachwieja et al. 1991; Munger et al. 1993; Furler et al. 1998; Nielsen et al. 2001). Thus, glycogen synthase activity may be increased by a mechanism linked to glycogen breakdown that counteracts the exercise-induced deactivation of glycogen synthase (possibly involving AMPK and PKA). However, the existence of such a scenario remains to be verified. Its confirmation would require discrimination of the effects of exercise from the effects of glycogen breakdown per se, which is a methodological problem not easily overcome. However, a potential model to resolve this issue is McArdle's disease (glycogen storage disease type V) first described in 1951 (McArdle, 1951), in which a genetic defect causes absence of glycogen phosphorylase exclusively in skeletal muscle, leading to high muscle glycogen concentrations and an inability to utilize muscle glycogen as a metabolic substrate.

Considerable progress in revealing components involved in muscle insulin signalling has been made, while the elucidation of the cellular machinery linking muscle contraction to glucose transport and alteration of glycogen synthase activity has been less successful. However, AMPK has recently emerged as a possible element mediating the effects of muscle contraction on glucose transport. AMPK is sensitive to rapid changes in cellular energy balance, switching off ATP-consuming anabolic pathways and activating ATP-producing catabolic processes (reviewed in Kemp et al. 1999; Winder & Hardie, 1999), making AMPK a candidate for mediating exercise-induced changes in glucose metabolism. Studies in rodents indicate that exercise-induced glucose uptake may be partly mediated by AMPK (Merrill et al. 1997; Hayashi et al. 1998; Bergeron et al. 1999; Kurth-Kraczek et al. 1999; Hayashi et al. 2000; Kawanaka et al. 2000), but it is also clear that other mechanisms must be involved (Derave et al. 2000; Mu et al. 2001; Richter et al. 2001a). In human skeletal muscle it has been demonstrated that AMPK is activated during exercise under conditions where glucose uptake is also expected to increase (Fujii et al. 2000; Wojtaszewski et al. 2000; Musi et al. 2001), but only one preliminary report has addressed AMPK activation and glucose uptake in the same experimental setup (Richter et al. 2001b).

Thus, we investigated the effect of deficient glycogenolysis and chronic high muscle glycogen level on skeletal muscle glycogen synthase activity and exercise-induced whole-body glucose utilization and the possible involvement of AMPK and other signalling intermediates in patients with McArdle's disease and in healthy control subjects.

	METHODS

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Subjects

A total of 12 volunteers (six patients with McArdle's disease and six matched control subjects) participated. The McArdle patients had a typical history of exercise intolerance and myoglobinuria. Furthermore, all patients had been histochemically and biochemically diagnosed with an absence of muscle glycogen phosphorylase activity. A control subject was matched to each of the patients according to age, sex, body weight, body mass index (BMI) and lean body mass, determined by hydrostatic weighing (Siri, 1956). The control subjects included in the study undertook very low levels of habitual physical exercise and did not participate in any physical exercise-training activities. However, it was not possible to match the maximal work capacity of the controls and the patients due to the abnormally low maximal work capacity of the latter group. The purpose and nature of the study was explained to all subjects, and written consent was obtained before their participation. The Copenhagen Ethics Committee approved the experimental protocol and all human experiments conformed to the Declaration of Helsinki.

Experimental protocol

All experiments began at 09.00 h after a 10 h overnight fast. Before the exercise experiment the subjects took part in a euglycaemic-hyperinsulinaemic (75 µU ml^-1) clamp procedure to investigate the insulin sensitivity of the patients. The results of that experiment are reported elsewhere (Nielsen et al. 2002). During the clamp [6,6-²H₂]glucose (99 % enriched, Cambridge Isotope Laboratories Inc., MA, USA) was infused to measure the rate of appearance and disappearance of glucose. After termination of the clamp procedure subjects rested fasting for 2 h while the [6,6-²H₂]glucose infusion was continued. The 2 h interval between termination of the clamp procedure and initiation of the exercise bout was sufficient to restore basal levels of all signalling parameters and biochemical endpoints studied. The subjects exercised on a cycle ergometer (CPE 2000, Medgraphics, Medical Graphics Corp., St Paul, MN USA) for 20 min. The workload for each McArdle patient had been assessed on a separate day prior to the experiment by determining the maximum workload that could be tolerated for 20 min without causing muscle pain or contracture. During exercise, the workload was adjusted according to the fluctuating exercise capacity of the patient (second wind phenomenon). The 'second wind' occurred approximately 7-8 min into the exercise bout. At that point the workload was increased by 10-15 W and was unchanged for the remaining part of the exercise bout. Each control subject followed the same protocol as the individually matched patient and thus the control subject worked at the same absolute workload. The resting rate of [6,6-²H₂]glucose infusion was 0.31 µmol kg^-1 min^-1 and this rate of infusion was doubled at the onset of exercise. Subjects had stable plasma glucose values for at least 1 h without glucose infusion before exercise was initiated. Pulmonary gas exchange (CPX, Medgraphics, Medical Graphics Corp., St Paul, MN USA) and heart rate (Vantage NV, Polar Electro, Finland) were measured continuously during the exercise bout. Arterialized blood samples were obtained at 5 min intervals from a catheter in a hand vein heated to 40 °C throughout the experiment and needle biopsies from m. vastus lateralis were excised and frozen in liquid nitrogen before and approximately 30 s after termination of exercise.

Measurement of glucose metabolism

From the enrichments of labelled [6,6-²H₂]glucose in deproteinized plasma samples (Roepstorff et al. 2002), the whole-body rate of appearance and disappearance of glucose was calculated by the non-steady-state equation of Steele (1959) modified for use with stable isotopes (Romijn et al. 1993). The percentage of enrichment of plasma by [6,6-²H₂]glucose was determined by gas chromatography-mass spectrometry as previously described (Bier et al. 1977), with some modifications (Roepstorff et al. 2002).

