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J Physiol. 2005 Apr 15; 564(Pt 2): 563–573.
Published online 2005 Feb 17. doi: 10.1113/jphysiol.2005.082669
PMCID: PMC1464439
PMID: 15718261

5′AMP activated protein kinase expression in human skeletal muscle: effects of strength training and type 2 diabetes

Jørgen F P Wojtaszewski,1 Jesper B Birk,1 Christian Frøsig,1 Mads Holten,2 Henriette Pilegaard,3 and Flemming Dela2
Author information Article notes Copyright and License information PMC Disclaimer

Abstract

Strength training enhances insulin sensitivity and represents an alternative to endurance training for patients with type 2 diabetes (T2DM). The 5′AMP-activated protein kinase (AMPK) may mediate adaptations in skeletal muscle in response to exercise training; however, little is known about adaptations within the AMPK system itself. We investigated the effect of strength training and T2DM on the isoform expression and the heterotrimeric composition of the AMPK in human skeletal muscle. Ten patients with T2DM and seven healthy subjects strength trained (T) one leg for 6 weeks, while the other leg remained untrained (UT). Muscle biopsies were obtained before and after the training period. Basal AMPK activity and protein/mRNA expression of both catalytic (α1 and α2) and regulatory (β1, β2, γ1, γ2a, γ2b and γ3) AMPK isoforms were independent of T2DM, whereas the protein content of α1 (+16%), β2 (+14%) and γ1 (+29%) was higher and the γ3 content was lower (−48%) in trained compared with untrained muscle (all P < 0.01). The majority of α protein co-immunoprecipitated with β2 and α2/β2 accounted for the majority of these complexes. γ3 was only associated with α2 and β2 subunits, and accounted for ∼ 20% of all α2/β2 complexes. The remaining α2/β2 and the α1/β2 complexes were associated with γ1. The trimer composition was unaffected by T2DM, whereas training induced a shift from γ3- to γ1-containing trimers. The data question muscular AMPK as a primary cause of T2DM whereas the maintained function in patients with T2DM makes muscular AMPK an obvious therapeutic target. In human skeletal muscle only three of 12 possible AMPK trimer combinations exist, and the expression of the subunit isoforms is susceptible to moderate strength training, which may influence metabolism and improve energy homeostasis in trained muscle.

The 5′AMP-activated protein kinase (AMPK) is a kinase fully activated as a heterotrimeric complex consisting of a catalytic (α) and two regulatory (β, γ) subunits. Two isoforms of the catalytic subunit (α1,α2), two of the β subunit (β1,β2), and three of the γ subunit (γ1, γ2, γ3) have been identified in mammalian cells (Stapleton et al. 1996; Thornton et al. 1998; Cheung et al. 2000). AMPK is activated by low intracellular energy levels and is therefore thought to serve as a fuel gauge to protect against energy deprivation, for example in skeletal muscle during exercise and other metabolically stressed conditions such as hypoxia (Winder & Hardie, 1996; Hayashi et al. 2000; Wojtaszewski et al. 2000).

Pharmacological activation of AMPK in skeletal muscle enhances expression of genes/proteins, for example GLUT4 and hexokinase II (Holmes et al. 1999), and regulates mitochondrial biogenesis (Bergeron et al. 2001; Zong et al. 2002). In skeletal muscle, AMPK regulates lipid and glucose metabolism, as well as gluconeogenesis, glycolysis, lipogenesis and cholesterol formation in the liver (reviewed by Winder & Hardie, 1999). Accordingly, the metabolic profile of the tissue is reflected in the AMPK isoform expression or functions as observed in humans and animals harbouring mutated or genetically modified forms of AMPK (Milan et al. 2000; Arad et al. 2002; Viollet et al. 2003; Barnes et al. 2004; Jørgensen et al. 2004).

Patients with type 2 diabetes mellitus (T2DM) have been reported to have normal AMPK protein expression (Musi et al. 2001; Højlund et al. 2003) and maintained responses to 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR), metformin and acute exercise (Musi et al. 2001, 2002; Koistinen et al. 2003). To our knowledge no data are available on the actual AMPK heterotrimetric subunit isoform composition in human skeletal muscle and accordingly, whether this is changed in individuals with T2DM is unknown.

