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Am J Physiol Endocrinol Metab 292: E331-E339, 2007. First published September 5, 2006; doi:10.1152/ajpendo.00243.2006 Free Article
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Role of AMPK{alpha}2 in basal, training-, and AICAR-induced GLUT4, hexokinase II, and mitochondrial protein expression in mouse muscle

Sebastian B. Jørgensen,1 Jonas T. Treebak,1 Benoit Viollet,2 Peter Schjerling,3 Sophie Vaulont,2 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, Denmark; 2Institute Cochin, Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, Rene Descartes University, Department of Genetic, Development, and Molecular Pathology, Paris, France; and 3Copenhagen Muscle Research Centre, Department for Molecular Muscle Biology, Rigshospitalet, Copenhagen, Denmark

Submitted 23 May 2006 ; accepted in final form 8 August 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We investigated the role of AMPK{alpha}2in basal, exercise training-, and AICAR-induced protein expression of GLUT4, hexokinase II (HKII), mitochondrial markers, and AMPK subunits. This was conducted in red (RG) and white gastrocnemius (WG) muscle from wild-type (WT) and {alpha}2-knockout (KO) mice after 28 days of activity wheel running or daily AICAR injection. Additional experiments were conducted to measure acute activation of AMPK by exercise and AICAR. At basal, mitochondrial markers were reduced by ~20% in {alpha}2-KO muscles compared with WT. In both muscle types, AMPK{alpha}2 activity was increased in response to both stimuli, whereas AMPK{alpha}1 activity was increased only in response to exercise. Furthermore, AMPK signaling was estimated to be 60–70% lower in {alpha}2-KO compared with WT muscles. In WG, AICAR treatment increased HKII, GLUT4, cytochrome c, COX-1, and CS, and the {alpha}2-KO abolished the AICAR-induced increases, whereas no AICAR responses were observed in RG. Exercise training increased GLUT4, HKII, COX-1, CS, and HAD protein in WG, but the {alpha}2-KO did not affect training-induced increases. Furthermore, AMPK{alpha}1, -{alpha}2, -beta1, -beta2, and -{gamma}3 subunits were reduced in RG, but not in WG, by 30–60% in response to exercise training. In conclusion, the {alpha}2-KO was associated with an ~20% reduction in mitochondrial markers in both muscle types and abolished AICAR-induced increases in protein expression in WG. However, the {alpha}2-KO did not reduce training-induced increases in HKII, GLUT4, COX-1, HAD, or CS protein in WG, suggesting that AMPK{alpha}2 may not be essential for metabolic adaptations of skeletal muscles to exercise training.

5'-adenosine monophosphate-activated protein kinase-{alpha}2; glucose transporter-4; 5-aminoimidazole-1-beta-D-ribofuranoside; exercise training; mitochondrial proteins; skeletal muscle

BIOCHEMICAL ADAPTATIONS of skeletal muscle to regular physical activity include an increase in mitochondrial oxidative enzyme capacity and an increase in key proteins involved in glucose uptake, such as glucose transporter-4 (GLUT4) and hexokinase II (HKII) (41, 51). The intracellular pathways involved in eliciting these exercise training-induced increases in protein expression and mitochondrial content remain largely unknown. The 5'-AMP-activated protein kinase (AMPK) has been proposed as a signaling molecule involved in transmitting an "exercise signal" to the nuclei of the muscle cell (15, 49). This is in part based on the finding that AMPK is activated by in vitro electrical induced contractions of rodent skeletal muscle (8, 44) and by in vivo exercise in human (11, 50) and rodent skeletal muscle (48).

Chronic activation of AMPK by the adenosine analog 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) or the creatine analog beta-guanadinopropionic acid (beta-GPA) in resting rat and mouse muscles increases transcription of metabolic genes and expression of metabolic enzymes as well as mitochondrial density mimicking effects of exercise training (4, 15, 20, 49, 52). The observation that pharmacologically induced upregulation of peroxisome proliferator-activated receptor-{gamma} coactivator-1{alpha} (PGC-1{alpha}) and {delta}-aminolevulinate synthase mRNA, cytochrome c (cyt c) protein, and mitochondrial density is abolished in mouse muscle overexpressing a kinase-dead AMPK construct suggests a causal role of AMPK in these responses (52). The AMPK-dependent increase in PGC-1{alpha} is of particular interest because this nuclear transcription modulator is shown to be important in coordinating muscular adaptations in lipid oxidation and mitochondrial function to exercise training (23, 24) and to be involved in regulating GLUT4 protein expression (28, 30).