Analysis of plasma substrates and hormones

Glucose and lactate concentrations in blood were determined in duplicate using a dual channel glucose-lactate analyser (YSI-2700 Select; Yellow Springs Instruments, Yellow Springs, OH, USA). Plasma insulin concentration was determined using a radioimmunoassay kit (Insulin Ria 100, Pharmacia, Sweden). Concentrations of plasma free fatty acids (FFA) and glycerol were determined in accordance with Shimizu et al. (1979) and Passonneau & Lowry (1993), respectively, using an automatic spectrophotometer (COBAS FARA 2, Roche Diagnostic, Switzerland). Plasma adrenaline and noradrenaline were analysed by means of a radioimmunoassay (KatCombi, Immuno-Biological Laboratories GmbH, Hamburg, Germany).

Muscle glycogen and phosphocreatine

Muscle glycogen content and the concentration of muscle phosphocreatine were measured in freeze-dried muscle tissue by fluorometry as previously described (Passonneau & Lowry, 1993).

Preparation of muscle lysates

For studies of enzyme activity and Western blotting, muscle lysates were prepared as follows. Approximately 40 mg of frozen muscle tissue was homogenized (model Omni 2000, Omni Int., Warrenton, VA, USA) in 10 volumes of buffer A (50 mM Hepes, 150 mM sodium chloride, 20 mM sodium pyrophosphate, 20 mM -glycerophosphate, 10 mM sodium fluoride, 2 mM sodium orthovanadate, 2 mM EDTA, 1 % Igepal CA630, 10 % glycerol, 2 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM magnesium chloride, 1 mM calcium chloride, 10 µg ml^-1 leupeptin, 10 µg ml^-1 aprotinin, 3 mM benzamidine, pH 7.5), except for measurements of protein phosphatase activities where buffer B was used (50 mM Tris HCl, 2 mM EDTA, 2 mM EGTA, 0.5 % Triton X-100, 5 % glycerol, 0.1 % -mercaptoethanol, 2 mM PMSF, 10 µg ml^-1 leupeptin, 10 µg ml^-1 aprotinin, 3 mM benzamidine, pH 7.5). Homogenates were rotated end over end at 4 °C for 60 min after which they were centrifuged at 4 °C for 30 min at 4000 g. The supernatants were harvested and total protein content was determined in the lysates by the BCA method (Pierce Inc., Rockford, IL, USA).

Glycogen synthase activity

Glycogen synthase activity was measured in triplicate on 5 µl of lysate in buffer A by a modification of the method of Thomas et al. (1968) described previously (Richter et al. 1989). Glycogen synthase activity at a maximally stimulating glucose 6-phosphate concentration of 8 mM was defined as total activity because the enzyme is fully activated, regardless of its phosphorylation status. The activity at a glucose 6-phosphate concentration of 0.17 mM divided by total activity is defined as fractional velocity.

Glycogen synthase kinase 3 (GSK3) activity

GSK3 and GSK3 were immunoprecipitated from 100 µg of protein from muscle lysate in buffer A using an anti-GSK3 (Upstate Biotechnology, Lake Placid, MA, USA) or an anti-GSK3 antibody (Transduction Laboratories, San Diego, CA, USA). A P81-filter paper assay, with a phospho GS2 peptide (Upstate Biotechnology) as substrate, was used to measure isoform-specific GSK3 activity, as previously described (Markuns et al. 1999).

Protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) activity

Activities of PP1 and PP2A were measured in muscle lysates in buffer B as glycogen phosphorylase phosphatase activity as previously described (Begum, 1995) using a protein phosphatase assay system (GibcoBRL Life Technologies). In short, 1 µg of lysate in buffer B was incubated with ³²P-phosphorylase A for 10 min at 30 °C. The reaction was stopped by adding 180 µl trichloroacetic acid and 10 µl of 3 % bovine serum albumin as a carrier protein. The tubes were left on ice for 10 min followed by centrifugation at 14 000 g for 3 min. Aliquots of supernatant were counted to determine the released ³²P_i. Discrimination between PP1 and PP2A activity was performed by the absence or presence of 1 nM okadaic acid in the assay. At 1 nM of okadaic acid, the remaining activity represents PP1 activity, whereas the activity inhibited represents PP2A activity.

5'AMP-activated protein kinase (AMPK) activity

-Isoform-specific AMPK activity was measured in immunoprecipitates from 100 µg of muscle lysate protein in buffer A using an anti-1AMPK and an anti-2AMPK antibody (Woods et al. 1996). A p81 filter paper assay, using SAMS-peptide (200 µM) as the substrate, was used to measure AMPK activity in the presence of 0.2 mM AMP as previously described (Wojtaszewski et al. 2000).

Acetyl CoA carboxylase (ACC) phosphorylation (Ser²²¹) and total ACC

In order to semi-purify ACC, 200 µg of lysate in buffer A was incubated with Avidin-immobilized agarose beads (Sigma Aldrich, St Louis, MO, USA) because ACC is a biotinylated enzyme. The beads were washed and the precipitated proteins were eluted with electrophoresis sample buffer for Western blotting (see below)

Western blotting

For measurements of ACC phosphorylation, total ACC, and GLUT4, muscle lysates were subjected to SDS-PAGE (5 or 7.5 % gel) and transferred to a polyvinylidene fluoride (PVDF) membrane by semi-dry blotting as previously described (Kristiansen et al. 1996). The membrane was probed with the primary antibody raised against the protein of interest (rabbit anti-phospho- ACC (Ser⁷⁹), goat anti-GLUT4, C-20 from Santa Cruz Biotechnology, CA, USA). The ACC phosphospecific antibody was raised against a peptide corresponding to the sequence in rat ACC containing the serine 79 phosphorylation site, but the antibody also recognised human ACC when phosphorylated at serine 221. An anti-rabbit or anti-sheep secondary antibody conjugated to alkaline phosphatase (Zymed Laboratories Inc., San Francisco, CA, USA) or Extravidin conjugated to alkaline phosphatase (Sigma Aldrich) for the detection of total ACC was applied to the membrane before incubation with an enhanced chemifluorescense substrate (Vistra ECF, Amersham Pharmacia Biotech Ltd, UK). Signal intensities were visualized and quantified using a Kodak image station 440CF and Kodak 1D software (Kodak Scientific Imaging Systems, New Haven, CT, USA), respectively.