Exercise training improves insulin action in skeletal muscle tissue (Dela et al. 1992, 1995; Holten et al. 2004). The improvement has been observed in both healthy subjects and in patients with T2DM, and is associated with changes in protein expression of elements in the insulin signalling cascade as well as proteins involved in the process of glucose uptake and storage in skeletal muscle (Dela et al. 1993, 1994; Holten et al. 2004). Both acute and chronic pharmacological activation of AMPK increases insulin action on glucose metabolism in skeletal muscle suggesting that AMPK may be a factor regulating insulin action (Buhl et al. 2001; Fisher et al. 2002; Iglesias et al. 2002; Jessen et al. 2003). In skeletal muscle of young, healthy individuals increased protein levels of the α1, β2 and γ1 as well as a decreased level of the γ3 AMPK subunit are found in response to endurance training (Langfort et al. 2003; Nielsen et al. 2003; Frosig et al. 2004). Exercise strength training increases insulin sensitivity (Holten et al. 2004) and is well tolerated by most people, including individuals with T2DM. However, whether strength training changes AMPK isoform subunit expression and whether an association between skeletal muscle insulin sensitivity and the content of the various isoforms of AMPK exists, have not been established.

Methods

This study is part of a larger study of which some data have been published previously (Holten et al. 2004). Ten patients with T2DM and seven healthy control subjects (control) participated in the study, which was approved by the ethical committee of Copenhagen and Frederiksberg (KF 01–204/99) and was performed in accordance with the Helsinki Declaration. Informed written consent was obtained from all subjects before participation. Time since diagnosis of T2DM ranged from 2 to 11 years. All the patients were treated with diet recommendations and in addition some patients were treated with tolbutamide 1000 mg day−1 (n = 2), glibenclamide 7 mg day−1 (n = 1), metformin 1700 mg day−1 (n = 1), amlodipin 5 mg day−1 (n = 1), and cerivastatin 200 μg day−1 (n = 1). On the experimental day no medication was taken. None of the control subjects took any medication or had a family history of T2DM. The patients were similar to the control subjects with respect to age (mean ± s.e.m., 62 ± 2 versus 61 ± 2 years) and body weight (85 ± 5 versus 78 ± 3 kg), but the patients were shorter than the control subjects (172 ± 1 versus 178 ± 2 cm; P < 0.05). Thus, body mass index (BMI) was different (P < 0.05) between control subjects (24.5 ± 0.8 kg m−2) and the patients (28.3 ± 1.2 kg m−2). Resting arterial blood pressure was 157 ± 10 mmHg (systolic) and 82 ± 6 mmHg (diastolic) in the patients and 147 ± 6 mmHg (systolic) and 74 ± 3 mmHg (diastolic) in the control subjects (n.s.).

Experimental design

On a screening day, before entering the study, basic physiological characteristics and fasting blood parameters were measured in all subjects (Table 1). The subjects participated in a 6-week strength-training programme. The aim of the programme was to perform strength-training exercise in one leg, while the other leg remained untrained. The leg to be trained was randomly chosen. All training sessions were supervised. Training took place three times a week with each training session lasting no more than 30 min. This included time for warm-up, which was light exercises for the upper body only, followed by a 2-min rest period. During the first and the last training session the three-repetition maximum (3 RM) was measured for each subject. One-repetition maximum (1 RM) was calculated as 106% of the measured 3 RM for each leg exercise (leg press, knee extension, hamstring curl). During the first 2 weeks of training, the subjects performed three sets of 10 repetitions, utilizing a load equivalent to 50% of 1 RM. During weeks 3–6, the subjects performed four sets of eight to 12 repetitions utilizing 70–80% of 1 RM. During the last 2 weeks, the load was adjusted so that all sets were exhaustive. The subjects rested for ∼ 90 s between sets, and for ∼ 2 min between different exercises. The subjects arrived to the laboratory after an overnight fast and 16–18 h after the last training session. After 30 min of supine rest, needle biopsies were obtained from the vastus lateralis muscle of both the trained (T) and the untrained leg (UT). A two-step sequential, hyperinsulinaemic, isoglycaemic clamp was then performed in order to estimate insulin sensitivity. Insulin was infused at rates of 28 and 480 mU min−1 m−2 for 2 h in each clamp step resulting in plasma insulin concentrations of 377 ± 26 pm (T2DM) and 270 ± 20 pm (control) (P < 0.05) and 12453 ± 856 pm (T2DM) and 11066 ± 874 pm (control) (P > 0.05) in the two clamp steps, respectively.

Table 1

Subject characteristics

Type 2 diabetes, n = 10Healthy control, n = 7
Age (year)61 ± 2;62 ± 2
Weight (kg)85 ± 578 ± 3
BMI (kg m−2)28.3 ± 1.2*24.5 ± 0.8
f-B-glucose (mm)7.9 ± 0.9*4.7 ± 0.3
f-S-Insulin (pm)72 ± 17*39 ± 5
W-B glucose clearance (ml min−1 kg−1)
 28 mU min−1 m−22.5 ± 0.6*5.7 ± 0.9
 480 mU min−1 m−28.9 ± 0.7*14.4 ± 0.7

BMI, Body mass index; f-B, fasting blood; f-S, fasting serum; W-B glucose clearance, whole body glucose clearance (i.e. glucose infusion rate (mg min−1 kg−1) during the final 30 min of 2-h sequential insulin infusions divided by the prevailing plasma glucose concentration (mg (100 ml)−1)).