Several studies indicate that AMPK increases content of target proteins by increasing transcriptional activity of their respective genes. For instance, arterial infusion of AICAR in rodent hindlimb muscle stimulates HKII and uncoupling protein-3 (UCP3) gene transcription (43), and an acute injection of AICAR increases muscle GLUT4 mRNA in rodents (5). Furthermore, a chronic treatment of rat with beta-GPA treatment is followed by an increase in nuclear respiratory factor-1 (NRF-1) DNA binding activity correlated by an increase in expression of NRF-1 targets and mitochondrial density in muscle (4). NRF-1 is activated by binding with PGC-1{alpha}, which in turn leads to increased transcription of nuclear genes encoding subunits of the mitochondrial respiratory chain and components of the mitochondrial transcription and replication machinery (for review, see Refs. 22 and 42).

Altogether, it seems evident that activation of AMPK in resting muscle specifically increases the protein content of several metabolic enzymes as well as mitochondrial density. Therefore, the purpose of the present study was to investigate the importance of the catalytic AMPK{alpha}2 subunit in expression of HKII and GLUT4 protein, as well as mitochondrial enzymes in skeletal muscle in the basal state, and after exercise training and chronic AICAR treatment. This was investigated in AMPK{alpha}2 whole body knockout ({alpha}2-KO) and corresponding wild-type (WT) mice that had undertaken a 28-day program of activity wheel exercise training or daily AICAR injections.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals

The {alpha}2-KO mice have been described previously (18, 19, 45, 46). Four-month-old {alpha}2-KO and AMPK{alpha}2 WT ({alpha}2-WT) mice were studied, and the mice were littermates produced by intercross breeding of heterozygote parents. Female animals were used in all experiments except for the acute AICAR control experiment, where both males and females were used. The genotype of the offspring was determined by southern blotting on DNA extracted from a tail piece. Mice were maintained on a 10:14-h light-dark cycle and received standard rodent chow (Altromin nr 1324; Chr. Pedersen, Ringsted, Denmark) and water ad libitum. All experiments were approved by the Danish Animal Experiments Inspectorate and complied with the European Convention for the Protection of Vertebrate Animals Used for Experiments and Other Scientific Purposes (Council of Europe no. 123, Strasbourg, France, 1985).

Experimental Protocols

Chronic AICAR treatment. Animals were randomly subdivided into four groups (WT saline, WT AICAR, {alpha}2-KO saline, {alpha}2-KO AICAR) and injected subcutaneously at the lateral distal part of the back with sterile isotonic saline solution, with or without AICAR (500 mg kg/body wt; Toronto Research Chemicals, Toronto, ON, Canada). Treatment was given for 28 days, with injection every second day for the first 6 days and every day for the remaining period. Animals were injected between 1000 and 1400. Muscles were removed from anesthetized (6 mg of pentobarbital 100 g/body wt) animals 24 h after last injection on day 29, quick-frozen by aluminum tongs precooled in liquid nitrogen, and stored at –80°C. To assess AICAR tolerance, expressed as changes in blood glucose concentration, tail blood was collected on days 1, 13, and 26 and measured for glucose content using a glucometer (Bayer, Leverkusen, Germany).

Acute AICAR experiment. To verify that a single AICAR injection increased muscle AMPK activity, fed WT and {alpha}2-KO animals were injected with sterile isotonic saline solution with or without AICAR as described above. Muscles were removed from animals 60 min after injection with saline and 30, 60, or 120 min after injection with AICAR, quick-frozen by aluminum tongs precooled in liquid nitrogen, and stored at –80°C.

Exercise training by activity wheel running. WT and {alpha}2-KO animals were either exercise trained or kept as nontrained controls. Animals in the trained groups were placed in individual cages, with free access to an activity wheel (Techniplast activity cage, wheel Ø: 23 cm; Techniplast, Buguggiate, Italy) and run on a voluntary basis for 28 days. Time spent in activity wheel, average running speed, and distance covered per day was measured online by computer (BC 1400; Sigma Sport, Neustadt, Germany). Muscles were removed from anesthetized animals on day 29 14–16 h after the last exercise bout, quick-frozen by aluminum tongs precooled in liquid nitrogen, and stored at –80°C.

Acute activity wheel running. To verify that activity wheel running increased muscle AMPK activity, WT and {alpha}2-KO animals were placed in cages with or without (controls) access to an activity wheel. After 4 wk of habituation to voluntary activity wheel running, muscles were removed from mice immediately after they completed 1 min of successive voluntary activity wheel running (3–4 h into the activity/dark period). Mice were pacified by cervical dislocation, and muscles were removed immediately after running, quick-frozen by aluminum tongs precooled in liquid nitrogen, and stored at –80°C.

Body Weight and Food Consumption

Food intake and body weight were recorded during the 28 days of intervention from the various experiments.