Statistical analysis

Data are expressed as means ± S.E.M. Student's two-tailed paired t test was applied for comparison of two normally distributed groups. Comparisons between more than two normally distributed groups were done by two-way ANOVA for repeated measures for the detection of main effects and interactions between the different groups. If interactions between groups were detected, two-way ANOVA for repeated measures was followed by a multiple comparison test (Student-Newman-Keuls method). The strength of association between parameters was analysed by Pearson product moment correlation analysis. P values equal to or less than 0.05 were considered statistically significant.

	RESULTS

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Physical characteristics of the subjects

As seen in Table 1, the matching of each McArdle patient to a control subject resulted in almost identical anthropometric parameters between groups, except for the body height, which was slightly but significantly lower in patients.

Cardiac and respiratory responses to exercise

The workload during the last minute of the exercise bout was 65 ± 5 W for the McArdle patients and 66 ± 5 W for the control subjects. Seated on the cycle ergometer at rest, no differences between the McArdle patients and control subjects could be observed in heart rate, pulmonary ventilation, pulmonary oxygen uptake or respiratory exchange ratio (Table 2). All these parameters increased with exercise (P < 0.05, main effect), and only heart rate in the McArdle patients was significantly higher than in controls during exercise (Table 2).

Plasma substrates and hormones

Before initiation of the exercise bout the basal concentrations of plasma insulin, FFA, glycerol, adrenaline, noradrenaline and blood glucose were similar in the McArdle patients and in the controls (Table 3). At the end of the exercise bout, glucose, glycerol, adrenaline and noradrenaline were increased compared to the resting condition (P < 0.01, main effect), but only adrenaline and noradrenaline were higher in McArdle patients than in control subjects during exercise (Table 3).

Expression of GLUT4 protein

Measured in resting biopsies, McArdle patients had 116 ± 26 % of the amount of GLUT4 protein present in control subjects. This was not significantly different between the two groups, but it should be noted that a large inter-subject variation in GLUT4 content was observed (data not shown).

Muscle glycogen

At rest the muscle glycogen concentration was almost twice as high in the McArdle patients compared to controls (835 ± 111 versus 436 ± 36 mmol (kg dry wt)^-1, respectively (Fig. 1A). After exercise, glycogen concentration was 774 ± 105 mmol (kg dry wt)^-1 and 388 ± 19 mmol (kg dry wt)^-1 in McArdle patients and control subjects, respectively. McArdle patients had a significantly higher glycogen level than controls (P < 0.01, main effect) and there was a significant effect of exercise on the glycogen level (P < 0.05, main effect). Although a main effect of exercise was present, there were no statistical changes in the glycogen concentrations when the McArdle patients and the control subjects were analysed separately.

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Figure 1. Effect of exercise on the concentration of glycogen in skeletal muscle (A) and blood lactate (B) in McArdle patients (, ) and control subjects (, ) Please refer to text for detailed statistical analysis. Data are presented as mean ± S.E.M. of n = 6 subjects in each group. * P < 0.01 versus corresponding control value. † P < 0.01 versus rest value (0 min).

Blood lactate

A rise in blood lactate concentration when going from the resting to the exercising condition is an indicator of glycogen breakdown. Blood lactate was not different between McArdle patients and control subjects in the resting state (Fig. 1B). In the controls, blood lactate was above the resting level at 10, 15 and 20 min of exercise (P < 0.01). Blood lactate in McArdle patients was unchanged from the resting condition and significantly lower than the corresponding values in controls at 10, 15 and 20 min of exercise (P < 0.05) (Fig. 1B).

Whole-body glucose metabolism

The rate of glucose disappearance (glucose utilization) was significantly above the resting level at 7.5, 12.5 and 17.5 min of exercise (main effect P < 0.001), and was significantly higher in the McArdle patients than in the controls (main effect, P < 0.05) (Fig. 2). The rate of glucose appearance (glucose production) was also significantly higher in McArdle patients and was at no time point different from the rate of disappearance (not shown).

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Figure 2. Whole-body rate of glucose disappearance (glucose utilization) in McArdle patients () and control subjects () before and during 20 min of exercise * P < 0.05 versus controls (main effect). † P < 0.001 versus rest (-5 min) (main effect). Data are presented as means ± S.E.M. of n = 6 subjects in each group.

Glycogen synthase activity

Fractional velocity of glycogen synthase was markedly lower in the McArdle patients than in the control subjects, both at rest (11 ± 1 versus 19 ± 2 %, respectively, P < 0.05) and after exercise (5 ± 1 versus 23 ± 2 %, respectively, P < 0.001) (Fig. 3). In response to exercise, the fractional velocity of glycogen synthase decreased in the patients and increased in the control subjects (P < 0.01 and P < 0.05, respectively). The total activity of glycogen synthase was similar in patients and controls before and after exercise and no changes in the total glycogen synthase activity in response to exercise were observed (not shown).

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Figure 3. Effect of exercise on glycogen synthase activity expressed as fractional velocity in skeletal muscle from McArdle patients () and healthy control subjects () * P < 0.05 and **P < 0.001 versus corresponding control value. † P < 0.05 and †† P < 0.01 versus corresponding rest value. Data are presented as means ± S.E.M. of n = 6 subjects in each group.