*Significantly different from control subjects (P < 0.05). Data are mean ± s.e.m.

Muscle lysate preparation

The muscle biopsies obtained were frozen in liquid nitrogen within 30 s and stored at −80°C. Approximately 25 mg frozen muscle was freeze-dried and dissected free of visible fat, blood and connective tissue before being homogenized as previously described by Markuns et al. (1999). The homogenates were then rotated end over end at 4°C for 1 h before being centrifuged for 30 min (17 500 g, 4°C). The supernatants were harvested, frozen in liquid nitrogen and stored at −80°C. Total muscle lysate protein content was analysed by the bicinchoninic acid method (Pierce Chem. Comp., Rockford, IL, USA).

SDS-PAGE and Western blotting

Equal amounts of muscle lysate proteins were separated using 10% Tris-HCl gels (Biorad, Denmark), and transferred (semi-dry) to polyvinylidene fluoride (PVDF)-membranes (Immobilion Transfer Membrane, Millipore A/S, Denmark). After blocking (10 mm Tris-Base (pH 7.4), 0.9% NaCl, 1% Tween 20 (TBST) + 2% skimmed milk), the membranes were incubated with primary antibodies (TBST + 2% skimmed milk) followed by incubation in horseradish peroxidase-conjugated secondary antibody (TBST + 2% skimmed milk) (Amersham Pharmacia Biotech Limited, UK). Following detection and quantification using a CCD-image sensor and Kodak 1D software (Kodak Image Station, E440CF, Kodak, Denmark), the protein content was expressed in arbitrary units relative to a human skeletal muscle standard.

Antibodies used for detecting the AMPK subunit isoforms

The primary antibodies used for detecting the AMPK subunit isoforms α1, α2, β2 and γ2a (γ2 long form) came from sheep as previously described (Woods et al. 1996b; Cheung et al. 2000; Durante et al. 2002). The γ2b (γ2 short form) was detected using a rabbit antibody as previously described (Mahlapuu et al. 2004). To detect γ1 and γ3, rabbit antibodies against the N-terminal region of the human isoforms were used (Zymed Laboratories Inc., South San Francisco, CA, US.). An antibody raised in rabbit against a N-terminal region identical in both human β-isoforms was used for the detection of β1 (Upstate Biotecnology Inc., Waltham, MA, USA). For all isoforms of AMPK, electrophoretic mobility corresponded with the expected molecular weights of the proteins, and the antibodies used detected a polypeptide of the expected mass when liver epithelial cell (CCL13) lysate containing recombinant rat (α1, α2, β1, β2 and γ1) or human (γ2b and γ3) protein isoforms were analysed. The anti-γ2b antibody has previously been shown to recognize the bacterial-expressed recombinant protein (Mahlapuu et al. 2004). In human muscle, a doublet was detected at 37 kDa, which may represent two different variants of the γ2b subunit (based on mass spectrometry measurements, D. Carling, personal communication). Phosphorylation of αAMPK-subunits (Thr 172) and acetyl-CoA-carboxylase-β (ACCβ) (Ser 221) was detected using phosphospecific antibodies from Cell Signalling Technology Inc., Beverly, MA, USA and Upstate Biotechnology, Waltham, MA, USA, respectively. ACCβ protein content was accessed using horseradish peroxidase-conjugated strepavidin (Dako, Denmark) as previously described by Chen et al. (2000).

Detection of AMPK heterotrimetic composition

Of the antibodies described above, the anti-α1, -α2, -β2 and -γ3 AMPK immunoprecipitated the respective subunits from human skeletal muscle to ∼ 100%, whereas the anti-γ1 only resulted in partial (∼ 50%) immunoprecipitation. We evaluated the AMPK heterotrimetric protein composition by performing co-immunoprecipitation experiments. The isoforms α1, α2, β2, γ1 and γ3 were immunopurified from 400 μg muscle lysate protein using sepharose-coupled G-protein (overnight at 4°C in lysate buffer), followed by immunoblotting using the antibodies recognizing the various AMPK subunits. The characterization of the heterotrimetric complexes was performed using a large pool of human muscle biopsies obtained in the resting non-stimulated state from 10 different healthy male subjects. Then to evaluate whether T2DM or strength training induces major changes in the heterotrimeric protein composition, we performed analyses in individual biopsies from four subjects before and after training. Due to limited tissue, these analyses were performed using α2 and γ3 coimmunprecipitation experiments only.