Protein Expression and Phosphorylation

Expression or phosphorylation of investigated proteins was determined in muscle lysate (AMPK subunits, HKII, ACCbeta Ser227-phos, cyt c AMPK{alpha}-Thr172-phos) or in total crude membrane (14) [GLUT4 and cyt c oxidase 1 (COX-1)]. Lysates were prepared in a buffer containing 20 mM Tris, 50 mM NaCl, 2 mM DTT, 50 mM NaF, 1% Triton X-100, 250 mM sucrose, 5 mM Na pyrophosphate, 4 µg/ml leupeptin, 3 mM benzamidine, 500 µM PMSF, and 50 µg/ml soybean trypsin inhibitor. Muscle samples were homogenized using a homogenizer (PT3100, Brinkman Instruments) for 20 s. Homogenates were rotated end over end for 1 h at 4°C and were then subjected to centrifugation (17,000 g, 60 min, 4°C). Lysates were quickly frozen in liquid nitrogen and stored at –80°C. Protein content in lysates was measured by the bicinchoninic acid method (Pierce, Rockford, IL). Total crude membrane preparations were done as previously described (19) in a buffer containing 30 mM HEPES (pH 7.5), 40 mM NaCl, 2 mM PMSF, 2 mM EGTA, 250 mM sucrose, 5 µM pepstatin A, and 10 µg/ml aprotinin. Following SDS-PAGE, immunoblotting was done using the following primary antibodies: anti-AMPK{alpha}1- and -{alpha}2 (kindly donated by D. G. Hardie, University of Dundee, Dundee, UK), anti-AMPK{alpha}-Thr172 phospho (Cell Signaling Technology, Beverly, MA), anti-pan-beta1- and -beta2-AMPK (kindly donated by D. Carling, Imperial College London, London, UK), anti-AMPK{gamma}3 AB (kindly donated by Arexis, Gothenburg, Sweden), anti-ACCbeta Ser227 phospho (Upstate Biotechnology), anti-HKII (Alpha Diagnostic international, San Antonio, TX), anti-cyt c (BD Biosciences Pharmingen, San Diego, CA), anti-GLUT4 (Chemicon International, Temecula, CA), and anti-COX-1 (Molecular Probes, Eugene, OR). Secondary antibodies used were all species-specific horseradish peroxidase-conjugated immunoglobulins (DakoCytomation, Glostrup, DK). Bands were visualized using a Kodak Image Station 440CF (Kodak, Glostrup, Denmark) and an enhanced chemoluminescence system (ECL Plus; Amersham Pharmacia Biotech, Uppsala, Sweden). Bands were quantified using Kodak 1D 3.5 software, and protein content was expressed in relative units compared with control samples loaded on each gel (Fig. 1, A and B). The antibody used to detect COX-1 protein resulted in a specific signal at ~35 kDa despite the predicted molecular mass of 57 kDa. This finding is, however, in concert with previous reports (12, 31) and is in addition supported by the fact that COX-1 was isolated in a mitochondrial-enriched fraction from rat muscle (Fig. 1A).


Figure 1
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  		Fig. 1. Representative immunoblots of investigated proteins. A: the antibody used to detect cytochrome c (cyt c) oxidase-1 (COX-1) protein resulted in a specific signal at ~35 kDa, as previously reported (12, 31), and was intensified in a mitochondrial-enriched fraction from rat muscle. Hom., homogenate; Sup., supernatant fraction; Mito., mitochondrial-enriched fraction. B: representative immunoblots from white gastrocnemius muscle lysates from untrained controls (U) and activity wheel-trained (T) wild-type (WT) and AMPK{alpha}2 whole body knockout ({alpha}2-KO) mice. The signal obtained using the cyt c antibody was verified against purified cyt c from rat heart as positive control (Sigma-Aldrich). The signal obtained using the {gamma}3-antibody has previously been verified against bacterially expressed His-tagged {gamma}3-protein (26), and the signal is abolished in muscle lysates from {gamma}3-KO mice (3). HKII, hexokinase II; GLUT4, glucose transporter 4.

 
Citrate Synthase and 3-Hydroxyacyl-CoA Dehydrogenase Maximal Activity

Maximal activity of citrate synthase (CS) was measured in reaction coupled to conversion of NAD+ to NADH, and 3-hydroxyacyl-CoA dehydrogenase (HAD) was measured as NADH production by spectrophotometric determination of NADH changes at 340 nm at 37°C, pH 7.0, in muscle lysates (1 µg/µl protein), using an automatic analyzer (Hitachi automatic analyzer 912; Boehringer Mannheim, Ingelheim, Germany). CS activity was measured with acetyl-CoA and oxaloacetate as substrate and HAD measured with acetoacetyl-CoA as substrate (25).

Statistics

Data are expressed as means ± SE. Statistical evaluations were performed by either Student's t-test or two-way ANOVA using the Student-Newman-Keuls method as a post hoc test when appropriate. Differences between groups were considered statistically significant if P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Exercise Training Performance Data

In the activity wheel-trained group, WT and {alpha}2-KO mice ran the same average distance per day (WT: 3.98 ± 0.34 km; {alpha}2-KO: 4.59 ± 0.36 km) at the same average speed (WT: 15.3 ± 0.67 m/min; {alpha}2-KO: 16.7 ± 0.67 m/min, P = 0.09) and the same average running duration (WT: 4.19 ± 0.25 h; {alpha}2-KO: 4.40 ± 0.26 h).

