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Am J Physiol Endocrinol Metab 292: E1308-E1317, 2007. First published January 9, 2007; doi:10.1152/ajpendo.00456.2006
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Possible CaMKK-dependent regulation of AMPK phosphorylation and glucose uptake at the onset of mild tetanic skeletal muscle contraction

Thomas E. Jensen,1 Adam J. Rose,1 Sebastian B. Jørgensen,1 Nina Brandt,1 Peter Schjerling,2,3 Jørgen F. P. Wojtaszewski,1 and Erik A. Richter1

1Department of Human Physiology, Copenhagen Muscle Research Centre, Institute of Exercise and Sport Sciences, University of Copenhagen, Copenhagen; 2Department of Molecular Muscle Biology, Copenhagen Muscle Research Centre, Rigshospitalet, Copenhagen; and 3Department of Medical Biochemistry and Genetics, Medical Muscle Research Cluster, Molecular Muscle Biology, University of Copenhagen, Copenhagen, Denmark

Submitted 29 August 2006 ; accepted in final form 8 January 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The Ca2+/calmodulin (CaM) competitive inhibitor KN-93 has previously been used to evaluate 5'-AMP-activated protein kinase (AMPK)-independent Ca2+-signaling to contraction-stimulated glucose uptake in muscle during intense electrical stimulation ex vivo. With the use of low-intensity tetanic contraction of mouse soleus and extensor digitorum longus (EDL) muscles ex vivo, this study demonstrates that KN-93 can potently inhibit AMPK phosphorylation and activity after 2 min but not 10 min of contraction while strongly inhibiting contraction-stimulated 2-deoxyglucose uptake at both the 2- and 10-min time points. These data suggest inhibition of Ca2+/CaM-dependent signaling events upstream of AMPK, the most likely candidate being the novel AMPK kinase CaM-dependent protein kinase kinase (CaMKK). CaMKK protein expression was detected in mouse skeletal muscle. Similar to KN-93, the CaMKK inhibitor STO-609 strongly reduced AMPK phosphorylation and activity at 2 min and less potently at 10 min. Pretreatment with STO-609 inhibited contraction-stimulated glucose uptake at 2 min in soleus, but not EDL, and in both muscles after 10 min. Neither KN-93 nor STO-609 inhibited 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside-stimulated glucose uptake, AMPK phosphorylation, or recombinant LKB1 activity, suggestive of an LKB1-independent effect. Finally, neither KN-93 nor STO-609 had effects on the reductions in glucose uptake seen in mice overexpressing a kinase-dead AMPK construct, indicating that the effects of KN-93 and STO-609 on glucose uptake require inhibition of AMPK activity. We propose that CaMKKs act in mouse skeletal muscle regulating AMPK phosphorylation and glucose uptake at the onset of mild tetanic contraction and that an intensity- and/or time-dependent switch occurs in the relative importance of AMPKKs during contraction.

KN-93; STO-609; calmodulin kinase kinase

CONTRACTION-INDUCED GLUCOSE uptake in isolated skeletal muscle is mediated by an insulin-independent mechanism that increases surface membrane glucose transporter 4 content. Studies in rodent muscles have outlined a working model in which signaling proteins activated by metabolic stress and Ca2+ govern contraction-stimulated glucose uptake (35).

Because the 5'-AMP-activated protein kinase (AMPK) initiates diverse homeostasis-preserving mechanisms in response to metabolic stress and is activated during contraction, its involvement in contraction-stimulated glucose uptake is an obvious hypothesis and has been studied extensively (17, 25). Although studies using various transgenic mouse models have demonstrated that glucose uptake stimulation by the adenosine analog 5-aminoimidazole- 4-carboxamide-1-beta-4-ribofuranoside (AICAR) requires expression of the catalytic {alpha}2-AMPK, the regulatory {gamma}3-subunit of AMPK, and the AMPK kinase (AMPKK) LKB1 (4, 26, 31, 37), the necessity of AMPK activity during contraction is more controversial. Hence, some transgenic mouse models present considerable effects of reduced total AMPK activity on contraction-stimulated glucose uptake (31, 37) while others do not (16, 26).

Increasing cytosolic Ca2+ concentration below the contraction threshold significantly enhances glucose uptake in isolated rat soleus and epitroclearis muscle without an apparent increase in AMPK phosphorylation (7, 56, 57, 59), suggesting that Ca2+ may also provide a signal to increase glucose uptake during contraction. Two studies utilizing the calmodulin (CaM)-competitive inhibitor of CaM-dependent protein kinase (CaMK) I, CaMKII, and CaMKIV (KN-62) or its more potent structural analog KN-93 (10, 19, 43, 49) have implicated the CaMKs in this respect (56, 57). Although KN compounds have been reported to inhibit AMPK activation in response to an intracellular Ca2+-increasing stimulus in pancreatic MIN-6 cells (29), KN-93 does not seem to affect AMPK phosphorylation in rat muscle during intense ex vivo contraction (57). It has been proposed (56, 57), based on the potency of the KN compounds and additivity studies using AICAR and caffeine, that AMPK and Ca2+ signaling each account for ~50% of the maximal contraction-stimulated glucose uptake response in the fast-twitch rat epitroclearis muscle, whereas CaMKs, but not AMPK, are the predominant mediators of contraction-stimulated glucose uptake in the slow-twitch rat soleus muscle.

Several recent studies have shown that Ca2+ signaling may activate AMPK by CaMK kinase (CaMKKs)-dependent phosphorylation (18, 21, 22, 54), and a growing number of studies indicate that AMPK activity may be coregulated by LKB1 and CaMKKs in various cell types (3, 5, 11, 29, 38, 42, 45). However, although CaMKK protein expression has been reported in human skeletal muscle (34), the striking lack of {alpha}2-AMPK activation and potent inhibition of glucose uptake during intense electrical stimulation of mouse muscles lacking LKB1 expression have so far suggested that LKB1 is the principal donor of AMPKK activity in mouse skeletal muscle during contraction (37, 48).