Oxidative enzymes

Samples of biopsies were freeze-dried and dissected free of visible connective and fat tissue before fluorometric measurements of citrate synthase (CS), hydroxyacyl-3-dehydrogenase (HAD) and lactate dehydrogenase (LDH) activity were performed in accordance with previously described protocols (Lowry & Passonneau, 1972).

mRNA

Total RNA was isolated by a modified guanidinium thiocyanate–phenol–chloroform extraction method adapted from the method of Chomczynski & Sacchi (1987) as previously described by Pilegaard et al. (2000). The total RNA content was estimated from the absorbance at 260 nm and 1.5 μg total RNA was reverse transcribed (RT) using the Superscript II RNase H- system (Invitrogen, Denmark) (Pilegaard et al. 2000). The mRNA content of the selected genes was determined by fluorescence-based real-time PCR as previously described (Pilegaard et al. 2003) except for the use of a ABI PRISM 7900 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) and a 10 μl reaction volume in the present study. The sequences given in Table 1 were used to amplify fragments of the AMPK subunits (γ2b was not measured). The probes were labelled with 5′ 6-carboxyfluorescein (FAM) and 3′ 6-carboxy-N,N,N′,N′-tetramethylrhodamine (TAMRA).

The ratio of mRNA:cDNA hybrids was determined for each sample using the PicoGreen reagent (Molecular Probes, the Netherlands) following the guidelines of the manufacturer.

To correct for differences in cDNA content between samples, the arbitrary amount of each target gene was normalized to the cDNA content obtained from the PicoGreen analysis.

Statistics

Comparing controls and individuals with T2DM, two-way analysis of variance for repeated measurements was applied for values obtained before and after training and for values obtained in the trained and untrained legs. When a significant main effect was observed, Tukey's post hoc test was used. A significance level of 0.05 was chosen. Presented values are expressed as means ± s.e.m.

Results

Insulin sensitivity

The patients with T2DM had fasting hyperglycaemia and hyperinsulinaemia and were insulin resistant during clamp conditions compared with the healthy, control subjects (Table 2) (Holten et al. 2004).

Table 2

Sequences used for real time PCR

AMPKForward primerProbeReverse primer
α15′-CAGGGACTGCTACTCCACAGAGA-3′5′TCAGTTAGCAACTATCGATCTTGCCAAA-GGAGT-3′5′-CCTTGAGCCTCAGCATCTGAA-3′
α25′-CAACTGCAGAGAGCCATTCACTT-3′5′-CTGGCTCTCTCACTGGCTCTTTGACCG-3′5′-GGTGAAACTGAAGACAATGTGCTT-3′
β15′-CTTTAATGGTGGATTCCCAAAAGT-3′5′-TCCGATGTGTCTGAGCTGTCCAGTTCTC-3′5′-AGACGTAGGGCTCCTGATGGT-3′
β25′-TGGAAAGTTCTGAGACATCTTGTAGAGA-3′5′-CTCACCCCCAGGGCCTTATGGTCA-3′5′-CCTCAGATCGAAACGCATACATT-3′
γ15′-GTTCCCCAAGCCAGAGTTCA-3′5′-CTCTGGAAGAGCTACAGATTGGCACCTATGC-3′5′-TGGTAGTGCGAACCATAGCAAT-3′
γ2a5′-GCGGTTATGGACACCAAGAAGA-3′5′-ACGCGCAGCGAACGCCTCTT-3′5′-AAGGAGCTCAGGTCCGGAAT-3′
γ35′-GGAAGTGATCGACAGGATTGC-3′5′-CGGGAGCAGGTACACAGGCTGGTG-3′5′-GAGATGCTGGGTCTCGTCCA-3′

CS, HAD, LDH activities and muscle strength

The enzyme activities in muscle of CS, HAD and LDH were similar in control subjects and those with T2DM in untrained legs (control: CS, 84 ± 7; HAD, 124 ± 7; LDH, 840 ± 62 μmol min−1 (g dry weight muscle tissue)−1; T2DM: CS, 70 ± 5; HAD, 119 ± 6; LDH, 966 ± 93 μmol min−1 (g dry weight muscle tissue)−1), and no effect of the strength training was seen (control: CS, 90 ± 7; HAD, 127 ± 6, LDH, 922 ± 75 μmol min−1(g dry weight muscle tissue)−1; T2DM: CS, 70 ± 5; HAD, 120 ± 9; LDH, 1069 ± 185 μmol min−1 (g dry weight muscle tissue)−1) (Holten et al. 2004). Muscle strength increased in all subjects and to a similar degree in the two groups. Thus, knee extension and leg press increased by 42 ± 8% (P < 0.05) and 75 ± 7% (P < 0.05) in subjects with T2DM and by 29 ± 1% (P < 0.05) and 77 ± 15% (P < 0.05) in control subjects, respectively (Holten et al. 2004).