Food Intake and Body Weight

The average daily food intake (WT: 6.7 ± 0.1 g; {alpha}2-KO: 7.1 ± 0.2 g) did not change in response to AICAR treatment or activity wheel running but was ~25% higher (P < 0.05, main effect) in activity wheel-trained WT and {alpha}2-KO mice compared with nontrained controls. There was no difference in body weight (WT: 24.2 ± 0.4 g; {alpha}2-KO: 23.3 ± 0.5 g) between WT and {alpha}2-KO animals before or after 28 days of activity wheel running or AICAR, and body weight was also not affected by either of the two interventions.

Acute AMPK Activation by AICAR and Activity Wheel Running

The acute experiments were performed to verify that AMPK was activated by the interventions and that the {alpha}2-KO was associated with a reduced AMPK signaling.

AICAR. AMPK{alpha}1-associated activity was not increased by a single injection AICAR at any time point in either red (RG) or white gastrocnemius (WG) muscles (Fig. 2, A and B). Furthermore, the {alpha}2-KO led to a compensatory increase of AMPK{alpha}1 activity in RG (~45%, P < 0.05, main effect) and strongly tended to do so in WG (P = 0.05) compared with WTs (Fig. 2, A and B). AICAR increased (P < 0.05) AMPK{alpha}2 activity after 30 (185 and 187%) and 60 min (144 and 57%) in RG and WG muscle, respectively, and was returned to basal in both muscles after 120 min compared with saline controls (Fig. 2, C and D). We also measured the phosphorylation (P) level of AMPK{alpha}-Thr172 by immunoblotting to estimate the impact of the {alpha}2-KO on the total level of AMPK activity in {alpha}2-KO muscles. AICAR increased AMPK{alpha}-P only in WT and not in {alpha}2-KO muscles (P < 0.05), and the {alpha}2-KO was associated with an ~70 and ~80% reduction (P < 0.05) in {alpha}-AMPK-P at basal and in response to AICAR, respectively, in both muscle types compared with WTs (Fig. 3, A and B). The phosphorylation level of the endogenous AMPK target ACCbeta Ser227 was also measured and displayed essentially the same pattern in phosphorylation as AMPK{alpha}-Thr172 in both muscles (data not shown). The inability of AICAR in activating AMPK{alpha}1 in the present study contrasts findings in ex vivo incubated mouse muscle, and this difference may be due to less intramyocellular accumulation of 5-aminoimidazole-4-carboxamide-1-beta-D-ribosyl-5-monophosphate in response to a subcutaneous AICAR injection than when exposed to a fixed concentration during ex vivo incubation.


Figure 2
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  		Fig. 2. {alpha}-Isoform-specific AMPK kinase activity in red (RG) and white gastrocnemius (WG) muscle lysates of WT and {alpha}2-KO mice after the designated intervention as well as in corresponding controls. A and B: AMPK{alpha}1 activity in RG and WG 30, 60, and 120 min after a single subcutaneous injection of 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) and 60 min after injection of saline; n = 5–7. C and D: AMPK{alpha}2 activity in RG and WG 30, 60, and 120 min after a single subcutaneous injection of AICAR and 60 min after injection of saline; n = 5–7. E and F: AMPK{alpha}1- and -{alpha}2 activity immediately after 1 min of voluntary activity wheel running was completed; n = 5–7. *{alpha}2-KO significantly different from WT (P < 0.05). {dagger}Significantly different from same genotype in basal group (P < 0.05). —, Main effect. AW, activity wheel running. Data are presented as means ± SE.

 

Figure 3
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  		Fig. 3. AMPK{alpha}-Thr172 phosphorylation (P) activity in red (RG) and white (WG) gastrocnemius muscle lysates of WT and {alpha}2-KO mice after the designated intervention as well as in corresponding controls. Protein phosphorylation was assessed by immunoblotting, using phosphospecific antibodies. A and B: AMPK{alpha}-Thr172-P 60 min after a single subcutaneous injection of either saline or AICAR solution; n = 5–7. C and D: AMPK{alpha}-Thr172-P immediately after 1 min of voluntary activity wheel running was completed; n = 5–7. *{alpha}2-KO significantly different from WT (P < 0.05). {dagger}Significantly different from same genotype in saline group (P < 0.05). —, Main effect. Data are presented as means ± SE relative to WT control.