In this study, the effects of KN-93 and STO-609 on AMPK activation and glucose uptake during low-intensity tetanic ex vivo contraction were evaluated. Our data suggest that CaMKKs act in mouse skeletal muscle regulation of AMPK phosphorylation and glucose uptake at the onset of mild tetanic contraction and that an intensity- and/or time-dependent switch may occur in the relative importance of AMPKKs during contraction.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Muscle-specific kinase-dead {alpha}2-AMPK mice. 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 (31) and were a kind gift from Morris J. Birnbaum, Pennsylvania School of Medicine. Female hemizygous transgenic mice and wild-type mice (8–20 wk of age) used were littermates from intercross breeding of hemizygous transgenic mice and wild-type 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. 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 and extensor digitorum longus (EDL) muscles were obtained from fed anesthetized mice (6 mg pentobarbital sodium/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-Ringer-Henseleit (KRH) buffer supplemented with 0.1% BSA, 2 mM pyruvate, and 8 mM mannitol at 30°C, as described previously (26). The muscles were incubated for 1 h with 10 µM KN-93 (Calbiochem, Nottingham, UK), 5 µM STO-609 (Calbiochem), or a corresponding amount of DMSO as a vehicle control. The incubation chambers were kept dark to avoid degradation of light-sensitive compounds. Following the first hour, contractions were induced by electrical stimulation every 15 s with 1-s trains of 0.2-ms impulses delivered at 100 Hz (~30 V) for up to 10 min. For AICAR stimulation, the muscles were further incubated for 40 min in KRH buffer containing 2 mM AICAR with or without the inhibitors. To evaluate the effect of hook up at resting tension in our incubation apparatus on muscle signaling, muscles were harvested immediately postexcission or after 1 h incubation with or without being suspended at resting tension. Force development was measured during incubations by a force transducer hooked to one end of the muscles. Total (integrated) force production during 10 min of electrical stimulation was used to evaluate fatigue development.

2-Deoxyglucose uptake. 2-Deoxyglucose (2-DG) uptake was measured for 10 min immediately after cessation of either 0, 2, or 10 min of electrical stimulation or 40 min of AICAR treatment using radioactive tracers as described previously (26).

Muscle analyses. At 0, 2, and 10 min of contraction or 40 min of AICAR treatment, muscles were frozen with aluminum tongs precooled in liquid nitrogen and stored at –80°C. Muscles were homogenized (PT 3100; Brinkmann Instruments) in ice-cold buffer (50 mM Tris·HCl, 10% glycerol, 1 mM EDTA, 5 mM EGTA, 50 mM NaF, 5 mM Na4P2O7, 2 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithithreitol, 1 mM benzamidine, 1% Nonidet P-40, and protease inhibitor cocktail, pH 7.4) and rotated end over end for 1 h at 4°C. Lysates were generated by centrifugation (17,500 g) for 1 h at 4°C.

Immunoblotting. Total protein and phosphorylation levels of relevant proteins were determined by standard immunoblotting techniques, as described previously (26). The primary antibodies used were sheep {alpha}1- and {alpha}2-AMPK sheep polyclonal antibodies kindly donated by D. G. Hardie (Dundee, UK; see Ref. 28), phospho-AMPK Thr172 (Cell Signaling Technology), phospho-CaMKI Thr177 (a gift from N. Nozaki, Kanawaga Dental College; see Ref. 40 for details), total CaMKI (Affinity Bioreagents), total CaMKII (BD Biosciences-Pharmingen), phospho-CaMKII Thr287 (Cell Signaling Technology), and non-isoform-specific (i.e., pan) CaMKK (BD Biosciences-Pharmingen). Polyvinylidene difluoride membranes (Immobilon Transfer Membrane; Millipore) were blocked in TBS-Tween 20 containing 2% skim milk protein for 1–2 h at room temperature. Membranes were incubated with primary antibodies overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature (DAKO). Bands were visualized using an Eastman Kodak Image Station 2000MM and enhanced chemoluminescence (ECL+; Amersham Biosciences).

{alpha}1- and {alpha}2-AMPK activity. Isoform-specific {alpha}-AMPK activity was measured in vitro in sequential immunoprecipitations (IPs) from 100 µg of muscle lysate protein using anti-{alpha}1- and anti-{alpha}2-antibodies. The protocol was identical to the one described previously (26), except that 200 µM AMARA peptide (Schafer-N) was used instead of SAMS peptide.

LKB1 and CaMKK activities. The activity of 30 ng of recombinant LKB1/MO25/STRAD complex (no. 14–596; Upstate Biotechnologies, Lake Placid, NY) against 10 µg of LKBtide (no. 12–540; Upstate Biotechnologies) was assessed using the buffers and procedures provided by the manufacturer, running the assay for 10 min at 30°C. Recombinant CaMKKbeta (50 ng/ml) was run against 10 µg of glutathione S-transferase-CaMKI (both a kind gift from A. M. Edelman, State University of New York) in assay buffer (50 mM HEPES, 5 mM MgCl2, 1 mM EGTA, 1 mM Na4P2O7, pH 7.4) with or without 2 mM CaCl2 and 0.5 µM CaM (no. 14–368; Upstate Biotechnologies) and was assayed for 30 min at 30°C. CaMKK{alpha} and -beta activities were measured on IPs of mouse brain lysate (20 µg) using two polyclonal antibodies from Santa Cruz Biotechnology [R-73 (sc11370) and L-19 (sc 9629)] in the same buffers and conditions as for recombinant CaMKKbeta. Recombinant {alpha}1beta1{gamma}1-AMPK was kindly provided by Dietbert Neumann, Swiss Federal Institute of Technology (Zurich, Switzerland).

CaMKK immunoenrichment/depletion. IPs of the two CaMKK isoforms were performed overnight on 50 µg of mouse brain using 0, 5, 10, or 15 µg of the CaMKK isoform-specific antibodies [R-73 (sc11370) and L-19 (sc 9629)]. Pre-IPs, IPs, and post-IPs were then analyzed by immunoblotting with the BD CaMKK antibody (BD Biosciences, Pharmingen).

CaM affinity precipitation. To assess Ca2+-dependent CaM binding of selected proteins, 500 µg of mouse quadriceps muscle were incubated with 20 µl of CaM-agarose with or without 1 mM CaCl2 overnight. Pre-IPs, IPs, and post-IPs were then analyzed by immunoblotting with selected antibodies, as previously described (34).

Statistical analyses. Results are means ± SE. Statistical testing was performed using unpaired t-tests or two-way ANOVA as appropriate. Interactions were evaluated by analyzing each time point separately using Tukey's honest significant difference post hoc test. An underlined symbol denotes bars that meet the criteria represented by that symbol. Statistical evaluation was performed using SPSS 13.0 (SPSS). The significance level was set at {alpha} = 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Hypothesizing that intense electrical stimulation protocols may have concealed or eliminated effects of KN compounds on AMPK signaling in previous studies, we investigated the effect of KN-93 on AMPK signaling and contraction-stimulated glucose uptake during relatively gentle (1 s/15 s) tetanic stimulation ex vivo (Fig. 1). In both soleus and EDL muscles, preincubation with KN-93 potently reduced AMPK phosphorylation at 2 min while having no significant effects at 10 min. The decrease in AMPK phosphorylation reflected reductions in both {alpha}1- and {alpha}2-AMPK activities (Fig. 1, B and C). Acetyl-CoA carboxylase (ACC)beta Ser221 phosphorylation mirrored these findings (data not shown). The contraction-stimulated increase in glucose uptake in both soleus and EDL muscles was strongly inhibited by KN-93 at both 2 and 10 min (Fig. 1D). These results show that KN-93 is a potent inhibitor of AMPK-dependent signaling and glucose uptake during low-intensity, short-duration (2 min) contraction ex vivo but suggest the KN-93 effect on contraction-stimulated glucose uptake during prolonged (10 min) contraction presumably cannot be explained by AMPK inhibition.


