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Am J Physiol Endocrinol Metab 293: E286-E292, 2007. First published April 3, 2007; doi:10.1152/ajpendo.00693.2006
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Caffeine-induced Ca2+ release increases AMPK-dependent glucose uptake in rodent soleus muscle

Thomas E. Jensen, Adam J. Rose, Ylva Hellsten, Jørgen F. P. Wojtaszewski, and Erik A. Richter

Department of Exercise and Sport Sciences, Copenhagen Muscle Research Center, Section of Human Physiology, University of Copenhagen, Copenhagen, Denmark

Submitted 19 December 2006 ; accepted in final form 19 March 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Previous studies have proposed that caffeine-induced activation of glucose transport in skeletal muscle is independent of AMP-activated protein kinase (AMPK) because {alpha}-AMPK Thr172 phosphorylation was not increased by caffeine. However, our previous studies, as well as the present, show that AMPK phosphorylation measured in whole muscle lysate is not a good indicator of AMPK activation in rodent skeletal muscle. In lysates from incubated rat soleus muscle, a predominant model in previous caffeine-studies, both acetyl-CoA carboxylase-beta (ACCbeta) Ser221 and immunoprecipitated {alpha}1-AMPK activity increased with caffeine incubation, without changes in AMPK phosphorylation or immunoprecipitated {alpha}2-AMPK activity. This pattern was also observed in mouse soleus muscle, where only ACCbeta and {alpha}1-AMPK phosphorylation were increased following caffeine treatment. Preincubation with the selective CaMKK inhibitor STO-609 (5 µM), the CaM-competitive inhibitor KN-93 (10 µM), or the SR Ca2+ release blocking agent dantrolene (10 µM) all inhibited ACCbeta phosphorylation and {alpha}1-AMPK phosphorylation, suggesting that SR Ca2+ release may work through a CaMKK-AMPK pathway. Caffeine-stimulated 2-deoxyglucose (2DG) uptake reflected the AMPK activation pattern, being increased with caffeine and inhibited by STO-609, KN-93, or dantrolene. The inhibition of 2DG uptake is likely causally linked to AMPK activation, since muscle-specific expression of a kinase-dead AMPK construct greatly reduced caffeine-stimulated 2DG uptake in mouse soleus. We conclude that a SR Ca2+-activated CaMKK may control {alpha}1-AMPK activation and be necessary for caffeine-stimulated glucose uptake in mouse soleus muscle.

adenosine 5'-monophosphate-activated protein kinase; STO-609; KN-93; dantrolene; calcium/calmodulin kinase kinase

SARCOPLASMIC RETICULUM (SR) Ca2+ release, a prerequisite for skeletal muscle contraction, may signal to increase skeletal muscle glucose uptake (8, 21). Although cell culture-based studies have suggested the involvement of conventional PKC isoforms, but not AMP-activated protein kinase (AMPK) or CaMKII in this process in L6 myotubes devoid of sarcomeres (15, 29), recent studies in incubated mature rat skeletal muscle have proposed a model in which two parallel signaling pathways, defined by CaMKII and AMPK, are necessary and sufficient to account for the entire contraction-stimulated increase in glucose uptake (32, 33). In those studies, the authors have suggested that CaMKII and AMPK each account for approximately one-half of the contraction increase in glucose uptake in glycolytic rat epitroclearis, whereas CaMKII accounts for all of the contraction increase in oxidative rat soleus muscle. A crucial control in these studies was that stimulation of SR Ca2+ release below the contraction threshold by caffeine increases glucose uptake without an apparent increase in {alpha}-AMPK Thr172 phosphorylation in incubated skeletal muscle strips from outbred rat (4, 32, 33), thus defining the two parallel pathways.

Meanwhile, a recent study by Koh et al. (16) demonstrated in muscle-specific LKB1 knockout (KO) mice that {alpha}1-AMPK is still partially activated by contraction in the absence of a change in total {alpha}-AMPK Thr172 phosphorylation measured in lysates. In agreement, in human skeletal muscle we recently estimated that {alpha}1-AMPK-containing complexes account for a small fraction, at most ~15%, of the total AMPK complexes and that changes in AMPK activity associated with different AMPK heterotrimers could be observed in the absence of changes in AMPK phosphorylation (3). Also, we presented evidence in our related study (11) that the CaM-competitive inhibitor KN-93, as well as the CaMKK inhibitor STO-609, inhibit {alpha}-AMPK Thr172 phosphorylation at 2 min of ex vivo contraction, particularly in the oxidative soleus muscle. Therefore, we decided to reinvestigate the effect of caffeine treatment on various measurements of AMPK activation, hypothesizing that SR Ca2+ release may cause an isoform-specific AMPK activation that was missed in earlier studies evaluating total {alpha}-AMPK Thr172 phosphorylation in lysates.