GSK3 activity

The GSK3 activity was not different between McArdle patients and control subjects, before or after exercise, but there was a significant fall in GSK3 activity in response to exercise in the McArdle patients (P < 0.05) (Table 4). The GSK3 activity was similar in patients and controls both before and after exercise (Table 4). No changes in the GSK3 activity in response to exercise were observed.

PP1 and PP2A activity

The PP1 activity was similar in patients and controls both before and after exercise (Table 4). No changes in the PP1 activity in response to exercise were observed. There was a significantly lower PP2A activity in the McArdle patients than in control subjects before exercise but not after exercise. PP2A activity did not change with exercise in McArdle patients, whereas PP2A activity decreased slightly in control subjects (P < 0.05) (Table 4).

1 and 2AMPK activity

The 1AMPK activity was similar in patients and controls both before and after exercise (Fig. 4A). No changes in the 1AMPK activity in response to exercise were observed. The exercise-induced 2AMPK activity tended to be higher in McArdle patients than in control subjects (1.9 ± 0.2 versus 1.3 ± 0.3 pmol min^-1 mg^-1, respectively, P = 0.08), but not at rest (1.3 ± 0.2 versus 1.0 ± 0.1 respectively, P < 0.37) (Fig. 4B). The activity of 2AMPK increased in response to exercise (main effect, P < 0.05). This was reflected as an increase in the McArdle patients (P = 0.05), but no change or tendency to a change in response to exercise was present in the control group (P = 0.32).

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Figure 4. Activity of 1 (A) and 2 (B) isoforms of the 5'AMP-activated protein kinase (AMPK) in skeletal muscle of McArdle patients () and control subjects () † P = 0.05 versus corresponding rest value. Data are presented as mean ± S.E.M. of n = 6 subjects in each group.

ACC (Ser²²¹) phosphorylation and total ACC protein expression

In the resting condition there was no difference in ACC phosphorylation between McArdle patients and control subjects, but in response to exercise, phosphorylation was significantly higher in the patients than in controls (Fig. 5A). A significant increase in ACC phosphorylation was observed in both McArdle patients and control subjects (P < 0.01 and P < 0.001, respectively). Total ACC protein was significantly lower in McArdle patients than in control subjects (main effect, P < 0.05) (Fig. 5B). Thus, the percentage of ACC protein, which was phosphorylated was even greater in McArdle patients than indicated by the phosphospecific Western blot per se (Fig. 5A).

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Figure 5. Phosphorylation of ACC (Ser221) (A) and total ACC protein (B) in skeletal muscle of McArdle patients () and healthy control subjects () Representative Western blots are shown. * P < 0.01 versus corresponding control value. † P < 0.01 and †† P < 0.001 versus corresponding rest value. § P < 0.05 versus control subjects (main effect). Data are presented as mean ± S.E.M. of n = 6 subjects in each group.

Phosphocreatine

The resting level of muscle phosphocreatine was similar in McArdle patients and controls (74 ± 5 versus 81 ± 3 mmol (kg muscle dry wt)^-1, respectively) and decreased significantly (P < 0.001) during exercise in the patients to 56 ± 5 mmol (kg muscle dry wt)^-1, whereas the levels were unchanged (78 ± 4 mmol (kg muscle dry wt)^-1) in the controls.

Correlation analysis

When pooling the data from McArdle patients and control subjects there was a significant negative correlation between the exercise-induced change in glycogen synthase activity (fractional velocity) on one hand and both the exercise-induced change in plasma adrenaline concentration (r² = 0.67, P < 0.01) and muscle ACC phosphorylation (r² = 0.36, P < 0.05) on the other hand. No correlations were observed between any measure of glucose metabolism and any measure of AMPK activity, but the exercise-induced increase in the rate of glucose disappearance tended to correlate with the increase in ACC phosphorylation (r² = 0.29, P = 0.07), when the rate of glucose disappearance was expressed per kg lean body mass.

	DISCUSSION

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In the present study we investigated the effect of blocked muscle glycogenolysis and chronic high glycogen levels on the response of AMPK to exercise at a given workload and the possible involvement of AMPK in regulation of glycogen synthase and glucose utilization during exercise. The principal new findings were that glycogen synthase activity is decreased by exercise in patients with McArdle's disease in contrast to significant increases in glycogen synthase activity in healthy subjects. Furthermore, glucose utilization during exercise was enhanced in McArdle patients compared to healthy subjects despite high muscle glycogen levels in the patients. The elevated mean glucose utilization and decrease in muscle glycogen synthase activity during exercise was paralleled by decreased phosphocreatine levels and by increased mean 2AMPK activity and ACC phosphorylation in skeletal muscle of the patients. These findings suggest that intact muscle glycogenolysis is a prerequisite for exercise-induced increases in glycogen synthase activity and that glycogen synthase activity is the result of the composite actions of inhibitory (AMPK and PKA) and enhancing (glycogen breakdown) stimuli. Furthermore, AMPK may be involved in the regulation of glucose utilization during exercise in humans, although the lack of correlation between individual AMPK activity or ACC phosphorylation values and individual glucose utilization during exercise implies that AMPK may not be an essential regulator.

Glycogen breakdown is very limited during short-term, low intensity exercise. Probably for this reason, significant muscle glycogenolysis could not be detected in healthy control subjects in the present study. A strong indication that muscle glycogen breakdown was actually occurring during exercise in the healthy controls, but not in McArdle patients, was the increase in blood lactate in controls and the absent blood lactate response in the patients.