 
Activity wheel. Mice were killed after 3–4 h of periodic wheel running completed by 1 min of continuous running. AMPK{alpha}1 activity was increased (P < 0.05, main effect) in WTs of RG and WG muscle by 185 and 60%, respectively, compared with nonexercised controls (Fig. 2E). The {alpha}2-KO was in both muscle types associated with an ~35 and ~45% higher (P < 0.05, main effect) AMPK{alpha}1 activity in resting and exercised muscles, respectively, compared with WT muscles (Fig. 2E). Activity of AMPK{alpha}2 increased (P < 0.05) in WTs of RG and WG muscle by 70 and 45%, respectively, compared with nonexercised controls (Fig. 2F). AMPK{alpha}-P was increased in response to running (P < 0.05, main effect) by 40 and 75% in RG and WG, respectively, compared with nonexercised controls (Fig. 3, C and D). Lack of the {alpha}2-isoform was associated with ~60 and ~70% reduction (P < 0.05, main effect) in AMPK{alpha} phosphorylation in RG and WG, respectively, compared with WT muscles (Fig. 3, C and D). Changes in AMPK{alpha}-P were mirrored by changes in ACCbeta Ser227-P in both muscles (data not shown). Last, during activity running, the in vitro-measured AMPK{alpha}1 activity was in general increased to a higher level than AMPK{alpha}2 activity regardless of the estimates of total endogenous AMPK signaling (AMPK{alpha}-P, ACCbeta-P), which suggests that the {alpha}2-isoform is the principal donator of AMPK activity in mouse muscle.

Blood Glucose in AICAR-Treated Animals

To monitor the effect of AICAR during the chronic treatment, blood glucose was measured 60 min after subcutaneous injection of AICAR on days 1, 13, and 26. Basal blood glucose was similar between groups on all 3 days (day 1: WT saline 5.9 ± 0.2 mM, WT AICAR 5.7 ± 0.2 mM; {alpha}2-KO saline 5.5 ± 0.2 mM, {alpha}2-KO AICAR 5.9 ± 0.2 mM) and decreased (P < 0.05, main effect) on average on days 1, 13, and 26 by 28.4 ± 5.1% 1 h after AICAR injection in both WT and {alpha}2-KO animals compared with basal values.

HKII and GLUT4 Protein Expression

Twenty-eight days of AICAR treatment increased (P < 0.05) GLUT4 protein by 45 and 85% in RG and WG WT muscles, respectively, compared with saline controls, and the {alpha}2-KO completely abolished the effect of AICAR on GLUT4 protein expression (Fig. 4, A and B). Furthermore, exercise training increased (P < 0.05, main effect) GLUT4 by 17 and 26% in RG and WG WT muscles, respectively, compared with nontrained controls (Fig. 4, A and B). Importantly, the training-induced increases of GLUT4 were normal in both {alpha}2-KO muscles despite the reduced AMPK signaling (Fig. 4, A and B). Finally, another novel finding was that GLUT4 protein in basal WG muscles was, in fact, upregulated (P < 0.05, main effect) in response to the {alpha}2-KO (Fig. 4B).


Figure 4
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  		Fig. 4. GLUT4 and HKII protein content in RG (A) and WG (B) muscle lysates of WT and {alpha}2-KO mice after a 28-day program of either saline/AICAR injections or activity wheel running was completed; n (AICAR) = 9–11, n (activity wheel) = 10–15. *{alpha}2-KO significantly different from WT (P < 0.05). {dagger}Significantly different from same genotype in control group (P < 0.05). —, Main effect. Data are presented as means ± SE relative to WT control.

 
Regarding HKII, the 28 days of AICAR treatment induced a solid 100 and 140% increase (P < 0.05) in RG and WG WT muscles, respectively, compared with saline controls, and the {alpha}2-KO completely abolished the effect of AICAR on HKII protein expression (Fig. 4, A and B). Exercise training increased (P < 0.05, main effect) HKII expression by 50% in WTs of WG, whereas no significant increase was observed in RG. As observed for GLUT4, the training-induced increase in HKII in {alpha}2-KO muscles was similar to WT muscle despite the lack of the {alpha}2-isoform (Fig. 4, A and B). Last, basal HKII expression was, in general (P < 0.05, main effect in RG), reduced by ~30% in response to the {alpha}2-KO in both RG and WG (Fig. 4, A and B).

Mitochondrial Marker Enzymes

Notably, the expression level of all four mitochondrial markers was ~20% lower (P < 0.05, main effect) in both {alpha}2-KO RG and {alpha}2-KO WG compared with WT muscles (Fig. 5, A and B). In RG muscles, no effect of the 28 days of AICAR treatment was observed on any of the mitochondrial marker enzymes measured (Fig. 5, A and B). In WG, AICAR increased (P < 0.05) cyt c and COX-1 protein as well as CS maximal activity by 115, 48, and 23%, respectively, whereas HAD maximal activity was unchanged compared with saline controls (Fig. 5B). The effect of AICAR treatment on mitochondrial marker enzymes was completely abolished in muscles not expressing the {alpha}2-subunit (Fig. 4B).