This study demonstrates that subcontraction caffeine treatment activates {alpha}1-AMPK, but not {alpha}2-AMPK, and glucose uptake in a STO-609-, KN-93-, and dantrolene-preventable manner in mouse soleus muscle. Furthermore, the effect of caffeine was almost absent in soleus muscle from mice overexpressing a kinase-dead AMPK construct, directly implicating a connection between Ca2+ and AMPK. Importantly, we have resolved the discrepancy between our current findings and previous caffeine incubation studies in outbred rats by demonstrating that assessments of only AMPK phosphorylation in lysate as an index of AMPK activation in outbred rat muscle hold the risk of committing a statistical type 2 error, i.e., not detecting a difference when present.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. C57BL/6 mice overexpressing a kinase-dead Lys45Arg mutant {alpha}2 protein, driven by the heart and skeletal muscle-specific creatine kinase promoter, have been described previously (19), and founder mice were a kind gift from Morris J. Birnbaum (University of Pennsylvania School of Medicine, Pittsburgh, PA). Female hemizygous transgenic mice and wild-type (WT) mice (12–16 wk of age) used were littermates from intercross breeding of hemizygous transgenic mice and WT mice. The animals were maintained on a 10:14-h light-dark cycle and received standard rodent diet (Altromin no. 1324; Chr. Pedersen, Ringsted, Denmark) and water ad libitum. Nontransgenic C57BL/6 female mice (12–16 wk old) or male Sprague-Dawley rats (70–90 g) from Taconic Europe were used for all other experiments. All experiments were approved by the Danish Animal Experimental Inspectorate and complied with the "European Convention for the Protection of Vertebrate Animals Used for Experiments and Other Scientific Purposes."

Muscle incubation. Soleus muscles were obtained from fed anesthetized mice or rats (6 mg pentobarbital/100 g body wt) and suspended at resting tension (4–5 mN) in incubation chambers (Multi Myograph system; Danish Myo-Technology, Aarhus, Denmark) in Krebs-Henseleit buffer supplemented with 2 mM pyruvate and 8 mM mannitol at 30°C, as described previously (11). The muscles were incubated for 1 h with 10 µM KN-93 (Calbiochem, Nottingham, UK), 5 µM STO-609 (Calbiochem), 10 µM dantrolene (Sigma, St. Louis, MO), or corresponding amounts of DMSO (0.1–0.15%) as a vehicle control. The incubation chambers were kept dark to avoid degradation of light-sensitive compounds. Muscles were then stimulated with 3 mM caffeine for 15 min, added from 100 mM caffeine stock solution (Sigma) to the chambers.

2-Deoxyglucose uptake. 2-Deoxyglucose (2DG) uptake was measured for 10 min after 0 or 15 min of caffeine treatment, as described previously (13).

Muscle analyses. At 0 min or after 15 min of caffeine treatment, muscles were frozen in liquid nitrogen and stored at –80°C. Muscles were processed into lysates, as described previously (11).

Immunoblotting and antibodies. Total protein and phosphorylation levels of relevant proteins were determined using standard immunoblotting techniques, as described previously (11). {alpha}1- and {alpha}2-AMPK were immunoprecipitated with sheep polyclonal antibodies kindly donated by D. G. Hardie (Dundee, UK) (31). Phosphorylation of {alpha}-AMPK Thr172 was measured using a phospho-specific antibody (Cell Signaling Technology, Beverly, MA). Acetyl-CoA carboxylase-beta (ACCbeta) Ser221 phosphorylation was determined using a phospho-specific antibody (Upstate Biotechnologies, Lake Placid, NY).