AMPK subunit isoform protein expression

Protein content of all isoforms of AMPK as well as ACCβ was similar in untrained muscle of control and subjects with T2DM (Fig. 1). The response to strength training was also similar in the two groups of subjects (Fig. 2). Significant increases in protein expression were observed for the α1 (16 ± 0%; main effect P < 0.009), β2 (14 ± 1%; main effect P < 0.01), γ1 (29 ± 2%; main effect P < 0.01) and ACCβ (49 ± 4%; main effect P < 0.01) in response to training (Fig. 2). The protein expression of α2 (5 ± 0%; n.s.), β1 (2 ± 0%; n.s.), γ2a (−8 ± 1%; n.s.) and γ2b (7 ± 1%; n.s.) was not altered significantly (Fig. 2). A marked training-induced decrease in protein expression of the γ3 isoform was observed (48 ± 5%; main effect P < 0.008; Fig. 2).

An external file that holds a picture, illustration, etc. Object name is tjp0564-0563-f1.jpg
Muscle protein expression of the various isoforms of the AMPK subunits and ACCβ was quantified in untrained muscle from seven control subjects (open bars) and 10 patients with T2DM (grey bars)

Values obtained in control subjects were set at 100 and the values obtained in subjects with T2DM were normalized to this. Values given are means ± s.e.m.

An external file that holds a picture, illustration, etc. Object name is tjp0564-0563-f2.jpg
Protein expression in untrained and trained muscle of the various isoforms of the AMPK subunits and ACCβ was quantified from seven control subjects (open bars) and 10 patients with T2DM (grey bars)

Values obtained after training are given as percentage changes compared with untrained values (indicated by the dashed line), calculated for each individual subject and presented as means ± s.e.m. The absolute values obtained in untrained muscle were given in Fig. 1. The effect of training was not different between controls and patients with T2DM, and P values given represent the main effect of training in the two groups. Representative immunoblots for the various AMPK subunit isoforms as well as ACCβ are given in the lower panel.

The basal level of ACCβ-Ser221 and αAMPK-Thr172 phosphorylation was not influenced by either T2DM or strength training (ACCβ phosphorylation in control subjects: UT, 16 ± 3; T, 15 ± 2 arbitrary scanning units; ACCβ-P in subjects with T2DM: UT, 13 ± 2; T, 16 ± 3 arbitrary scanning units; αAMPK phosphorylation in control subjects; UT, 12 ± 2; T, 11 ± 2 phosphorylation arbitrary scanning units; αAMPK-P in subjects with T2DM: UT, 11 ± 1; T, 11 ± 1 arbitrary scanning units; Western blots not shown).

mRNA content

In muscle, the mRNA content of AMPK subunit isoforms was similar in control and subjects with T2DM (Table 3; γ2b was not measured). In all subjects investigated, γ3 mRNA was decreased (average 42 ± 7%, n = 11, P = 0.008) after training and this effect was independent of T2DM (Table 3). The mRNA content of the remaining AMPK subunits was not significantly changed by training (Table 3).

Table 3

AMPK mRNA expression

Type 2 diabetes (n = 5–7)Healthy control (n = 6–9)
AMPKUT legT legUT legT leg
α10.13 ± 0.050.11 ± 0.040.18 ± 0.050.13 ± 0.06
α20.17 ± 0.080.16 ± 0.110.21 ± 0.070.19 ± 0.08
β10.10 ± 0.030.12 ± 0.030.14 ± 0.020.15 ± 0.02
β20.14 ± 0.060.18 ± 0.070.23 ± 0.060.22 ± 0.06
γ10.11 ± 0.030.09 ± 0.020.14 ± 0.020.16 ± 0.03
γ20.28 ± 0.140.30 ± 0.100.32 ± 0.050.29 ± 0.05
γ30.29 ± 0.070.15 ± 0.04*0.19 ± 0.020.11 ± 0.02*

AMPK subunit isoform mRNA content in untrained (UT) or trained (T) human muscle biopsies from patients with type 2 diabetes or control subjects. Values are presented as the ratio between the mRNA of interest and cDNA content, and are given as mean ± s.e.m.

*Significant difference (main effect) between UT and T (P = 0.008).

Heterotrimeric AMPK complex composition

Due to a limitation in the amount of muscle material from the main study, we performed detailed analysis of the AMPK heterotrimeric complex in a human muscle sample pooled from several different healthy subjects obtained in a resting non-stimulated state.