A striking observation is that glycogen synthase activity is decreased by exercise in McArdle patients at the same time as glycogen synthase activity is increased in control subjects. As a high level of glycogen has been shown to inhibit glycogen synthase activity in healthy human skeletal muscle (Yan et al. 1992; Munger et al. 1993) it is not surprising, that McArdle patients have low activities of glycogen synthase at rest, although the underlying mechanism awaits definition. GSK3, which is known to be involved in insulin regulation of glycogen synthase (reviewed in Yeaman et al. 2001), could not explain the low basal level of glycogen synthase activity in McArdle patients. Neither could total PP1 activity, which was similar in McArdle patients and control subjects and unchanged by exercise, which has previously been observed (Bak & Pedersen, 1990). This does not, however, exclude PP1 as an important activator of glycogen synthase as discussed below. Surprisingly, in McArdle patients, exercise induced a decrease in GSK3 activity, which if anything would increase the activity of glycogen synthase - the opposite of what was actually observed. It could be speculated that the abnormal GSK3 activation is a compensatory response without which the GS deactivation would be even greater. In general, the existence of compensatory mechanisms in the patients cannot be excluded. In healthy muscle from the control subjects, GSK3 activity was unaffected by exercise as observed in the present and a previous study from our laboratory (Wojtaszewski et al. 2001). As also observed in the control subjects, glycogen synthase activity is increased by exercise in healthy skeletal muscle at various exercise intensities and different glycogen levels (Kochan et al. 1979; Bak & Pedersen, 1990; Yan et al. 1992; Katz & Raz, 1995), so the abnormal response to exercise in McArdle patients is likely to be directly or indirectly associated with the absence of glycogenolysis. This suggests that intact glycogenolysis is required for exercise to activate glycogen synthase. In vitro studies (Carling & Hardie, 1989; Chen et al. 1999) and observations in rat skeletal muscle (Wojtaszewski et al. 2002), suggest that glycogen synthase is a substrate of AMPK, but this has not been investigated in human skeletal muscle. Interestingly, a significant negative correlation between the change in glycogen synthase activity and the change in ACC phosphorylation is seen in the present study supporting the view that AMPK is involved in regulation of exercise-induced glycogen synthase activity. Whereas GSK3 is unlikely to play a role as discussed above, it cannot be ruled out that the activity of cAMP-dependent protein kinase (PKA) can account for the decreased activity of glycogen synthase in McArdle patients. Circulating adrenaline activates PKA by increasing the cellular level of cAMP. Support for a role of PKA in regulation of glycogen synthase activity was provided by a significant negative correlation between the change in plasma adrenaline concentration and the change in glycogen synthase activity in the present study.

Recent work in mouse skeletal muscle suggests that the PP1 regulatory subunit G_M (R_Gl) is required for exercise-induced glycogen synthase activation (Aschenbach et al. 2001). Extrapolating these results to the present findings could mean that somehow activation of G_M-associated PP1 is hampered in McArdle patients. In the present study, PP1 activity is measured as total activity, meaning that changes in activity of targeted subpopulations of PP1 is not picked up. Also, as the homogenizing buffer does not have protein phosphatase inhibitors any effect due to change in phosphorylation of PP1 could be lost. It could be hypothesized that the chronic high muscle glycogen level present in McArdle patients constitutes a spatial hindrance for the interaction between G_M, PP1 and glycogen synthase, which could be essential for the exercise-induced increase in glycogen synthase activity. In this scenario, when glycogen is broken down during exercise in healthy muscle, G_M may be exposed to interaction with PP1 and glycogen synthase, leading to dephosphorylation and activation of glycogen synthase.

During exercise, glucose utilization was higher in McArdle patients than in controls working at the same absolute workload, as has been described previously (Vissing et al. 1992). The higher glucose utilization was not associated with higher levels of total GLUT4 protein. However, this does not exclude the possibility that an increased amount of GLUT4 is localized to the plasma membrane in the patients, as this was not evaluated. From a teleological standpoint it seems reasonable that plasma glucose utilization is increased in the patients since the utilization of muscle glycogen, the major carbohydrate source is abolished in the patients. The understanding of the mechanism by which the glucose utilization is increased is, however, ambiguous. In some, but not all (Hargreaves et al. 1995), studies, it has been observed that glucose uptake during exercise is inhibited by high glycogen concentrations (Gollnick et al. 1981; Blomstrand & Saltin, 1999; Richter et al. 2001b) but from the present study it is clear that the possible effect of high glycogen was overridden by absent glycogenolysis and the ensuing 'energy crisis' in McArdle patients. The observation that 2AMPK activity in vitro and in vivo, as reflected by ACC phosphorylation, was enhanced in McArdle patients lends support to the idea that AMPK mediates part of the exercise-induced glucose utilization signal. However, it cannot be excluded that both AMPK and glucose uptake are reacting to a common stimulus such as the cellular energy charge. Thus, the two parameters could be causally unrelated, although they co-vary. Activation of AMPK by muscle contraction and other interventions has been associated with increased glucose uptake in rodent skeletal muscle (reviewed in Goodyear, 2000). On the other hand our findings also show that the inhibitory effect that glycogen exerts on AMPK activity during exercise, observed in rodents (Derave et al. 2000; Kawanaka et al. 2000) and humans (Richter et al. 2001b) is offset when glycogenolysis is blocked. Although the different mean measures of AMPK activity and glucose utilization co-vary in the present study, the lack of a significant correlation between individual values of glucose utilization (expressed per kg body wt or per kg lean body mass) and AMPK activity (1 or 2) or ACC phosphorylation argues against AMPK as an important regulator of exercise-induced glucose uptake. It could be argued that the lack of correlation is due to glucose utilization being measured at the whole-body level and not as muscle-specific uptake. However the lack of correlation is in concordance with observations from our laboratory where leg and not whole-body glucose uptake was measured. In that study it was observed that a wide difference in muscle glycogen level in exercising healthy humans was indeed associated with wide differences in both leg glucose uptake and AMPK activity, but also in this situation there was no correlation between these parameters (Richter et al. 2001b). Furthermore, we previously also observed a dissociation of AMPK activity and contraction-induced glucose transport in perfused slow-twitch rat muscle (Derave et al. 2000). Interestingly, a recent study in transgenic animals demonstrated that ~40 % of the glucose transport in mice skeletal muscle induced by electrical stimulation was abolished by expression of a dominant inhibitory mutant of AMPK, whereas hypoxia-stimulated glucose transport was totally absent (Mu et al. 2001). Thus, based on the study by Mu et al. (2001) AMPK seems to play a role in contraction-induced glucose uptake, but it is also clear that other regulatory mechanisms are involved.