Figure 5
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  		Fig. 5. Cyt c and COX-1 protein content and citrate synthase (CS) and 3-hydroxyacyl-CoA dehydrogenase (HAD) maximal activity in RG (A) and WG (B) muscle lysates of WT and {alpha}2-KO mice after a 28-day program of saline or AICAR injections was completed. Basal CS and HAD activity (µmol·µg lysate protein–1·min–1) in WT RG muscles were 0.41 ± 0.02 and 0.38 ± 0.02, respectively. Basal CS and HAD activity in the WT WG muscles were 0.27 ± 0.001 and 0.34 ± 0.01, respectively; n = 9–11. * {alpha}2-KO significantly different from WT (P < 0.05). {dagger}Significantly different from same genotype in control group (P < 0.05). —, Main effect. Data are presented as means ± SE relative to WT control.

 
In exercise-trained muscles, increases in mitochondrial marker were mainly evident in WG, as also observed for AICAR treatment (Fig. 6, A and B). In RG, COX-1 protein was increased (P < 0.05, main effect) by 60% in response to exercise training, whereas the cyt c protein and HAD maximal activity was unchanged and the CS maximal activity was slightly lowered (P < 0.05, main effect; Fig. 6B). In WG, COX-1 protein, CS, and HAD maximal activity were increased in response to exercise training (P < 0.05, main effect; Fig. 6B) by 50, 25, and 15%, respectively, compared with nontrained muscles. Importantly, the training-induced increases in COX-1, CS, and HAD levels were maintained in WG {alpha}2-KO muscle despite the reduced AMPK signaling in muscles lacking the {alpha}2-isoforms.


Figure 6
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  		Fig. 6. Cyt c and COX-1 protein content and CS and HAD maximal activity in RG (A) and WG (B) muscle lysates of WT and {alpha}2-KO mice after a 28-day program of activity wheel running was completed; n = 10–15. *{alpha}2-KO significantly different from WT (P < 0.05). {dagger}Significantly different from same genotype in control group (P < 0.05). —, Main effect. Data are presented as means ± SE relative to WT control.

 
AMPK Subunit Protein Expression

We furthermore measured protein expression of five of the seven known AMPK isoforms ({alpha}1, {alpha}2, beta1, beta2, {gamma}3) to investigate whether a chronic AICAR treatment or exercise training had an effect on AMPK protein expression. The general pattern in AMPK protein in {alpha}2-KO muscles was a compensatory increase in {alpha}1-protein (53–73%) and a decrease in beta1- (23–32%), beta2- (60–66%), and {gamma}3-protein (24–37%) compared with WT muscles (Fig. 7, A and B). Interestingly, in response to exercise training, {alpha}1-, {alpha}2-, beta1-, beta2-, and {gamma}3-proteins were all decreased by ~30–60% in RG muscles (Fig. 7A), whereas expression of AMPK subunits remained unchanged in WG muscles in response to exercise training (Fig. 7B). AICAR treatment did not have any effect on AMPK protein expression in either of the two muscles, indicating that activation of AMPK does not initiate a direct feedback regulation on its own expression (data not shown).


Figure 7
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  		Fig. 7. Protein content of AMPK{alpha}1-, -{alpha}2, -beta1, -beta2, and -{gamma}3 subunit isoforms in RG (A) and WG (B) muscle lysates of WT and {alpha}2-KO mice. Only values from activity wheel training study are shown; n = 10–15. *{alpha}2-KO significantly different from WT (P < 0.05). {dagger}Significantly different from same genotype in untrained control group (P < 0.05). —, Main effect. Data are presented as means ± SE relative to WT control. ND, nondetectable.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The purpose of the present study was to investigate the role of the catalytic AMPK{alpha}2 isoform in basal and exercise training- and AICAR-induced upregulation of metabolic proteins in skeletal muscles. The main findings were that the {alpha}2-KO was associated with a general ~20% reduced content of all mitochondrial markers measured in nonexercised red and white gastrocnemius, implying that AMPK has a mitochondriogenic function in basal nonexercised skeletal muscle. Furthermore, increases in GLUT4, hexokinase II, COX-1, CS, and HAD with exercise training were normal in white skeletal muscle lacking the {alpha}2-isoform. This was seen even though the {alpha}2-KO markedly reduced AMPK signaling by ~60–70% in response to acute AICAR injection and activity wheel running. The difference in AMPK signaling between genotypes was, in fact, seen despite ~45% higher AMPK{alpha}1 activity in {alpha}2-KO muscle during running. This suggests that the {alpha}2-isoform is important in expression of mitochondrial enzymes in muscles subjected to normal locomotor activity but is not essential in training-induced expression of hexokinase II, GLUT4, COX-1, CS, and HAD protein, indicating that, if AMPK is involved in metabolic adaptations of muscles to training, the role is probably of only minor importance. The findings further suggest either that signaling pathways other than AMPK are involved in training adaptations or that the remaining {alpha}1-isoform by itself is sufficient in this respect despite its inability to convey the adaptations to repeated AICAR injections. On the other hand, the knockout of the {alpha}2-isoform completely abolished AICAR-induced increases in hexokinase II, GLUT4, cyt c, COX-1 protein, and CS maximal activity, showing that the {alpha}2-subunit is the principal catalytic isoform in AMPK-mediated expression of metabolic enzymes in response to this type of stimulus.