α2-containing AMPK complexes (Fig. 3)

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AMPK heterotrimeric complexes in human skeletal muscle were investigated by co-immunoprecipitation

In the upper panel the degree of precipitation/coprecipitation is shown using anti-α1, -α2, -β2, -γ1 or -γ3 as precipitating antibody (IP) and antibodies recognizing the various AMPK isoforms by western blotting (WB). The degree of precipitation/coprecipitation was evaluated both by comparing signals in the immunoprecipitates (obtained from 100 μg lysate protein) to that of the incoming lysate (represents 20 μg lysate protein) as well as by comparing the remaining signal in the post-immunoprecipitation lysate (represents 20 μg lysate protein) to that of the incoming lysate. The results were categorized into 5 levels: ++++, complete precipitation; +++, 80% or more precipitated; ++, ∼ 50% precipitated; +, 20% or less precipitated; -, no precipitation. Representative immunoblots are shown in the lower panel. Each picture consists of three lanes representing, the immunoprecipitates (IP), the post-immunoprecipitation lysate (Supern.) and the incoming lysate (Lysate). Analyses were performed on a pooled human muscle (vastus lateralis) sample from 10 healthy subjects. In addition, α2 and γ3 co-immunoprecipitation analyses were also performed on eight individual biopsies as described in Method and Results. Analyses were run in duplicates. Some secondary bands at ∼ 50 KDa are visual at some of the IP blots. These represent the heavy chain from the immunoprecipitating antibody. ND, not detected.

The majority (> 80%) of β2 was co-immunoprecipitated with α2, and correspondingly nearly all α2 was immunoprecipitated with β2. In both α2 and β2 immunoprecipitates, γ1 was co-immunoprecipitated to a large extent. All γ3 was co-immunoprecipitated with α2, and correspondingly ∼ 20% of α2 was co-immunoprecipitated with γ3. γ2a, γ2b and β1 were not co-immunoprecipitated with α1, α2, β2, γ3 or γ1. Thus, all α2 subunits were associated with β2 subunits. In ∼ 20% of these α2/β2 complexes γ3 was the third isoform. Due to the incomplete (50%) immunoprecipitation of γ1 it could not be directly verified by the present data whether the remaining 80% of the α2/β2 complexes were associated with γ1. However, it is likely as neither γ2a nor γ2b co-immunopecipitated with the α2 or the β2 isoform. Alternatively, α2/β2 complexes may exist to some extent as a dimer.

α1-containing AMPK complexes (Fig. 3)

No γ3 was co-immunoprecipitated with α1, and correspondingly no α1 was co-immunoprecipitated with γ3. β2 co-immunoprecipitated to a low extent with α1, whereas all α1 co-immunoprecipitated with β2. Only a small fraction of γ1 co-immunoprecipitated with α1 and, correspondingly only a minor fraction of α1 co-immunoprecipitated with γ1. Whether γ1 is associated with all α1/β2 complexes was difficult to evaluate due to the incomplete immunoprecipitation of the γ1, and thus some α1/β2 dimers may exist. However, as α1 does not associate with γ2a, γ2b or γ3, it is likely that γ1 is the major γ isoform in α1 heterotrimeric complexes. The data therefore suggest that the α1/β2/γ1 complex contributes 20% or less of all α-containing complexes in human skeletal muscle.

When comparing muscle samples from two control and two subjects with T2DM, we were not able to identify any major differences in the composition of the α2- and γ3-containing complexes. As expected from the protein expression measurements in muscle lysate, less γ3 was co-immunoprecipitated with α2 and more γ1 co-immunoprecipitated with α2 after training. Thus, training induces an increase in α2/β2/γ1 complexes, a result that is probably due to a decrease in α2/β2/γ3 complexes. Although, we do not provide the evidence by co-immunoprecipitation experiments, the isoform expression data would also suggest that training induces a small increase in the amount of α1/β2/γ1 complexes.

Correlations between AMPK protein expression and muscle characteristics

As reported elsewhere (Holten et al. 2004), insulin sensitivity increased specifically in the trained muscles in both groups, but there was no significant correlation between this variable and the content of AMPK proteins (data not shown). Another feature of the muscles was the substantial difference in muscle strength between the two legs. Muscle strength showed a significant correlation to protein content of the AMPK β2 isoform (Fig. 4), but not to any of the other isoforms.

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Relationship between muscle strength and expression of β2 AMPK protein

Relationship between muscle strength (knee extension) and expression of β2 AMPK protein in human vastus lateralis before (filled symbol) and after strength training (open symbols) in control subjects (circles) and patients with T2DM (squares).

Discussion

Defects in the AMPK system may be a primary and/or secondary cause of T2DM. In addition, AMPK may be a therapeutic target in diseases associated with insulin resistance. To evaluate these possibilities, knowledge about the human AMPK heterotrimeric complex is important. Besides the possibility that different AMPK complexes serve different roles within a given tissue, the possibility also exists that different complex compositions are present in different tissues, allowing pharmacological targeting of AMPK in a tissue-specific manner.