{alpha}1- and {alpha}2-AMPK activity. Isoform-specific {alpha}-AMPK activity was measured in vitro in sequential immunoprecipitations from 200 µg of muscle lysate protein, as described previously (11). The AMPK activities were normalized to a basal and in situ-stimulated rat gastrocnemius muscle standard.

{alpha}1- and {alpha}2-AMPK-specific phosphorylation. Isoform-specific {alpha}-AMPK phosphorylation was measured in sequential immunoprecipitations from 75 µg of muscle lysate protein using 1 µg of anti-{alpha}1- or anti-{alpha}2-AMPK antibody. Immunoprecipitates were washed as for measurements of AMPK activity, after which 30 µl of Laemmli buffer was added to each tube. {alpha}-AMPK Thr172 phosphorylation was then assessed using standard immunoblotting. One-third of the solubilized immunoprecipitate was loaded for {alpha}2-AMPK, and all of the immunoprecipitate for {alpha}1-AMPK, to get comparable signal.

Nucleotide measurement. Frozen muscles from the incubation experiments were freeze-dried, extracted with perchloric acid, and neutralized with KOH, after which ATP, ADP, AMP, and IMP were determined using reverse-phase high-performance liquid chromatography, as described previously (26).

Statistical analysis. Results are means ± SE. Blot data were normalized by assigning the mean of the reference group the value of 1, expressing values for the other groups relative to the reference group, and then adding the relative standard error. Statistical testing was performed using unpaired or paired Student's t-tests or two-way ANOVA as appropriate. Statistical evaluation was performed using SPSS 13.0. The significance level was set at {alpha} = 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Similar to prior studies, incubating with 3 mM caffeine for 15 min did not significantly increase AMPK phosphorylation in rat soleus muscle (Fig. 1A). However, in our study, some individual pairs of muscles clearly displayed a higher {alpha}-AMPK Thr172 phosphorylation (in Fig. 1A, the blot shows 2 of the 8 pairs of rat soleus muscles with clear increases in AMPK phosphorylation), leading us to assess other measures of AMPK activation as well. ACCbeta Ser221 phosphorylation, a measure of endogenous AMPK activity, was higher with caffeine treatment (Fig. 1B). Interestingly, this elevation was attributable to higher {alpha}1-AMPK activity but not {alpha}2-AMPK activity (Fig. 1C). These data indicate that previous studies may have missed an increase in AMPK activity as the result of measurements of total {alpha}-AMPK Thr172 phosphorylation alone.


Figure 1
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  		Fig. 1. Caffeine increases {alpha}1-AMP-activated protein kinase (AMPK) activity and acetyl-CoA carboxylase-beta (ACCbeta) phosphorylation in rat soleus muscle. Basal and caffeine-stimulated (3 mM, 15 min) {alpha}-AMPK Thr172 phosphorylation (pAMPK; A), ACCbeta Ser221 phosphorylation (pACC; B) and {alpha}1- and {alpha}2-AMPK activities (C) were determined in rat soleus muscles (n = 8). Representative blot shows 2 of 8 pairs of muscles. *P < 0.05, caffeine vs. basal.

 
Because CaMKKs have been shown to act as AMPKKs in various cell types, we next assessed the effect of the selective CaMKK inhibitor STO-609, as well as the CaM-competitive inhibitor KN-93 or the SR Ca2+ release blocker dantrolene, on measures of AMPK activation in C57BL/6 mouse soleus muscle. Our initial caffeine dose-response and time-response trials revealed that caffeine had more pronounced effects on AMPK activation in mouse soleus than in extensor digitorum longus (EDL) muscle (data not shown), leading us to focus on the former. Resting tension was not increased in incubated mouse muscles at the 3-mM caffeine concentration used (data not shown). {alpha}-AMPK Thr172 phosphorylation showed a more consistent trend toward a higher level with caffeine treatment in inbred mice than in outbred rats (Fig. 2A, P = 0.07). In addition, this increment appeared to be reduced by all three inhibitors used (Fig. 2A). As in rats, ACCbeta phosphorylation was higher with caffeine treatment, with a varying effect of the inhibitors on ACCbeta phosphorylation (Fig. 2B). When measured in immunoprecipitates, a higher {alpha}1-AMPK Thr172 phosphorylation, but not {alpha}2-AMPK Thr172 phosphorylation, was observed with caffeine treatment alone, but not after preincubation with STO-609, KN-93, or dantrolene (Fig. 2C). These findings imply that a SR Ca2+-activated kinase, most likely CaMKK, plays a role in activating {alpha}1-AMPK, but not {alpha}2-AMPK, during caffeine stimulation.