Although the observed exercise-induced increases in the measured proteins were not as pronounced as findings in humans, the increases are generally in concert with other exercise training studies using voluntary activity wheel running (6, 17, 32). It could be argued that this type of training is insufficient in intensity to induce robust increases of metabolic proteins. However, if instead WT and {alpha}2-KO mice are trained by treadmill running for 28 days by a protocol progressively increasing in speed and duration, essentially similar results are obtained (unpublished data). These observations, therefore, lead us to believe that the mouse as species is characterized by less metabolic muscle adaptability to exercise training than, e.g., rat or human.

The understanding of how exercise training enhances metabolic performance of skeletal muscle is that metabolic proteins are upregulated by cumulative effects of transient increases in gene transcription in response to each exercise bout (33, 35). AMPK has been proposed as a candidate in coordinating metabolic adaptations of muscle to exercise training, because AMPK is activated in exercising/contracting muscle (11, 14, 48, 50) and because pharmacological activation of AMPK is correlated with increased transcription of genes encoding metabolic proteins as well as increased mitochondrial content (4, 5, 20, 43). The signaling pathway(s) downstream of AMPK leading to gene activation is not clear, but several lines of evidence suggest that transcriptional coactivator PGC-1{alpha} is linking AMPK to mitochondrial biogenesis and GLUT4 expression via NRF- and MEF2 isoforms, respectively (22, 28). For instance, activation of AMPK in muscle with beta-GPA or AICAR increases PGC-1 expression, and abrogating AMPK signaling by overexpressing a kinase-dead AMPK construct or knocking out the {alpha}2-isoform abolishes the effect of AMPK activation on PGC-1 expression (20, 52). The latter finding could, in addition, suggest that the {alpha}2-isoform is the main catalytic AMPK isoform in regulating gene activity in muscle, which in part is supported by the fact that only {alpha}2 and not {alpha}1 is found in the nucleus of mammalian cell types, including rat muscle fibers (1, 40), and that {alpha}2 content in the nucleus is further enriched in response to a single bout of exercise in human muscle (29). Therefore, the finding that knockout of the {alpha}2-isoform did not affect training adaptations in protein expression of hexokinase II and GLUT4 and mitochondrial markers was somewhat unexpected. Still, these findings extend recent findings showing that knocking out the {alpha}2-isoform does not prohibit transcriptional activation of several exercise responsive genes, such as the hexokinase II or PGC-1{alpha} gene by a single exercise bout (20). The findings are supported by the fact that GLUT4 RNA messenger increases normally after treadmill running in mouse muscle expressing the kinase-dead AMPK construct (16).

We and others have previously shown that muscles with impaired AMPK signaling are recognized by a higher degree of metabolic stress and AMP buildup than WT muscles during treadmill running (20) and contraction (39), and this could be expected to induce a more potent allosteric activation of the remaining {alpha}1-isoform in {alpha}2-KO muscle than in WT. Furthermore, in conjunction with the 50–80% upregulation of the {alpha}1-isoform in the investigated {alpha}2-KO muscles, it could be argued that the total level of AMPK signaling would be normalized in {alpha}2-KO muscles by the elevated {alpha}1-signaling, which in addition could then explain the normal training-induced increase in metabolic proteins in white {alpha}2-KO muscle. However, both AMPK{alpha}-P and ACCbeta-P, measures believed to reflect total cellular AMPK signaling, were substantially lower in {alpha}2-KO muscles than in WT. In fact, AMPK signaling during AICAR exposure and activity wheel running never exceeded the basal level in resting WT muscles, which suggests that the remaining {alpha}1-isoform was not able to normalize AMPK signaling in {alpha}2-KO muscles. Thus, if AMPK is a key player in exercise-induced metabolic adaptation of skeletal muscle, the estimated 60–70% reduction in AMPK signaling should be expected to be manifested by some means, which was not the case. Together, these findings may then indicate that activation of AMPK during exercise only has a minor role in regulation of the expression of metabolic enzymes in muscle. One reason for the different results in resting and exercising muscles could be explained by signaling other than AMPK induced by exercise, e.g., CaMK- (37) or MAPK-dependent signaling (2), which could make AMPK dispensable during exercise. However, it cannot be ruled out that the remaining {alpha}1-isoform in {alpha}2-KO muscles substitutes for the {alpha}2-isoform, since AMPK{alpha}1 was also found to be activated by running.