Based on the present findings, the majority of AMPK complexes in human muscle contains both α2 and β2. Of these α2/β2 complexes, ∼ 20% were associated with γ3 and, assuming that α2/β2 dimers do not exist, the remaining was most probably associated with γ1. Although some controversy exist regarding the γ3 expression profile in rodent muscle (Cheung et al. 2000; Durante et al. 2002), our observations are in agreement with recent data from mouse skeletal muscle in which γ3 was also found to be associated with α2 and β2, but not with α1 and β1 (Mahlapuu et al. 2004). Based on mRNA measurements, the γ3 isoform is the predominant γ isoform expressed in mouse muscle representing glycolytic fibres (Mahlapuu et al. 2004; Yu et al. 2004), indicating that the α2/β2/γ3 complexes are the major AMPK complex in this fibre type of mouse muscle. The smaller contribution of γ3-containing AMPK complexes in the present study may relate to the mixed fibre type composition in the human vastus lateralis muscle as well as species differences.

β2 is probably the only β-isoform in AMPK complexes in human skeletal muscle, and our co-immunoprecipitation experiments indicate that α2/β2 complexes exceed (> 80%) the amount of α1/β2 complexes. Although this suggests an important role for α2/β2 complexes, it is interesting that when α1- and α2-AMPK activities are measured in vitro these are approximately equal in resting non-stimulated human muscles (Wojtaszewski et al. 2000; Fujii et al. 2000). Among several plausible explanations concerning the kinase assay (antibody interference, AMP dependency and substrate specificity), it could be argued that the α-phosphorylation (Thr172) stoichiometry is different between α1 and α2 in human muscle. This idea is supported by the observation that α1- and α2-AMPK activities in resting mouse skeletal muscle are around the same magnitude yet deletion of the α2 isoform eliminates the majority of α-AMPK Thr172 phosphorylation (Jørgensen et al. 2004).

The β1 isoform was not co-immunoprecipitated with the α1, α2, γ1 or γ3 isoform. This seems to contradict findings in rodent muscle where association between β1 and α1 and/or α2 has been reported (Chen et al. 1999; Yu et al. 2004), although the quantitative importance of β1 was unclear from those studies. It is expected that the AMPK isoforms are stabilized when associated in complexes, thus a decreased β1 (∼ 20%) and β2 (∼ 75%) expression in muscle of the α2 AMPK knockout mouse (Viollet et al. 2003) when compared with the wild-type mouse may suggest some importance for β1 in mouse skeletal muscle (S.B. Jørgensen, J.F.P. Wojtaszewski & E.A. Richter, unpublished observation). Nevertheless, even if present in human skeletal muscle, the amount of β1-containing AMPK complexes must be very limited, because nearly all α1 and α2 co-immunoprecipitated with β2. The present data also suggest that neither γ2a nor γ2b are in AMPK complexes in human skeletal muscle. However, the proteins (β1, γ2a and γ2b) are apparently present in human skeletal muscle, suggesting alternative roles of these proteins.

The present study does not provide evidence as to what cellular consequences the changes in AMPK isoform expression may have. However, several possibilities exist. Based on previous measurements in vitro, a shift from α2/β2/γ3- to α2/β2/γ1-containing complexes in addition to an increased abundance of α1/β2/γ1 complexes would probably provide an AMPK system more sensitive to changes in cellular AMP concentration (Cheung et al. 2000). Also, it is now well established that the content of γ3 protein and/or the functionality of this isoform influences important metabolic processes, for example muscular glucose transport and glycogen synthesis (Milan et al. 2000; Barnes et al. 2004). Thus, the γ3-containing AMPK complexes probably have significant metabolic influence perhaps due to specialized cellular localization. In what way a decreased abundance of α2/β2/γ3 complexes may contribute to a phenotype of a trained muscle, if at all, remains to be elucidated. Although the present data do not provide evidence that AMPK activity is elevated, the increased expression of the α1 catalytic subunit, together with the elevated ACCβ protein expression makes it tempting to speculate that these adaptations are important for the increased fatty acid oxidation both at rest and during exercise in trained muscle, and this could be a particular benefit for patients with T2DM.

Moderate strength training induced changes in AMPK subunit isoform expression. Because a one-legged training model was used it is clear that the training-induced adaptations are phenomena strictly related to the muscles performing the exercise and thus, must be dependent on local contraction-induced factors. It is intriguing that a low volume training regimen that is well tolerated by most people, including elderly people, elicited a response at the AMPK protein expression level quite similar to that seen after a much more intense endurance training regimen (Nielsen et al. 2003; Langfort et al. 2003; Frosig et al. 2004).

