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

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 (alpha1 and alpha2) and regulatory (beta1, beta2, gamma1, gamma2a, gamma2b and gamma3) AMPK isoforms were independent of T2DM, whereas the protein content of alpha1 (+16%), beta2 (+14%) and gamma1 (+29%) was higher and the gamma3 content was lower (-48%) in trained compared with untrained muscle (all P < 0.01). The majority of alpha protein co-immunoprecipitated with beta2 and alpha2/beta2 accounted for the majority of these complexes. gamma3 was only associated with alpha2 and beta2 subunits, and accounted for approximately 20% of all alpha2/beta2 complexes. The remaining alpha2/beta2 and the alpha1/beta2 complexes were associated with gamma1. The trimer composition was unaffected by T2DM, whereas training induced a shift from gamma3- to gamma1-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.

Figure 1. 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.

Figure 2. 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.

Figure 3. 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.

Figure 4. 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).