Dissociation of AMP-activated protein kinase and p38 mitogen-activated protein kinase signaling in skeletal muscle

Abstract

AMP-activated protein kinase (AMPK) is widely recognized as an important regulator of glucose transport in skeletal muscle. The p38 mitogen-activated protein kinase (MAPK) has been proposed to be a component of AMPK-mediated signaling. Here we used several different models of altered AMPK activity to determine whether p38 MAPK is a downstream intermediate of AMPK-mediated signaling in skeletal muscle. First, L6 myoblasts and myotubes were treated with AICAR, an AMPK stimulator. AMPK phosphorylation was significantly increased, but there was no change in p38 MAPK phosphorylation. Similarly, AICAR incubation of isolated rat extensor digitorum longus (EDL) muscles did not increase p38 phosphorylation. Next, we used transgenic mice expressing an inactive form of the AMPKalpha2 catalytic subunit in skeletal muscle (AMPKalpha2i TG mice). AMPKalpha2i TG mice did not exhibit any defect in basal or contraction-induced p38 MAPK phosphorylation. We also used transgenic mice expressing an activating mutation in the AMPKgamma1 subunit (gamma1R70Q TG mice). Despite activated AMPK, basal p38 MAPK phosphorylation was not different between wild type and gamma1R70Q TG mice. In addition, muscle contraction-induced p38 MAPK phosphorylation was significantly blunted in the gamma1R70Q TG mice. In conclusion, increasing AMPK activity by AICAR and AMPKgamma1 mutation does not increase p38 MAPK phosphorylation in skeletal muscle. Furthermore, AMPKalpha2i TG mice lacking contraction-stimulated AMPK activity have normal p38 MAPK phosphorylation. These results suggest that p38 MAPK is not a downstream component of AMPK-mediated signaling in skeletal muscle.

Figure 1. Activation of AMPK by AICAR is not associated with an increase in p38 MAPK phosphorylation in L6 cells

L6 myoblasts (A) or myotubes (B) were treated with 2 mM AICAR for 10 min. Phosphorylation of AMPK and p38 MAPK were determined by immunoblotting using phospho-specific antibodies. Data are the means ± SEM of three independent experiments and each data point was assayed in duplicate.

Figure 2. Activation of AMPK by AICAR is not associated with an increase in p38 MAPK phosphorylation in skeletal muscle

Rat EDL muscles were isolated and preincubated in KRB buffer for 20 min. The muscles were then incubated with 2 mM AICAR for 40 min. Phosphorylation of AMPK (A) and p38 MAPK (B) were determined by immunoblotting using phospho-specific antibodies. Data are the means ± SEM. n = 4/group. (C); Mice were injected intraperitoneal with AICAR (1mg/g). Gastrocnemius muscles were harvested at each time point indicated. Phosphorylation of AMPK and p38 MAPK were determined by immunoblotting using phosphospecific antibodies. Representative images are shown.

Figure 3. p38 MAPK phosphorylation in γ1R70Q TG mice

Wild type and γ1R70Q TG mice were anesthetized, and the sciatic nerves were attached to electrodes. One leg was electrically stimulated for 10 min to induce muscle contractions, and the other leg served as sham control. Gastrocnemius muscles were dissected. In vitro kinase assay was done to determine total AMPK activity after immunoprecipitation with an antibody that recognizes both α1 and α2 AMPK isoforms (A). The same protein samples used for the kinase assays were used for immunoblotting of phospho-ACC (B), phospho-p38 MAPK (C), and phospho-MKK3/6 (D). Data are the means ± SEM. n = 6/group.

Figure 4. Muscle contraction-induced p38 MAPK phosphorylation in AMPK activity in α2i TG mice

Wild type and α2i TG mice were anesthetized, and sciatic nerves were exposed. One leg was electrically stimulated for 10 min to induce muscle contractions, and the other leg served as sham control. Gastrocnemius muscles were dissected. In vitro kinase assay was done to determine AMPKα1 (A) and α2 (B) activities after immunoprecipitation with their specific antibodies as described in Material and Methods. The same protein samples used for the kinase assays were used for immunoblotting of phospho-p38 MAPK (C) and phospho-ATF2 (D). Data are the means ± SEM. n = 5-9/group.