Figure 1
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  		Fig. 1. Inhibition of contraction-stimulated 5'-AMP-activated protein kinase (AMPK) signaling and glucose uptake by KN-93. A: contraction-stimulated AMPK phosphorylation in soleus and extensor digitorum longus (EDL) muscles pretreated with KN-93 at 0, 2, and 10 min of electrical stimulation. B and C: {alpha}1-AMPK (B) and {alpha}2-AMPK (C) activity in same muscles (n = 5–16). D: contraction-stimulated 2-deoxyglucose (2-DG) uptake after 0, 2, and 10 min of electrical stimulation; n = 8–23 experiments. {dagger}P < 0.05 and {dagger}{dagger}P < 0.01, inhibitor effect.

 
Because KN compounds are Ca2+/CaM-competitive inhibitors, our KN-93 data suggested that the activation of AMPK at 2 min was Ca2+/CaM-dependent. Because of their Ca2+/CaM dependency and confirmed role as AMPKKs, CaMKKs seemed a probable target of KN compounds. Therefore, we attempted to confirm protein expression of CaMKKs in mouse skeletal muscle (Fig. 2). Using two commercially available antibodies against CaMKK{alpha} and -beta, both capable of immunoprecipitating Ca2+/CaM-dependent CaMKI kinases (Fig. 2A), we could occasionally, but not consistently, detect CaMKK{alpha} and -beta in mouse skeletal muscle by Western blotting (data not shown). Instead, we showed that IP enrichment/post-IP depletion of the immunoprecipitated CaMKI kinases is detected by another pan-CaMKK antibody (Fig. 2B), showing that the latter antibody recognizes the same antigen as the former. Unlike the isoform-specific antibodies, this pan-CaMKK antibody consistently detects bands around the expected molecular weight in skeletal muscle (compare Fig. 2, B and C, with Fig 2D). Also, as previously reported for human skeletal muscle (34), we show in mouse skeletal muscle that CaM affinity precipitation (CaM-AP) enhances the signal detected by the pan-CaMKK antibody in a Ca2+-dependent manner, showing that it is a Ca2+/CaM-binding protein (Fig. 2D). A separate experiment confirmed that recombinant CaMKI alone is unable to autophosphorylate in the presence of Ca2+/CaM (data not shown).


Figure 2
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  		Fig. 2. Calmodulin-dependent protein kinase kinase (CaMKK) protein expression in mouse skeletal muscle. A: immunoprecipitated CaMKK{alpha} and -beta from mouse brain (20 µg) or recombinant CaMKKbeta was incubated with calmodulin-dependent protein kinase (CaMK) I in vitro in the presence or absence of 2 mM Ca2+ and 0.5 µM calmodulin (CaM), for 30 min at 30°C, and phospho-Thr177 CaMKI was assessed by Western blotting. B and C: isoform-specific immunodepletion/enrichment of CaMKK{alpha} (B) and -beta (C) from 50 µg of mouse brain (Br), immunoblotted using the Becton-Dickinson and Company CaMKK antibody. D: CaM-agarose precipitation on 500 µg of mouse skeletal muscle with or without Ca2+ immunoblotted using antibodies against pan-CaMKK, total CaMKI, phospho-Thr177 CaMKI, or total CaMKII, as indicated.

 
We tried to evaluate CaMKI signaling in mouse skeletal muscle as a measure of CaMKK activity using a phosphospecific antibody. However, although this antibody clearly detects recombinant CaMKI phoshorylation, neither this antibody nor a total CaMKI antibody detects enrichment of CaMKI in CaM-APs, suggesting very little CaMKI protein in skeletal muscle (Fig. 2D). This is consistent with our previous work showing CaM-AP enrichment of CaMKI in rat brain, but not human skeletal muscle, using an antibody against total CaMKI (34).

CaMKII is likely an important CaMK in skeletal muscle, and CaMKII phospho-Thr287 is clearly increased by contraction in rats ex vivo and in vivo (41, 56, 57) and during human exercise (33, 34). We confirmed CaMKII expression and CaM-AP enrichment of CaMKII in mouse skeletal muscle (Fig. 2D). Surprisingly, however, we had difficulties confirming a contraction-induced increase in CaMKII phosphorylation in our incubated mouse muscles. On further investigation, this was likely because 1 h of incubation suspended in our incubation apparatus at resting tension increased basal CaMKII phosphorylation (Fig. 3A). Interestingly, the increase in CaMKII phosphorylation was not seen when the muscles were allowed to hang slack in the incubation chambers (Fig. 3A), suggesting that the increase in CaMKII phosphorylation was stretch-dependent. Importantly, the increased activation of signaling proteins was not a general phenomenon, since AMPK phosphorylation was not increased during 1 h of incubation (Fig. 3B). Because of the uncertain nature of the rise in CaMKII phosphorylation, we decided not to evaluate CaMKII phosphorylation during contraction in our model. Meanwhile, the rise in CaMKII phosphorylation in the face of similar or even decreasing 2-DG uptake during the 1st h of incubation (data not shown), if anything, implies that CaMKII activation is not sufficient to increase glucose uptake. However, CaMKII Thr287 phosphorylation may not be a good measure of in vivo CaMKII activity, as suggested previously by our group (34).


Figure 3
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  		Fig. 3. Muscle incubation at resting tension increases CaMKII phosphorylation. A: CaMKII Thr287 phosphorylation in soleus and EDL muscles harvested immediately postexcission or incubated 1 h with or without hook up at resting tension, as indicated. B: AMPK Thr172 phosphorylation in the same muscles; n = 4. *P < 0.05 and **P < 0.01, different from no incubation.

 
To further explore whether the KN-93 inhibition of AMPK phosphorylation was CaMKK dependent, the effect of the CaMKK inhibitor STO-609 on AMPK signaling and contraction-stimulated glucose uptake was investigated. This experiment revealed that STO-609 had inhibitory effects on AMPK signaling and contraction-stimulated glucose uptake similar to those seen with KN-93 (Fig. 4). Hence, in both soleus and EDL muscles, AMPK phosphorylation was potently inhibited at 2 min and less potently at 10 min (Fig. 4A). As with KN-93, this reflected inhibition of both {alpha}1- and {alpha}2-AMPK activity, which were significantly inhibited at 2 and 10 min (Fig. 4, B and C). Phosphorylation of ACCbeta largely mirrored the AMPK phosphorylation data but only reached significance at 2 min (data not shown). STO-609 significantly inhibited the contraction stimulation of glucose uptake at 2 min in soleus muscle, but not in EDL, while having a partial effect on contraction-stimulated glucose uptake at 10 min in both muscles (Fig. 4D). Importantly, neither KN-93 nor STO-609 affected basal 2-DG uptake (data not shown).