Figure 2
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  		Fig. 2. Caffeine increases {alpha}1-AMPK activity and ACCbeta phosphorylation in mouse soleus in a STO-609-, KN-93-, and dantrolene-sensitive manner. Basal and caffeine-stimulated (3 mM, 15 min) {alpha}-AMPK Thr172 phosphorylation (A), ACCbeta Ser221 phosphorylation (B), and immunoprecipitated (IP) {alpha}1- and {alpha}2-AMPK-specific phosphorylation (C) were determined in mouse soleus pretreated with 5 µM STO-609, 10 µM KN-93, or 10 µM dantrolene (n = 8–9). *P < 0.05, caffeine vs. basal. Caff, caffeine; STO, STO-609; KN, KN-93; Dan, dantrolene.

 
To assess the functional consequence of AMPK inhibition, we measured 2DG uptake in muscles pretreated with the inhibitors assessed above. STO-609 largely prevented the increase in 2DG uptake with caffeine, as did KN-93 and dantrolene (Fig. 3A). The KN-93 and dantrolene data confirm previous findings in rat soleus muscle (32). Next, we attempted to confirm a causal link between AMPK and caffeine-stimulated glucose uptake by evaluating caffeine-stimulated glucose uptake in muscles that overexpress a kinase-dead AMPK (KD AMPK) construct. In soleus muscle from the AMPK KD mice, a large reduction in caffeine-stimulated 2DG uptake was observed compared with WT soleus muscles (Fig. 3B). This indicates that SR Ca2+ release increases glucose uptake through a mechanism requiring AMPK activation, likely by CaMKKs.


Figure 3
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  		Fig. 3. Inhibition of caffeine-stimulated 2-deoxyglucose (2DG) uptake by STO-609, KN-93, dantrolene, and kinase-dead (KD) AMPK expression. Basal and caffeine-stimulated (3 mM, 15 min) 2DG uptake were determined in mouse soleus muscles pretreated with 5 µM STO-609, 10 µM KN-93, or 10 µM dantrolene (A) and in wild-type vs. KD AMPK soleus (n = 6–13). **P < 0.01, caffeine effect. {dagger}P < 0.05, caffeine x genotype interaction.

 
Caffeine treatment has previously been shown not to affect nucleotide status in incubated rat muscle (32). To test whether caffeine treatment affected nucleotide status in incubated WT and KD AMPK-expressing mouse soleus muscles, we measured nucleotides in muscles incubated with or without caffeine (Fig. 4). Importantly, in WT soleus muscles, caffeine had no significant effect on any of the nucleotide levels measured. In contrast, KD AMPK-expressing soleus muscles showed significant increases in AMP and IMP with caffeine, suggesting that AMPK signaling-deficient muscles are less proficient at maintaining energy homeostasis when challenged with a constant low-level Ca2+ release from the SR.


Figure 4
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  		Fig. 4. Caffeine increases AMP and IMP in KD AMPK-expressing but not wild-type mouse soleus muscles. Basal and caffeine-stimulated (3 mM, 15 min) changes in ATP (A), ADP (B), AMP (C), and IMP (D) were determined in wild-type vs. KD AMPK soleus (n = 7). *P < 0.05, caffeine vs. basal. d.w., Dry weight.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study suggests that in rodent skeletal muscle, a SR Ca2+ release-dependent kinase, most likely one or more CaMKK isoforms, signals upstream of AMPK to increase glucose transport. The evidence that KD AMPK expression greatly reduced the caffeine-induced glucose uptake indicates that AMPK is causally linked to the caffeine-stimulated glucose uptake response. The present study also demonstrates that evaluation of AMPK activation by measuring total {alpha}-AMPK Thr172 phosphorylation in whole muscle lysates is prone to statistical type 2 errors and thus may have resulted in false conclusions in previous caffeine incubation studies that caffeine does not activate AMPK (4, 28, 32, 33). With regard to the present study, it is important to note that probably neither STO-609 nor KN-93 are specific inhibitors, as discussed in our earlier report (11). Furthermore, another kinase, TAK1, which may act directly (18) or indirectly through LKB1 (34) on AMPK, has been shown to be activated downstream of CaMKII in a KN-93-preventable manner in HEK-293 cells (10). Clearly, genetic models of CaMKK and TAK1 deficiency are needed to clarify the importance of these kinases in the observed inhibitory profile in skeletal muscle.