A unifying hypothesis would be that glucose uptake in human skeletal muscle during exercise cannot be explained by AMPK activation alone, but AMPK may play a role in inhibition of glycogenesis by phosphorylation of glycogen synthase. As exercise ceases, AMPK activity and glucose uptake is sustained for a short period, but the inhibition of glycogen synthase is rapidly overridden, possibly by an increase in activity of G_M-associated PP1. The decreased glycogen level may cause a redistribution of the key enzymes in glycogen metabolism, which enables this. In support of this, glycogen synthase has been shown to translocate in skeletal muscle in response to glycogen depletion (Nielsen et al. 2001).

In conclusion, exercise-induced activity of 2AMPK and ACC phosphorylation are increased in McArdle patients characterized by chronic high glycogen stores and blocked glycogenolysis. Based on previous findings in vitro, the increased glucose utilization during exercise in these patients could be suggested to be due to the higher activation of AMPK. However, the lack of correlation between AMPK activity and ACC phosphorylation on one hand and glucose utilization on the other hand indicates the involvement of additional mechanisms in exercise-induced muscle glucose uptake. The data also suggest that glycogen synthase activity is depressed during exercise by AMPK and furthermore, that an increase in glycogen synthase activity with exercise requires functional glycogenolysis.

	REFERENCES

Top Abstract Introduction Methods Results Discussion References

ASCHENBACH, W. G., SUZUKI, Y., BREEDEN, K., PRATS, C., HIRSHMAN, M. F., DUFRESNE, S. D., SAKAMOTO, K., VILARDO, P. G., STEELE, M., KIM, J. H., JING, S. S., GOODYEAR, L. J. & DEPAOLI-ROACH, A. A. (2001). The muscle-specific protein phosphatase PP1G/RGL (GM) is essential for activation of glycogen synthase by exercise. Journal of Biological Chemistry 276, 39959-39967	[Abstract/Full Text]
BAK, J. F. & PEDERSEN, O. (1990). Exercise-enhanced activation of glycogen synthase in human skeletal muscle. American Journal of Physiology 258, E957-963	[Medline]
BEGUM, N. (1995). Stimulation of protein phosphatase-1 activity by insulin in rat adipocytes. Evaluation of the role of mitogen-activated protein kinase pathway. Journal of Biological Chemistry 270, 709-714	[Abstract/Full Text]
BERGERON, R., RUSSELL, R. R. III, YOUNG, L. H., REN, J. M., MARCUCCI, M., LEE, A. & SHULMAN, G. I. (1999). Effect of AMPK activation on muscle glucose metabolism in conscious rats. American Journal of Physiology 276, E938-944	[Medline]
BIER, D. M., LEAKE, R. D., HAYMOND, M. W., ARNOLD, K. J., GRUENKE, L. D., SPERLING, M. A. & KIPNIS, D. M. (1977). Measurement of 'true' glucose production rates in infancy and childhood with 6,6-dideuteroglucose. Diabetes 26, 1016-1023	[Abstract]
BLOMSTRAND, E. & SALTIN, B. (1999). Effect of muscle glycogen on glucose, lactate and amino acid metabolism during exercise and recovery in human subjects. Journal of Physiology 514, 293-302	[Abstract/Full Text]
BOGARDUS, C., THUILLEZ, P., RAVUSSIN, E., VASQUEZ, B., NARIMIGA, M. & AZHAR, S. (1983). Effect of muscle glycogen depletion on in vivo insulin action in man. Journal of Clinical Investigation 72, 1605-1610	[Medline]
CARLING, D. & HARDIE, D. G. (1989). The substrate and sequence specificity of the AMP-activated protein kinase. Phosphorylation of glycogen synthase and phosphorylase kinase. Biochimica et Biophysica Acta 1012, 81-86	[Medline]
CHEN, Z., HEIERHORST, J., MANN, R. J., MITCHELHILL, K. I., MICHELL, B. J., WITTERS, L. A., LYNCH, G. S., KEMP, B. E. & STAPLETON, D. (1999). Expression of the AMP-activated protein kinase beta1 and beta2 subunits in skeletal muscle. FEBS Letters 460, 343-348	[Medline]
DANFORTH, W. H. (1965). Glycogen synthase activity in skeletal muscle. Interconversion of two forms and control of glycogen synthesis. Journal of Biological Chemistry 240, 588-593
DERAVE, W., AI, H., IHLEMANN, J., WITTERS, L. A., KRISTIANSEN, S., RICHTER, E. A. & PLOUG, T. (2000). Dissociation of AMP-activated protein kinase activation and glucose transport in contracting slow-twitch muscle. Diabetes 49, 1281-1287	[Abstract]
FUJII, N., HAYASHI, T., HIRSHMAN, M. F., SMITH, J. T., HABINOWSKI, S. A., KAIJSER, L., MU, J., LJUNGQVIST, O., BIRNBAUM, M. J., WITTERS, L. A., THORELL, A. & GOODYEAR, L. J. (2000). Exercise induces isoform-specific increase in 5'AMP-activated protein kinase activity in human skeletal muscle. Biochemical and Biophysical Research Communications 273, 1150-1155	[Medline]
FURLER, S. M., GOLDSTEIN, M., COONEY, G. J. & KRAEGEN, E. W. (1998). In vivo quantification of glucose uptake and conversion to glycogen in individual muscles of the rat following exercise. Metabolism: Clinical and Experimental 47, 409-414	[Medline]