Figure 4
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  		Fig. 4. Inhibition of contraction-stimulated AMPK signaling and glucose uptake by STO-609. A: contraction-stimulated AMPK phosphorylation in soleus and EDL muscles pretreated with STO-609 at 0, 2, and 10 min of electrical stimulation. B and C: {alpha}1-AMPK (B) and {alpha}2-AMPK (C) activity in same muscles (n = 5–16). D: contraction-stimulated 2-DG uptake after 0, 2, and 10 min of electrical stimulation; n = 5–23. {dagger}P < 0.05 and {dagger}{dagger}P < 0.01, inhibitor effect.

 
To confirm that the inhibition of AMPK phosphorylation by KN-93 and STO-609 is not because of inhibition of LKB1, two approaches were taken (Fig. 5). First, because AICAR-stimulated glucose uptake critically depends on LKB1 (37), we reasoned that AICAR stimulation of glucose uptake should not be inhibited by either inhibitor ex vivo. Indeed, neither KN-93 nor STO-609 significantly inhibited the AICAR stimulation of glucose uptake in either soleus or EDL muscle (Fig. 5A). Furthermore, we confirmed that neither KN-93 nor STO-609 had discernible effects on AICAR-stimulated AMPK phosphorylation ex vivo (Fig. 5B). Also, no significant effect of KN-93 or STO-609 on AICAR-stimulated ACCbeta Ser221 phosphorylation was found (data not shown). Second, we assessed the effect of KN-93 and STO-609 on the activity of recombinant LKB1/MO25/STRAD against LKBtide in vitro (Fig. 5C). Although neither KN-93 nor STO-609 significantly inhibited LKB1 activity at the 10 and 5 µM concentrations used during incubation in this study, STO-609 did significantly inhibit LKB1 activity at 10 µM and above. Because the effect of allosteric regulators on LKB1 activity differs markedly depending on the substrate chosen (13, 46), we also confirmed that phosphorylation of recombinant {alpha}1beta1{gamma}1-AMPK by recombinant LKB1 is not affected by 5 µM STO-609 or 10 µM KN-93 (data not shown).


Figure 5
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  		Fig. 5. LKB1-independent effects of KN-93 and STO-609 on AMPK activity. 5-Aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR)-stimulated (2 mM, 40 min) 2-DG uptake (A) and AMPK phosphorylation (B) in soleus and EDL muscles pretreated with either vehicle, KN-93, or STO-609; n = 5–11. EDL is shown in the representative blot. *P < 0.05, contraction vs. basal. B: recombinant LKB1/MO25/STRAD was run against LKBtide in vitro in the presence or absence of either DMSO or various concentrations of STO-609 or KN-93 as indicated; n = 3–4/condition. {dagger}{dagger}P < 0.01, inhibitor effect.

 
Finally, to evaluate the possible AMPK dependency of the KN-93 and STO effects on contraction-stimulated glucose uptake, the effect of the inhibitors on glucose uptake was evaluated in mice overexpressing a muscle-specific kinase dead AMPK construct (Fig. 6). The KD AMPK construct displaces endogenous {alpha}2-AMPK, thereby greatly reducing total {alpha}2-AMPK activity in basal (82 and 89% lower vs. wild-type soleus and EDL, respectively) and contracted (70 and 89% lower vs. wild-type soleus and EDL at 10 min, respectively) muscles (Fig. 6A). This occurs concomitantly with large reductions in {alpha}1-AMPK activity in basal (68 and 59% lower vs. wild-type soleus and EDL, respectively) and contracted (42 and 65% lower vs. wild-type soleus and EDL at 10 min, respectively) muscle as well (Fig. 6B). To what extent the remaining {alpha}1- and {alpha}2-AMPK activities stem from extramuscular tissues cannot be evaluated using the current technique but has been estimated to be a significant proportion (16). Compared with the corresponding wild-type glucose uptakes in Fig. 1, the increase in glucose uptake in muscles expressing KD AMPK was 74% lower in soleus at 2 min and 76 and 29% lower in soleus and EDL, respectively, at 10 min (contraction x genotype interaction; P < 0.01 in soleus and P < 0.05 in EDL). Inhibition of glucose uptake was significantly greater in KD AMPK-expressing soleus compared with EDL. Interestingly, neither KN-93 nor STO-609 further reduced glucose uptake in KD AMPK muscles at 2 or 10 min (Fig. 6, C and D). The lack of inhibition in muscles, which already have greatly reduced AMPK activity, suggests that the inhibitory effects of KN-93 and STO-609 on glucose uptake require inhibition of AMPK, at least in mouse skeletal muscle.


Figure 6
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  		Fig. 6. Nonadditive effect of KN-93 or STO-609 in muscles from mice expressing a muscle-specific kinase-dead AMPK construct. A and B: {alpha}2-AMPK (A) and {alpha}1-AMPK (B) activities measured after 0, 2, and 10 min of electrical stimulation in soleus and EDL KD AMPK muscles (n = 5–16). Stippled lines, mean soleus and EDL wild-type basal and 10 min contraction AMPK activity. C and D: contraction-stimulated 2-DG uptake measured after 0, 2, and 10 min of electrical stimulation in soleus and EDL KD AMPK muscles pretreated with either KN-93 (C) or STO-609 (D); n = 5–26. *P < 0.05, contraction vs. basal.

 
A recent study in a kinase-dead AMPK-overexpressing mouse model similar to KD AMPK mice used in this study, the {alpha}2i transgenic mice, has suggested than the reduction in glucose uptake seen in these mice may be related to reduced total force production (16). Using the current protocol, an ~15% lower total force development was observed in EDL muscle, but not soleus, from KD AMPK compared with wild-type mice (soleus wild type vs. KD AMPK, DMSO: 43.2 ± 3.8 vs. 48.0 ± 8.1 N·min, EDL wild type vs. KD AMPK, DMSO: 70.7 ± 5.2 vs. 59.4 ± 26 N·min, P < 0.01, n = 16–22). No differences in initial (peak) force production were observed between wild-type and KD AMPK mice in either muscle (data not shown). Neither KN-93 nor STO-609 influenced peak force or total force development (soleus DMSO: 43.2 ± 3.8 N·min, KN-93: 45.0 ± 4.6 N·min, STO-609: 42.0 ± 7.9 N·min; EDL DMSO: 70.7 ± 5.2 N·min, KN-93: 66.6 ± 5.1 N·min, STO-609: 68.8 ± 4.8 N·min; n = 16–22).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study demonstrated that inhibition of Ca2+/CaM-dependent signaling in skeletal muscle potently inhibits AMPK phosphorylation and activation at the onset of mild tetanic contraction. Furthermore, our approach combining pharmacological inhibition and AMPK-deficient mice suggests that Ca2+ and AMPK signaling to glucose uptake may be partly serial connected in mice rather than parallel as proposed for rats (56, 57). This is particularly evident in soleus muscle, suggesting that AMPK is a major mediator of contraction-stimulated glucose uptake in oxidative mouse muscle ex vivo.