An interesting question is why only {alpha}1-AMPK activity, not {alpha}2-AMPK activity, increases with caffeine stimulation. During contraction, there appears to be an intensity and time dependency in isoform-specific {alpha}-AMPK activation. Thus short-duration low-intensity twitch contraction has been shown to increase {alpha}1-AMPK activity but not AMP/ATP ratio or {alpha}2-AMPK activity, whereas tetanic contraction increases all three parameters (24). It is possible that activation of {alpha}2-AMPK requires a more global increase in Ca2+ levels than is provided by caffeine stimulation, low-intensity twitch contraction, and/or other changes associated with increased AMPK activation, likely an increase in AMP/ATP ratio or a decrease in glycogen (12).

LKB1 has been shown to be the major {alpha}2-AMPK kinase in mouse skeletal muscle during intense tetanic contraction in situ and ex vivo (13, 22, 23), In contrast, {alpha}1-AMPK activation assessed in mouse EDL muscle ex vivo is only partially affected by LKB1 KO (16). This is consistent with a working model in which multiple kinases act on {alpha}1- and {alpha}2-AMPK during contraction in an intensity- and/or time-dependent fashion, as discussed in our earlier report (11), with a kinase distinct from LKB1 playing a major role in rodent soleus muscle during caffeine stimulation and during low-intensity, short-duration contraction. If so, this kinase is probably able to act on both {alpha}1- and {alpha}2-AMPK early on during tetanic contraction, since both {alpha}1- and {alpha}2-AMPK activation at 2 min of tetanic contraction was reduced by STO-609 and KN-93 in the previously reported study (11). Because siRNA-mediated reductions in CaMKK inhibit Ca2+ activation of AMPK in LKB1-deficient HeLa cells (7, 9, 30), and because CaMKK is detectable in skeletal muscle (20) and STO-609 inhibits {alpha}-AMPK Thr172 phosphorylation during caffeine (present study) and short-term contraction stimulation ex vivo (11), we currently consider CaMKK the most likely non-LKB1 AMPKK.

A concern with caffeine stimulation and other methods potentially used to dissect out the Ca2+ dependency of various processes in skeletal muscle, e.g., W-7 (35), N-benzyl-p-toluene sulfonamide inhibition of cross-bridge cycling (36), or low-intensity twitch contraction (24), is that ATP-consuming SR Ca2+ pumping is likely to account for a large proportion of energy turnover (36). Hence, although many of these methods, including caffeine stimulation in our study and that of others (35), do not cause measurable changes in nucleotides, local increases in ATP turnover could potentially drive activation of AMPK. An increase in ATP turnover with caffeine is supported by the observed increase in AMP and IMP in KD AMPK-expressing muscles (Fig. 4). Also, this finding supports reports by our group and others showing that AMPK-deficient mouse models are less capable of maintaining energy homeostasis during exercise/contraction (14, 22). Thus, although these methods are valuable tools, they may not unequivocally distinguish between Ca2+- and energy turnover-activated signaling. Specifically, the increase in ACCbeta phosphorylation observed presently could be due to covalent {alpha}1-AMPK activation or allosteric activation of either catalytic {alpha}-AMPK isoform. Therefore, data obtained using these methods should be interpreted with caution.