GOLLNICK, P. D., PERNOW, B., ESSÉN, B., JANSSON, E. & SALTIN, B. (1981). Availability of glycogen and plasma FFA for substrate utilization in leg muscle of man during exercise. Clinical Physiology 1, 27-42
GOODYEAR, L. J. (2000). AMP-activated protein kinase: a critical signaling intermediary for exercise-stimulated glucose transport? Exercise and Sport Science Reviews 28, 113-116
HAYASHI, T., HIRSHMAN, M. F., FUJII, N., HABINOWSKI, S. A., WITTERS, L. A. & GOODYEAR, L. J. (2000). Metabolic stress and altered glucose transport: activation of AMP-activated protein kinase as a unifying coupling mechanism. Diabetes 49, 527-531	[Abstract]
HAYASHI, T., HIRSHMAN, M. F., KURTH, E. J., WINDER, W. W. & GOODYEAR, L. J. (1998). Evidence for 5' AMP-activated protein kinase mediation of the effect of muscle contraction on glucose transport. Diabetes 47, 1369-1373	[Abstract]
KATZ, A. & RAZ, I. (1995). Rapid activation of glycogen synthase and protein phosphatase in human skeletal muscle after isometric contraction requires an intact circulation. Pflügers Archiv 431, 259-265	[Medline]
KAWANAKA, K., NOLTE, L. A., HAN, D. H., HANSEN, P. A. & HOLLOSZY, J. O. (2000). Mechanisms underlying impaired GLUT-4 translocation in glycogen-supercompensated muscles of exercised rats. American Journal of Physiology - Endocrinology and Metabolism 279, E1311-1318	[Medline]
KEMP, B. E., MITCHELHILL, K. I., STAPLETON, D., MICHELL, B. J., CHEN, Z. P. & WITTERS, L. A. (1999). Dealing with energy demand: the AMP-activated protein kinase. Trends in Biochemical Sciences 24, 22-25	[Medline]
KIDA, Y., KATZ, A., LEE, A. D. & MOTT, D. M. (1989). Contraction-mediated inactivation of glycogen synthase is accompanied by inactivation of glycogen synthase phosphatase in human skeletal muscle. Biochemical Journal 259, 901-904	[Medline]
KOCHAN, R. G., LAMB, D. R., LUTZ, S. A., PERRILL, C. V., REIMANN, E. M. & SCHLENDER, K. K. (1979). Glycogen synthase activation in human skeletal muscle: effects of diet and exercise. American Journal of Physiology 236, E660-666	[Medline]
KRISTIANSEN, S., ASP, S. & RICHTER, E. A. (1996). Decreased muscle GLUT-4 content and contraction-induced glucose transport after eccentric contractions. American Journal of Physiology 271, R477-482	[Medline]
KURTH-KRACZEK, E. J., HIRSHMAN, M. F., GOODYEAR, L. J. & WINDER, W. W. (1999). 5' AMP-activated protein kinase activation causes GLUT4 translocation in skeletal muscle. Diabetes 48, 1667-1671	[Abstract]
MARKUNS, J. F., WOJTASZEWSKI, J. F. & GOODYEAR, L. J. (1999). Insulin and exercise decrease glycogen synthase kinase-3 activity by different mechanisms in rat skeletal muscle. Journal of Biological Chemistry 274, 24896-24900	[Abstract/Full Text]
MCARDLE, B. (1951). Myopathy due to a defect in muscle glycogen breakdown. Clinical Science 10, 13-33
MERRILL, G. F., KURTH, E. J., HARDIE, D. G. & WINDER, W. W. (1997). AICA riboside increases AMP-activated protein kinase, fatty acid oxidation, and glucose uptake in rat muscle. American Journal of Physiology 273, E1107-1112	[Medline]
MU, J., BROZINICK, J. T. JR, VALLADARES, O., BUCAN, M. & BIRNBAUM, M. J. (2001). A role for AMP-activated protein kinase in contraction- and hypoxia-regulated glucose transport in skeletal muscle. Molecular cell 7, 1085-1094	[Medline]
MUNGER, R., TEMLER, E., JALLUT, D., HAESLER, E. & FELBER, J. P. (1993). Correlations of glycogen synthase and phosphorylase activities with glycogen concentration in human muscle biopsies. Evidence for a double- feedback mechanism regulating glycogen synthesis and breakdown. Metabolism: Clinical and Experimental 42, 36-43
MUSI, N., FUJII, N., HIRSHMAN, M. F., EKBERG, I., FROBERG, S., LJUNGQVIST, O., THORELL, A. & GOODYEAR, L. J. (2001). AMP-activated protein kinase (AMPK) is activated in muscle of subjects with type 2 diabetes during exercise. Diabetes 50, 921-927	[Abstract/Full Text]
NIELSEN, J. N., DERAVE, W., KRISTIANSEN, S., RALSTON, E., PLOUG, T. & RICHTER, E. A. (2001). Glycogen synthase localization and activity in rat skeletal muscle is strongly dependent on glycogen content. Journal of Physiology 531, 757-769	[Abstract/Full Text]
NIELSEN, J. N., VISSING, J., WOJTASZEWSKI, J. F. P., HALLER, R. G., BEGUM, N. & RICHTER, E. A. (2002). Decreased insulin action in skeletal muscle from patients with McArdle's disease. American Journal of Physiology - Endocrinology and Metabolism 282, E1267-1275	[Abstract/Full Text]
PASSONNEAU, J. V. & LOWRY, O. H. (1993). Enzymatic Analysis - a Practical Guide. Humana Press Inc., Totowa, NJ, USA
RICHTER, E. A., DERAVE, W. & WOJTASZEWSKI, J. F. (2001a). Glucose, exercise and insulin: emerging concepts. Journal of Physiology 535, 313-322	[Abstract/Full Text]