Although several studies have shown that Ca2+ signaling to AMPK can occur through the CaMKKs in various cell types (18, 21, 22, 54), Sakamoto and colleagues (37) showed a striking lack of {alpha}2-AMPK phosphorylation during intense in situ electrical stimulation in EDL muscles from LKB1-deficient mice, suggesting that LKB1 is the principal donor of AMPKK activity in mouse skeletal muscle during contraction. However, they also recently reported that mice lacking LKB1 expression in heart retain the ability to activate the {alpha}1-AMPK isoform (38), leading to the proposal that skeletal muscle {alpha}1-AMPK may also be regulated by a kinase distinct from LKB1. We concur with this notion, because the skeletal muscle AMPK activity data from LKB1-deficient mice (37) were produced in muscles contracted in situ, where little to no changes occurred in {alpha}1-AMPK activity, in contrast to the rapid increases in both {alpha}1- and {alpha}2-AMPK activity seen with ex vivo contraction (1, 26, 32, 36, 52, 53). Furthermore, the AMPK activities in mice lacking LKB1 were measured in EDL muscle, where we generally see less potent KN-93/STO-609 inhibition of AMPK phosphorylation and activity compared with soleus muscle. Finally, a recent paper by Koh and colleagues (28) directly demonstrated that muscle-specific LKB1 KO mice retain some ability to phosphorylate and activate {alpha}1-AMPK in response to contraction and AICAR in skeletal muscle (28).

Unlike an earlier study (18), we can detect CaMKK protein in mouse skeletal muscle lysates (34). CaMKKs fit the criteria of being inhibited by STO-609 and presumably the CaM-competitive KN-compounds (9, 29, 50), although a direct effect on CaMKK by the latter remains to be confirmed by in vitro studies. Therefore, we propose the involvement of CaMKK(s) in the regulation of Thr172 {alpha}-AMPK phosphorylation in mouse skeletal muscle. A working model in which an intensity- and/or time-dependent switch occurs in the relative importance of AMPKKs during contraction may explain why AMPK phosphorylation during ex vivo contraction is only potently inhibited at 2 min compared with 10 min. Alternatively, signaling through LKB1 could be regulated in an intensity- and/or time-dependent manner. Interestingly, TAK1, also known as MAP3K7, was recently suggested to regulate AMPK activity either directly (30) or indirectly by regulating LKB1 activity (58). K+ depolarization has been shown to increase TAK1 phosphorylation in a KN-93-sensitive manner in HEK293 cells, and dominant-negative CaMKII expression reduces Wnt-5a-stimulated TAK1 phosphorylation (23), suggesting that CaMKII signals upstream of TAK1. Furthermore, a range of AMPK-activating stimuli, including oligomycin, metformin, AICAR, and ischemia, increase TAK1 activity in cardiomyocyte cell culture (58). Therefore, an alternative model, consistent with available data, is that a CaMKII-TAK1 pathway regulates AMPK phosphorylation directly or through LKB1, with the KN effect observed in the present study being on TAK1 and an unspecific effect of STO-609 on either TAK1 or LKB1. However, small-interfering RNA-mediated reduction in CaMKK protein largely reduces Ca2+-activated AMPK phosphorylation in LKB1-deficient HeLa cells, suggesting that the Ca2+-mediated AMPK activation, at least in these cells, requires CaMKK (18, 22, 54). Therefore, we presently consider CaMKK the more likely non-LKB1 AMPKK candidate in skeletal muscle. Genetically modified models will be needed to clarify the importance of CaMKKs, LKB1, and TAK1 in {alpha}-AMPK isoform-specific activation during various stimulation regimens in skeletal muscle.

Based on studies using KN compounds, Wright and co-workers (56, 57) have proposed the involvement of CaMKII in contraction-stimulated glucose uptake in rat skeletal muscle. Unlike the present study, they did not find a reduced AMPK phosphorylation by KN compounds following 10 min of intense ex vivo contraction. Meanwhile, this is consistent with our model of an intensity- and/or time-dependent switch in AMPKK. Then again, fiber-type composition of mouse and rat muscles is not directly comparable, for instance rat soleus is of a more oxidative phenotype (~80% type I fibers; see Ref. 44) compared with mouse soleus (37% type I fibers; see Ref. 2). Also, a genuine species difference in AICAR-stimulated AMPK signaling to glucose uptake may exist, since AICAR potently stimulates glucose uptake in mouse soleus (26), but not in most studies using rat soleus (12, 15, 27), despite potent AMPK activation by AICAR in both species. Regardless, the inhibition of AMPK activation by KN-93 reported here suggests that KN-93 may not be a suitable tool when attempting to dissect out the relative importance of CaMKs and AMPK.

Previous studies in isolated rat soleus and epitroclearis muscles (7, 56, 57) have shown that increases in sarcoplasmatic reticulum (SR) Ca2+ release by subcontraction caffeine concentrations increases 2-DG uptake without stimulating AMPK phosphorylation, providing a strong argument against Ca2+-dependent AMPK activation. However, in our hands, AMPK phosphorylation and activity of {alpha}1-AMPK, but not {alpha}2-AMPK, consistently increase in isolated rodent soleus muscles after subcontraction caffeine treatment (24), suggesting type 2 errors because of a low number of observations in the previous studies. Interestingly, hypoxia increases intracellular Ca2+ concentrations in rat epitroclearis muscle (47), and hypoxia-stimulated glucose uptake is inhibited by both KN compounds and the SR-Ca2+ release inhibitor dantrolene in rat soleus and epitroclearis muscles (6, 8) as well as by KD AMPK expression in mouse muscles (31). Furthermore, H2O2-treatment of muscle cells increases Ca2+ release from the SR (14, 20) and activates {alpha}1-AMPK and glucose uptake, independent of {alpha}2-AMPK, in isolated rat epitroclearis (51), and N-acetylcysteine, a nonspecific antioxidant, inhibits contraction-stimulated AMPK phosphorylation and activity in mouse EDL (39). Together, these findings suggest that hypoxia and H2O2 could signal to AMPK through a SR-Ca2+ release-dependent pathway.

Although the effect of STO-609 on AMPK activation generally mirrored the inhibition of glucose uptake, KN-93 still inhibited glucose uptake at the 10-min time point at which KN inhibition of AMPK activation was no longer apparent, suggesting an AMPK-independent inhibition of glucose uptake. This observation is hard to reconcile with the finding that KN-93 and STO-609 do not reduce contraction-stimulated glucose uptake further in KD AMPK-expressing mice. Therefore, an involvement of other Ca2+/CaM-dependent signaling proteins, including CaMKII, in contraction-stimulated glucose uptake cannot be ruled out at present. It is worth mentioning that the specificity of KN compounds has been questioned, based on their ability to inhibit insulin-stimulated glucose uptake in rat soleus and epitroclearis muscle in some (55), but not all, studies (57). However, the observation that KN-93 and STO-609 reduce contraction-stimulated glucose uptake in wild-type muscle, while having no effect in the KD AMPK mice or on AICAR-stimulated glucose uptake, suggests that the KN inhibition seen is not a direct effect on the glucose uptake machinery.