5-Aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), an often used AMP-mimicking AMPK activator, clearly increases both {alpha}1- and {alpha}2-AMPK activity in mouse skeletal muscle (1, 13), but this increase is insufficient to increase glucose uptake in the absence of LKB1 or {alpha}2-AMPK (13, 22), suggesting that {alpha}1-AMPK is at most necessary, but not sufficient, to increase glucose uptake. In this study, since expression of the KD AMPK construct reduced both {alpha}1- and {alpha}2-AMPK expression and activity (11), it is not possible to discriminate between the importance of each isoform to the observed reduction in caffeine-stimulated glucose uptake. Interestingly, however, soleus muscles from {alpha}1-AMPK KO mice display ~20% reduction in 2DG uptake compared with WT during intense tetanic contraction ex vivo, suggesting that {alpha}1-AMPK may be partially required to increase glucose uptake in this muscle type (13). Our attempts to evaluate the specific role of {alpha}1-AMPK in caffeine-stimulated glucose uptake in {alpha}1-AMPK KO mice were unsuccessful, since in the SV/129 background, caffeine was not able to increase glucose transport even in WT (data not shown), despite its clear effect in the C57BL/6 background. Although unfortunate in regard to answering the scientific question at hand, a varying responsiveness to different chemical compounds is a well-described trait of inbred mouse strains (6). However, studies in the {alpha}1-AMPK KO mice using other means of raising intracellular Ca2+ levels in the absence of detectable changes in nucleotide status, for instance, the low-intensity, short-duration twitch contraction protocol utilized by Toyoda et al. (24), may be able to clarify the exact role of {alpha}1- vs. {alpha}2-AMPK to glucose uptake in rodent soleus muscle.

The inclination toward {alpha}1-AMPK activation in the absence of {alpha}2-AMPK activation with caffeine or low-intensity contraction (23) is very dissimilar to the AMPK isoform activation profile seen during in vivo exercise in humans (3, 5, 17, 25, 27), where {alpha}2-AMPK is more readily activated than {alpha}1-AMPK. Apart from species differences, there are obvious differences between the ex vivo contraction and in vivo exercise setting, including lack of perfusion as well as the integrated extramuscular milieu ex vivo, which may explain this. However, one intriguing possibility is that human quadriceps, the predominant muscle biopsy donor, is not representative of all human muscles, particularly the profound postural muscles, such as soleus muscle. Furthermore, the current study suggests that fiber-type composition per se is not a good predictor of caffeine responsiveness, because caffeine activation of {alpha}1-AMPK is conserved between mouse and rat soleus muscle, despite a markedly higher oxidative fiber-type percentage in rat soleus (1, 2). Rather, this could relate to muscle activation pattern and function.

Various Ca2+-activated signaling proteins, including conventional PKCs and CaMKs, have been proposed to be required for contraction-stimulated glucose uptake, as discussed previously (21). More recently, caffeine-stimulated glucose uptake in incubated rat soleus and EDL muscles was shown to require auto/paracrine activation of ErbB4 receptors by neuregulins (4). Interestingly, ErbB4 inhibition affected in situ contraction-stimulated glucose uptake more in soleus compared with EDL muscle, consistent with findings by others suggesting that Ca2+ signaling to glucose uptake is more predominant in soleus muscles (11, 32). Further studies are needed to chart the interrelationship between the various Ca2+-activated components.

In conclusion, this study showed increased {alpha}1-AMPK activation and ACCbeta phosphorylation in the absence of total {alpha}-AMPK Thr172 phosphorylation and {alpha}2-AMPK activation in response to caffeine stimulation. As we have previously reported in human muscle in vivo (3), this implies that {alpha}-AMPK Thr172 phosphorylation alone should not be used to evaluate AMPK activation in isolated muscle. Furthermore, in conjunction with the data in our earlier, related report (11), we propose that a SR Ca2+-activated kinase, most likely CaMKK, signals upstream of AMPK to increase glucose uptake in rodent soleus muscle.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by The Copenhagen Muscle Research Center, the Novo Nordisk Research Foundation, The Danish Diabetes Association, an Integrated Project (LSHM-CT-2004-005272) funded by the European Commission, The Lundbeck Foundation, and The Danish Medical and Natural Sciences Research Councils. J. F. P. Wojtaszewski was supported by a Hallas Møller Fellowship from The Novo Nordisk Foundation. A. J. Rose was supported by postdoctoral grants from the European Commission and the Carlsberg Foundation.


   ACKNOWLEDGMENTS
 
We acknowledge Karina Olsen for skilled technical assistance in measuring nucleotides.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. A. Richter, Dept. of Exercise and Sport Sciences, Copenhagen Muscle Research Center, Univ. of Copenhagen, Universitetsparken 13, 2100 Copenhagen, Denmark (e-mail: ERichter{at}ifi.ku.dk)

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.


   REFERENCES
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 DISCUSSION
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