The marked decrease in γ3 protein content in response to training was associated with a similar decrease in γ3 mRNA content, suggesting that the training-induced changes in AMPK γ3 expression may involve regulation at the transcriptional level. There were no significant changes in mRNA for the other subunit isoforms, which may explain why there were little or no changes in protein expression of these subunits.

In mouse skeletal muscle, the γ3 isoform is highly differentially expressed among the different fibre types (Yu et al. 2004; Mahlapuu et al. 2004) and this raises the possibility that changes in γ3 expression in the present study could follow a shift in fibre type composition. However, as no fibre type alterations were observed (Holten et al. 2004), the training-induced changes in AMPK subunit expression profile are probably related to changes in protein expression profile within a given myosin heavy chain (MHC) fibre type.

AMPK function may be linked to the contractile properties of the muscle, as indicated by the close relationship between the β2 isoform expression and knee extensor strength. Of course it cannot be excluded that the changes are purely associative but an interesting note is that in mouse muscle, expressing a kinase dead α2 AMPK construct, contractile properties are impaired (Mu et al. 2003).

In the present study, mRNA and protein expression of all the seven AMPK subunit isoforms and ACCβ as well as the isoform composition of the α2- and β2-containing AMPK complexes were similar in skeletal muscle of control subjects and patients with T2DM. This is in agreement with previous findings in individuals with T2DM and in insulin-resistant obese subjects (Musi et al. 2001; Højlund et al. 2004; Steinberg et al. 2004). Thus, no simple relationship seems to exist between muscle insulin sensitivity and AMPK expression levels. This is evident because expression levels in muscle from individuals with T2DM and controls were similar despite markedly different insulin sensitivity. In line with this, no correlation between the training-induced changes in AMPK isoform expression and muscle insulin sensitivity (Holten et al. 2004) was found (data not shown). Also, defects in the muscle AMPK system are unlikely to be the primary cause of T2DM. This may be argued because in T2DM the AMPK system in muscle is expressed normally, has normal levels of the major trimeric complexes, has normal basal activity levels (Musi et al. 2001; Højlund et al. 2004; present study), has normal activation by acute exercise and by metformin in vivo (Musi et al. 2002) and by AICAR in vitro (Koistinen et al. 2003) and has normal changes in AMPK isoform expression profile in response to exercise training (present study). However, this does not exclude the possibility that defects in the AMPK system in other tissues are of primary importance in T2DM or that diminished AMPK activation in muscle (e.g. by physical inactivity) is a secondary factor in T2DM.

The knowledge today with regard to beneficial metabolic effects of AMPK makes it an obvious target for the treatment of T2DM, both through activation by exercise and by pharmacological interventions. As human skeletal muscle does not seem to have β1-, γ2a- and γ2b-containing complexes to any major extent it may be possible to target AMPK in a tissue-specific manner from a pharmacological perspective, thereby avoiding effects in the liver as this tissue contains AMPK complexes (α1/β1/γ1, α2/β1/γ1, from rodent studies) different from those found in muscle (Stapleton et al. 1994, 1997; Woods et al. 1996a; Thornton et al. 1998).

In conclusion, protein and mRNA expression of AMPK subunit isoforms and ACCβ are susceptible to moderate strength training. In human skeletal muscle, α2/β2/γ1-containing AMPK complexes are the most dominant. The γ3 subunits are only associated with α2/β2 complexes and are present in ∼ 20% of all α2/β2 complexes. The normal AMPK activity and expression of AMPK isoforms as well as lack of association with insulin sensitivity, probably excludes skeletal muscle AMPK as a primary cause of T2DM, whereas the maintained function makes AMPK an obvious therapeutic target. The limited number of trimer combinations present in human skeletal muscle (three out of 12 possible) raises the possibility of being able to target AMPK pharmacologically in a tissue-specific manner.

Acknowledgments

We thank Professors David Carling and Grahame Hardie for the kind donation of CCL-13 cells expressing recombinant AMPK isoforms, and Professor Grahame Hardie for providing the anti α1, α2, β2 and γ2a antibodies. We also thank Dr Margit Mahlapuu (Arexis) for the kind donation of the anti-γ2b antibody, and Dr Morten Zacho for his advice on the training programme. The study was supported by grants from the Danish National Research Foundation (No. 504-14), The Copenhagen Muscle Research Centre, the Danish Diabetes Association, the Danish Heart Foundation, the Novo Nordisk Foundation, The Foundation of 1870, Jacob and Olga Madsens Foundation, Else and Mogens Wedell-Wedellsborg Foundation, the Danish Medical Research Council and by a Research & Technological Development Projects (QLG1-CT-2001-01488) funded by the European Commission. J.F.P.W. was supported by a Hallas Møller fellowship from the Novo Nordisk Foundation.

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