Previous ex vivo studies have generally used very intense protocols designed to elicit a maximal response in glucose uptake (16, 37, 56). As shown here, this may conceal physiologically important signaling mechanisms because of redundant signaling and/or time effects, in particular during the intricate multipathway contraction-signaling process. In addition, it is not known how well the reductionistic ex vivo contraction approach extrapolates to in vivo exercise in mice and ultimately in humans. Much to our surprise, we have recently observed no difference in exercise-stimulated 2-DG uptake in various muscles from catherized running KD AMPK mice compared with wild-type mice (S. B. Jørgensen, J. T. Treebak, T. E. Jensen, E. A. Richter, and J. P. F. Wojtaszewski, unpublished observation), even in soleus muscle, suggesting that glucose uptake regulation may differ markedly between the ex vivo and in vivo setting. Therefore, although the ex vivo contraction model remains a valuable tool in the attempt to dissect out the crucial components of contraction signaling, it remains imperative that the ex vivo findings are followed up with studies in more physiological in vivo systems as well.

In conclusion, the current study proposes as a working model that one or more CaMKKs act as AMPKKs at the onset of mild tetanic contraction and play a role in the regulation of AMPK-dependent contraction-stimulated glucose uptake in mouse skeletal muscle ex vivo.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by The Copenhagen Muscle Research Centre, 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 from the Carlsberg Foundation.