In basal muscle, the {alpha}2-KO was associated with an ~20% reduction in mitochondrial marker enzymes. Cyt c and COX-1 are important components in the electron transport chain, whereas CS and HAD are key enzymes in the TCA cycle and beta-oxidation, respectively. In line with this finding, we have previously reported that RNA messenger content of the mitochondrial fatty acid transporter carnitine palmitoyl transferase I is ~25% lower in {alpha}2-KO muscles (20). Since other studies (7) have shown that increased mitochondrial capacity is achieved by an increase in mitochondrial volume with a simultaneous upregulation of mitochondrial enzymes, the reduction of mitochondrial markers may reflect a reduced mitochondrial content in muscle lacking the {alpha}2-isoform. It could be hypothesized that the decrease in mitochondrial markers was driven by a shift in fiber type composition toward a more fast-twich muscle phenotype as a result of reduced basal AMPK signaling in the {alpha}2-KO muscle. This seems, however, not to be the case, because investigations of the quadriceps muscle have shown that the relative content of myosin heavy chain (MHC) I and MHC II isoforms are normal in {alpha}2-KO muscle (unpublished data). A functional consequence of reduced mitochondrial capacity might be decreased oxidative metabolism in muscles during exercise. In accordance, muscle ATP is reduced followed by a comparable increase in inosine monophosphate in response to prolonged treadmill running when the {alpha}2-isoform is missing (20). This view is supported by the fact that knocking out of the upstream AMPK kinase LKB1 is associated with impaired adenosine nucleotide balance during ex vivo contraction (39).

Expression of {alpha}1-protein was 50–70% higher in {alpha}2-KO muscles, in agreement with previous reports (19, 20), which probably reflects that the investigated muscles tried to restore total AMPK activity by upregulating {alpha}1-protein. Since AMPK signaling was still substantially lower in {alpha}2-KO muscles, the compensatory increase in {alpha}1-protein apparently did not restore the total level of {alpha}-protein back to normal. A final, interesting finding was that the five measured AMPK subunits all decreased by 30–60% in response to exercise training in red gastrocnemius, whereas AMPK isoform expression was unchanged by exercise training in white gastrocnemius. A consistent finding in response to strength and endurance training in human muscle is an increase in {alpha}1-protein content (10, 21, 34), and in rat muscles a tendency to an increase in {alpha}1-protein and a reduction in {alpha}2-protein in response to treadmill training has also been reported (9). The reason for different findings in the aforementioned studies and the present study could be due to, besides species difference, the relatively high training volume of the activity wheel-trained mice. Since the overall function of AMPK is to preserve the ATP level and redox state within the cell (13, 36, 47), the present findings could indicate less reliance on a metabolic "safeguard" in trained than in untrained mouse muscle for metabolic control in response to the vast (>100-fold) increase in muscle energy turnover during a bout of exercise (38). In support of this notion is the recent finding that AMPK kinase activity is increased to a lesser extent in trained than in untrained human muscle during exercise (27, 34).

In summary, knockout of AMPK{alpha}2 was associated with a general ~20% reduction in mitochondrial marker content in nonexercised red and white muscle, in concert with the substantial reduction in AMPK signaling. The exercise training program was associated with an increase in several (hexokinase II, GLUT4, COX-1, CS, HAD), but not all (cyt c), of the investigated proteins in WT white gastrocnemius, whereas WT red gastrocnemius was characterized by minor or no responses despite AMPK activation during activity wheel running. Interestingly, in {alpha}2-KO white gastrocnemius, hexokinase II, GLUT4, COX-1, CS, and HAD increased normally in response to exercise-trained muscles, regardless of the lowered AMPK signaling. Therefore, although pharmacological activation of AMPK{alpha}2 increases expression of these metabolic enzymes in resting muscle, AMPK{alpha}2 seems to not be essential for metabolic adaptation of skeletal muscles to exercise training, which furthermore suggest at least a partial reliance of other exercise-sensitive signaling pathways in this respect. Still, AMPK{alpha}2 activity is important for expression of mitochondrial enzymes in the basal state.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
The study was supported by grants from the Danish Diabetes Association, the Danish Medical Research Council, the Danish Natural Science Research Council, the Novo Nordisk Foundation, the Lundbeck Foundation, the Copenhagen Muscle Research Centre, an integrated project from the European Union (contract LSHM-CT-2004-005272), by Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, and by the French Ministry of Research. J. F. P. Wojtaszewski was supported by a Hallas Møller fellowship from the Novo Nordisk Foundation.


   ACKNOWLEDGMENTS
 
We thank Prof. D. Grahame Hardie, Dundee University, Dundee, Scotland, for the kind donation of AMPK{alpha}1 and -{alpha}2 antibodies, Professor David Carling, Imperial College London, London, UK, for the kind donation of the pan beta-AMPK antibody, and Dr. Margit Mahlapuu, Arexis, Gothenburg, Sweden, for the kind donation of the AMPK{gamma}3 antibody. Vigdis Hoel Christie, Merete Vannby, and Betina Bolmgren are acknowledged for skilled technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. B. Jørgensen, Dept. of Human Physiology, Copenhagen Muscle Research Centre, Inst. of Exercise and Sport Sciences, 13-Universitetsparken, Univ. of Copenhagen, DK-2100 Copenhagen, Denmark. (e-mail: SBJorgensen{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.


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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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