RICHTER, E. A., GARETTO, L. P., GOODMAN, M. N. & RUDERMAN, N. B. (1984). Enhanced muscle glucose metabolism after exercise: modulation by local factors. American Journal of Physiology 246, E476-482	[Medline]
RICHTER, E. A., MACDONALD, C., KIENS, B., HARDIE, D. G. & WOJTASZEWSKI, J. F. P. (2001b). Dissociation of 5'AMP-activated protein kinase activity and glucose clearance in human skeletal muscle during exercise. Diabetes 50, A62
RICHTER, E. A., MIKINES, K. J., GALBO, H. & KIENS, B. (1989). Effect of exercise on insulin action in human skeletal muscle. Journal of Applied Physiology 66, 876-885	[Medline]
ROEPSTORFF, C., STEFFENSEN, C. H., MADSEN, M., STALLKNECHT, B., KANSTRUP, I. L., RICHTER, E. A. & KIENS, B. (2002). Gender differences in substrate utilization during submaximal exercise in endurance-trained subjects. American Journal of Physiology - Endocrinology and Metabolism 282, E435-447	[Abstract/Full Text]
ROMIJN, J. A., COYLE, E. F., SIDOSSIS, L. S., GASTALDELLI, A., HOROWITZ, J. F., ENDERT, E. & WOLFE, R. R. (1993). Regulation of endogenous fat and carbohydrate metabolism in relation to exercise intensity and duration. American Journal of Physiology 265, E380-391	[Medline]
SHIMIZU, S., INOUE, K., TANI, Y. & YAMADA, H. (1979). Enzymatic microdetermination of serum free fatty acids. Analytical Biochemistry 98, 341-345	[Medline]
SIRI, W. E. (1956). The gross composition of the body. Advances in Biological and Medical Physics 4, 239-280
STEELE, R. (1959). Influences of glucose loading and of injected insulin on hepatic glucose output. Annals of the New York Academy of Sciences 82, 420-430
THOMAS, J. A., SCHLENDER, K. K. & LARNER, J. (1968). A rapid filter paper assay for UDPglucose-glycogen glucosyltransferase, including an improved biosynthesis of UDP-14C-glucose. Analytical Biochemistry 25, 486-499	[Medline]
VISSING, J., LEWIS, S. F., GALBO, H. & HALLER, R. G. (1992). Effect of deficient muscular glycogenolysis on extramuscular fuel production in exercise. Journal of Applied Physiology 72, 1773-1779	[Medline]
WINDER, W. W. & HARDIE, D. G. (1999). AMP-activated protein kinase, a metabolic master switch: possible roles in type 2 diabetes. American Journal of Physiology 277, E1-10	[Medline]
WOJTASZEWSKI, J. F., JORGENSEN, S. B., HELLSTEN, Y., HARDIE, D. G. & RICHTER, E. A. (2002). Glycogen-Dependent Effects of 5-aminoimidazole-4-carboxamide (AICA)-riboside on AMP-activated protein kinase and glycogen synthase activities in rat skeletal muscle. Diabetes 51, 284-292	[Abstract/Full Text]
WOJTASZEWSKI, J. F., NIELSEN, P., HANSEN, B. F., RICHTER, E. A. & KIENS, B. (2000). Isoform-specific and exercise intensity-dependent activation of 5'-AMP- activated protein kinase in human skeletal muscle. Journal of Physiology 528, 221-226	[Abstract/Full Text]
WOJTASZEWSKI, J. F. P., NIELSEN, P., KIENS, B. & RICHTER, E. A. (2001). Regulation of glycogen synthase kinase-3 in human skeletal muscle: effects of food intake and bicycle exercise. Diabetes 50, 265-269	[Abstract/Full Text]
WOODS, A., SALT, I., SCOTT, J., HARDIE, D. G. & CARLING, D. (1996). The alpha1 and alpha2 isoforms of the AMP-activated protein kinase have similar activities in rat liver but exhibit differences in substrate specificity in vitro. FEBS Letters 397, 347-351	[Medline]
YAN, Z., SPENCER, M. K., BECHTEL, P. J. & KATZ, A. (1993). Regulation of glycogen synthase in human muscle during isometric contraction and recovery. Acta Physiologica Scandinavia 147, 77-83
YAN, Z., SPENCER, M. K. & KATZ, A. (1992). Effect of low glycogen on glycogen synthase in human muscle during and after exercise. Acta Physiologica Scandinavia 145, 345-352
YEAMAN, S. J., ARMSTRONG, J. L., BONAVAUD, S. M., POINASAMY, D., PICKERSGILL, L. & HALSE, R. (2001). Regulation of glycogen synthesis in human muscle cells. Biochemical Society Transactions 29, 537-541	[Medline]
ZACHWIEJA, J. J., COSTILL, D. L., PASCOE, D. D., ROBERGS, R. A. & FINK, W. J. (1991). Influence of muscle glycogen depletion on the rate of resynthesis. Medicine and Science in Sports and Exercise 23, 44-48	[Medline]

Acknowledgements

This study was supported by a Research and Technological Development Project (QLG1-CT-2001-01488) funded by the European Commission and grant no. 504-14 from the Danish National Research Foundation and the Media and Grants Secretariat of the Danish Ministry of Culture. J.F.P.W. was supported by a postdoctoral fellowship from the Danish Medical Research Council. A Programme Grant from the Wellcome Trust supported D.G.H. Betina Bolmgren, Eva Rathkens Nielsen and Irene Beck-Nielsen are acknowledged for excellent technical contributions.

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