   ACKNOWLEDGMENTS
 
We thank Morris J. Birnbaum (Howard Hughes Medical Institute and The Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA), Arthur M. Edelman (Department of Pharmacology and Toxicology, State University of New York, Buffalo, NY), Dietbert Neumann (Institute of Cell Biology, Swiss Federal Institute of Technology, Zurich, Switzerland), and Naohito Nozaki (Department of Biochemistry and Molecular Biology, Kanagawa Dental College, Inaoka-cho, Yokosuka, Kanagawa, Japan) for donating materials essential to this study. We thank Jesper Birk for skilled technical assistance on optimization of the recombinant LKB1 assay.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. A. Richter, Dept. of Human Physiology, Institute of Exercise and Sport Sciences, Copenhagen Muscle Research Centre, Univ. of Copenhagen, Universitetsparken 13, Copenhagen 2100, 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
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Ai H, Ihlemann J, Hellsten Y, Lauritzen HPMM, Hardie DG, Galbo H, Ploug T. Effect of fiber type and nutritional state on AICAR- and contraction-stimulated glucose transport in rat muscle. Am J Physiol Endocrinol Metab 282: E1291–E1300, 2002.[Abstract/Free Full Text]
  2. Akimoto T, Ribar TJ, Williams RS, Yan Z. Skeletal muscle adaptation in response to voluntary running in Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) deficient mice. Am J Physiol Cell Physiol 287: C1311–C1319, 2004.[Abstract/Free Full Text]
  3. Altarejos JY, Taniguchi M, Clanachan AS, Lopaschuk GD. Myocardial ischemia differentially regulates LKB1 and an alternate 5'-AMP-activated protein kinase kinase. J Biol Chem 280: 183–190, 2005.[Abstract/Free Full Text]
  4. Barnes BR, Marklund S, Steiler TL, Walter M, Hjalm G, Amarger V, Mahlapuu M, Leng Y, Johansson C, Galuska D, Lindgren K, Abrink M, Stapleton D, Zierath JR, Andersson L. The 5'-AMP-activated protein kinase gamma3 isoform has a key role in carbohydrate and lipid metabolism in glycolytic skeletal muscle. J Biol Chem 279: 38441–38447, 2004.[Abstract/Free Full Text]
  5. Baron SJ, Li J, Russell RR, III, Neumann D, Miller EJ, Tuerk R, Wallimann T, Hurley RL, Witters LA, Young LH. Dual mechanisms regulating AMPK kinase action in the ischemic heart. Circ Res 96: 337–345, 2005.[Abstract/Free Full Text]
  6. Brozinick JT Jr, Reynolds TH, Dean D, Cartee G, Cushman SW. 1-[N,O-bis-(5-isoquinolinesulphonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62),an inhibitor of calcium-dependent camodulin protein kinase II, inhibits both insulin- and hypoxia-stimulated glucose transport in skeletal muscle. Biochem J 339: 533–540, 1999.[CrossRef][ISI][Medline]
  7. Canto C, Chibalin AV, Barnes BR, Glund S, Suarez E, Ryder JW, Palacin M, Zierath JR, Zorzano A, Guma A. Neuregulin mediate calcium-induced glucose transport during muscle contraction. J Biol Chem 281: 21690–21697, 2006.
  8. Cartee GD, Douen AG, Ramlal T, Klip A, Holloszy JO. Stimulation of glucose transport in skeletal muscle by hypoxia. J Appl Physiol 70: 1593–1600, 1991.[Abstract/Free Full Text]
  9. Chen BC, Wu WT, Ho FM, Lin WW. Inhibition of interleukin-1beta-induced NF-kappa B activation by calcium/calmodulin-dependent protein kinase kinase occurs through Akt activation associated with interleukin-1 receptor-associated kinase phosphorylation and uncoupling of MyD88. J Biol Chem 277: 24169–24179, 2002.[Abstract/Free Full Text]
  10. Corcoran EE, Means AR. Defining Ca2+/calmodulin-dependent protein kinase cascades in transcriptional regulation. J Biol Chem 276: 2975–2978, 2001.[Free Full Text]
  11. da Silva CG, Jarzyna R, Specht A, Kaczmarek E. Extracellular nucleotides and adenosine independently activate AMP-activated protein kinase in endothelial cells: involvement of P2 receptors and adenosine transporters. Circ Res 98: e39–e47, 2006.[Abstract/Free Full Text]
  12. Derave W, Ai H, Ihlemann J, Witters LA, Kristiansen S, Richter EA, Ploug T. Dissociation of AMP-activated protein kinase activation and glucose transport in contracting slow-twitch muscle. Diabetes 49: 1281–1287, 2000.[Abstract]
  13. Ellingson WJ, Chesser DG, Winder WW. The effects of 3-phosphoglycerate and other metabolites on the activation of AMP-activated protein kinase by LKB1-STRAD-MO25. Am J Physiol Endocrinol Metab 292: E400–E407, 2007.[Abstract/Free Full Text]
  14. Espinosa A, Leiva A, Pena M, Muller M, Debandi A, Hidalgo C, Angelica CM, Jaimovich E. Myotube depolarization generates reactive oxygen species through NAD(P)H oxidase; ROS-elicited Ca(2+) stimulates ERK, CREB, early genes. J Cell Physiol 209: 379–388, 2006.
  15. Fryer LG, Hajduch E, Rencurel F, Salt IP, Hundal HS, Hardie DG, Carling D. Activation of glucose transport by AMP-activated protein kinase via stimulation of nitric oxide synthase. Diabetes 49: 1978–1985, 2000.[Abstract]
  16. Fujii N, Hirshman MF, Kane EM, Ho RC, Peter LE, Seifert MM, Goodyear LJ. AMP-activated protein kinase alpha2 activity is not essential for contraction-and hyperosmolarity-induced glucose transport in skeletal muscle. J Biol Chem 280: 39033–39041, 2005.
  17. Hardie DG. The AMP-activated protein kinase pathway–new players upstream and downstream. J Cell Sci 117: 5479–5487, 2004.[Abstract/Free Full Text]
  18. Hawley SA, Pan DA, Mustard KJ, Ross L, Bain J, Edelman AM, Frenguelli BG, Hardie DG. Calmodulin-dependent protein kinase kinase-beta is an alternative upstream kinase for AMP-activated protein kinase. Cell Metab 2: 9–19, 2005.[CrossRef][ISI][Medline]
  19. Hidaka H, Yokokura H. Molecular and cellular pharmacology of a calcium/calmodulin-dependent protein kinase II (CaM kinase II) inhibitor, KN-62, and proposal of CaM kinase phosphorylation cascades. Adv Pharmacol 36: 193–219, 1996.[Medline]
  20. Hidalgo C, Sanchez G, Barrientos G, Racena-Parks P. A transverse tubule NOX activity stimulates calcium release from isolated triads via RYR1 S-glutathionylation. J Biol Chem M600451200, 2006.
  21. Hong SP, Momcilovic M, Carlson M. Function of mammalian LKB1 and Ca2+/calmodulin-dependent protein kinase kinase alpha as Snf1-activating kinases in yeast. J Biol Chem 280: 21804–21809, 2005.[Abstract/Free Full Text]
  22. Hurley RL, Anderson KA, Franzone JM, Kemp BE, Means AR, Witters LA. The Ca2+/calmodulin-dependent protein kinase kinases are AMP-activated protein kinase kinases. J Biol Chem 280: 29060–29066, 2005.[Abstract/Free Full Text]
  23. Ishitani T, Kishida S, Hyodo-Miura J, Ueno N, Yasuda J, Waterman M, Shibuya H, Moon RT, Ninomiya-Tsuji J, Matsumoto K. The TAK1-NLK mitogen-activated protein kinase cascade functions in the Wnt-5a/Ca2+ pathway to antagonize Wnt/beta-catenin signaling. Mol Cell Biol 23: 131–139, 2003.[Abstract/Free Full Text]
  24. Jensen TE, Rose Adam J, Wojtaszewki Jorgen FP, Richter EA. Caffeine-induced Ca2+ release increases AMPK-dependent glucose uptake in rodent soleus muscle. Am J Physiol Endocrinol Metab 292: E000–E000, 2007.
  25. Jorgensen SB, Richter EA, Wojtaszewski JFP. Role of AMPK in skeletal muscle metabolic regulation and adaptation in relation to exercise. J Physiol (Lond) 574: 17–31, 2006.[Abstract/Free Full Text]
  26. Jørgensen SB, Viollet B, Andreelli F, Frosig C, Birk JB, Schjerling P, Vaulont S, Richter EA, Wojtaszewski JFP. Knockout of the alpha2 but not alpha1 5'-AMP-activated protein kinase isoform abolishes 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside but not contraction-induced glucose uptake in skeletal muscle. J Biol Chem 279: 1070–1079, 2004.[Abstract/Free Full Text]
  27. Kaushik VK, Young ME, Dean DJ, Kurowski TG, Saha AK, Ruderman NB. Regulation of fatty acid oxidation and glucose metabolism in rat soleus muscle: effects of AICAR. Am J Physiol Endocrinol Metab 281: E335–E340, 2001.[Abstract/Free Full Text]
  28. Koh HJ, Arnolds DE, Fujii N, Tran TT, Rogers MJ, Jessen N, Li Y, Liew CW, Ho RC, Hirshman MF, Kulkarni RN, Kahn CR, Goodyear LJ. Skeletal muscle-selective knockout of LKB1 increases insulin sensitivity, improves glucose homeostasis, and decreases TRB3. Mol Cell Biol 26: 8217–8227, 2006.[Abstract/Free Full Text]
  29. Leclerc I, Rutter GA. AMP-activated protein kinase: a new beta-cell glucose sensor? Regulation by amino acids and calcium ions. Diabetes 53: S67–S74, 2004.[Abstract/Free Full Text]
  30. Momcilovic M, Hong SP, Carlson M. Mammalian TAK1 activates Snf1 protein kinase in yeast and phosphorylates AMP-activated protein kinase in vitro. J Biol Chem 281: 25336–25343, 2006.[Abstract/Free Full Text]
  31. Mu J, Brozinick JT Jr, Valladares O, Bucan M, Birnbaum MJ. A role for AMP-activated protein kinase in contraction- and hypoxia-regulated glucose transport in skeletal muscle. Mol Cell 7: 1085–1094, 2001.[CrossRef][ISI][Medline]
  32. Musi N, Hayashi T, Fujii N, Hirshman MF, Witters LA, Goodyear LJ. AMP-activated protein kinase activity and glucose uptake in rat skeletal muscle. Am J Physiol Endocrinol Metab 280: E677–E684, 2001.[Abstract/Free Full Text]
  33. Rose AJ, Hargreaves M. Exercise increases Ca2+-calmodulin-dependent protein kinase II activity in human skeletal muscle. J Physiol (Lond) 553: 303–309, 2003.[Abstract/Free Full Text]
  34. Rose AJ, Kiens B, Richter EA. Ca2+-calmodulin-dependent protein kinase expression and signalling in skeletal muscle during exercise. J Physiol (Lond) 574: 889–903, 2006.[Abstract/Free Full Text]
  35. Rose AJ, Richter EA. Skeletal muscle glucose uptake during exercise: how is it regulated? Physiology 20: 260–270, 2005.[Abstract/Free Full Text]
  36. Sakamoto K, Goransson O, Hardie DG, Alessi DR. Activity of LKB1 and AMPK-related kinases in skeletal muscle: effects of contraction, phenformin, and AICAR. Am J Physiol Endocrinol Metab 287: E310–E317, 2004.[Abstract/Free Full Text]
  37. Sakamoto K, McCarthy A, Smith D, Green KA, Grahame HD, Ashworth A, Alessi DR. Deficiency of LKB1 in skeletal muscle prevents AMPK activation and glucose uptake during contraction. EMBO J 24: 1810–1820, 2005.[CrossRef][ISI][Medline]
  38. Sakamoto K, Zarrinpashneh E, Budas GR, Pouleur AC, Dutta A, Prescott AR, Vanoverschelde JL, Ashworth A, Jovanovic A, Alessi DR, Bertrand L. Deficiency of LKB1 in heart prevents ischemia-mediated activation of AMPK{alpha}2 but not AMPK{alpha}1. Am J Physiol Endocrinol Metab 290: E780–E788, 2006.[Abstract/Free Full Text]
  39. Sandstrom ME, Zhang SJ, Bruton J, Silva JP, Reid MB, Westerblad H, Katz A. Role of reactive oxygen species in contraction-mediated glucose transport in mouse skeletal muscle. J Physiol (Lond) 575: 251–262, 2006.[Abstract/Free Full Text]
  40. Schmitt JM, Wayman GA, Nozaki N, Soderling TR. Calcium activation of ERK mediated by calmodulin kinase I. J Biol Chem 279: 24064–24072, 2004.[Abstract/Free Full Text]
  41. Smith JAH, Collins M, Grobler LA, Magee CJ, Ojuka EO. Exercise and CaMK activation both increase the binding of MEF2A to the Glut4 promoter in skeletal muscle in vivo. Am J Physiol Endocrinol Metab 292: E413–E420, 2007.[Abstract/Free Full Text]
  42. Stahmann N, Woods A, Carling D, Heller R. Thrombin activates AMP-activated protein kinase in endothelial cells via a pathway involving Ca2+/calmodulin-dependent protein kinase kinase beta. Mol Cell Biol 26: 5933–5945, 2006.[Abstract/Free Full Text]
  43. Sumi M, Kiuchi K, Ishikawa T, Ishii A, Hagiwara M, Nagatsu T, Hidaka H. The newly synthesized selective Ca2+/calmodulin dependent protein kinase II inhibitor KN-93 reduces dopamine contents in PC12h cells. Biochem Biophys Res Commun 181: 968–975, 1991.[CrossRef][ISI][Medline]
  44. Suwa M, Nakano H, Kumagai S. Effects of chronic AICAR treatment on fiber composition, enzyme activity, UCP3, and PGC-1 in rat muscles. J Appl Physiol 95: 960–968, 2003.[Abstract/Free Full Text]
  45. Suzuki A, Kusakai G, Kishimoto A, Shimojo Y, Ogura T, Lavin MF, Esumi H. IGF-1 phosphorylates AMPK-alpha subunit in ATM-dependent and LKB1-independent manner. Biochem Biophys Res Commun 324: 986–992, 2004.[CrossRef][ISI][Medline]
  46. Taylor EB, Ellingson WJ, Lamb JD, Chesser DG, Winder WW. Long-chain acyl-CoA esters inhibit phosphorylation of AMP-activated protein kinase at threonine-172 by LKB1/STRAD/MO25. Am J Physiol Endocrinol Metab 288: E1055–E1061, 2005.[Abstract/Free Full Text]
  47. Terada S, Muraoka I, Tabata I. Changes in [Ca2+]i induced by several glucose transport-enhancing stimuli in rat epitrochlearis muscle. J Appl Physiol 94: 1813–1820, 2003.[Abstract/Free Full Text]
  48. Thomson DM, Porter BB, Tall JH, Kim HJ, Barrow JR, Winder WW. Skeletal muscle and heart LKB1 deficiency causes decreased voluntary running and reduced muscle mitochondrial marker enzyme expression in mice. Am J Physiol Endocrinol Metab 292: E196–E202, 2007.[Abstract/Free Full Text]
  49. Tokumitsu H, Chijiwa T, Hagiwara M, Mizutani A, Terasawa M, Hidaka H. KN-62, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II. J Biol Chem 265: 4315–4320, 1990.[Abstract/Free Full Text]
  50. Tokumitsu H, Inuzuka H, Ishikawa Y, Ikeda M, Saji I, Kobayashi R. STO-609, a specific inhibitor of the Ca(2+)/calmodulin-dependent protein kinase kinase. J Biol Chem 277: 15813–15818, 2002.[Abstract/Free Full Text]
  51. Toyoda T, Hayashi T, Miyamoto L, Yonemitsu S, Nakano M, Tanaka S, Ebihara K, Masuzaki H, Hosoda K, Inoue G, Otaka A, Sato K, Fushiki T, Nakao K. Possible involvement of the alpha1 isoform of 5'AMP-activated protein kinase in oxidative stress-stimulated glucose transport in skeletal muscle. Am J Physiol Endocrinol Metab 287: E166–E173, 2004.[Abstract/Free Full Text]
  52. Toyoda T, Tanaka S, Ebihara K, Masuzaki H, Hosoda K, Sato K, Fushiki T, Nakao K, Hayashi T. Low-intensity contraction activates the {alpha}1-isoform of 5'-AMP-activated protein kinase in rat skeletal muscle. Am J Physiol Endocrinol Metab 290: E583–E590, 2006.[Abstract/Free Full Text]
  53. Vavvas D, Apazidis A, Saha AK, Gamble J, Patel A, Kemp BE, Witters LA, Ruderman NB. Contraction-induced changes in acetyl-CoA carboxylase and 5'-AMP-activated kinase in skeletal muscle. J Biol Chem 272: 13255–13261, 1997.[Abstract/Free Full Text]
  54. Woods A, Dickerson K, Heath R, Hong SP, Momcilovic M, Johnstone SR, Carlson M, Carling D. Ca2+/calmodulin-dependent protein kinase kinase-beta acts upstream of AMP-activated protein kinase in mammalian cells. Cell Metab 2: 21–33, 2005.[CrossRef][ISI][Medline]
  55. Wright DC, Fick CA, Olesen JB, Lim K, Barnes BR, Craig BW. A role for calcium/calmodulin kinase in insulin stimulated glucose transport. Life Sci 74: 815–825, 2004.[CrossRef][ISI][Medline]
  56. Wright DC, Geiger PC, Holloszy JO, Han DH. Contraction- and hypoxia-stimulated glucose transport is mediated by a Ca2+-dependent mechanism in slow-twitch rat soleus muscle. Am J Physiol Endocrinol Metab 288: E1062–E1066, 2005.[Abstract/Free Full Text]
  57. Wright DC, Hucker KA, Holloszy JO, Han DH. Ca2+ and AMPK both mediate stimulation of glucose transport by muscle contractions. Diabetes 53: 330–335, 2004.[Abstract/Free Full Text]
  58. Xie M, Zhang D, Dyck JRB, Li Y, Zhang H, Morishima M, Mann DL, Taffet GE, Baldini A, Khoury DS, Schneider MD. A pivotal role for endogenous TGF-beta-activated kinase-1 in the LKB1/AMP-activated protein kinase energy-sensor pathway. Proc Natl Acad Sci USA 0604708103, 2006.
  59. Youn JH, Gulve EA, Holloszy JO. Calcium stimulates glucose transport in skeletal muscle by a pathway independent of contraction. Am J Physiol Cell Physiol 260: C555–C561, 1991.[Abstract/Free Full Text]



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