When phosphorylated at Thr148, the β2-subunit of AMP-activated kinase does not associate with glycogen in skeletal muscle

The 5'AMP-activated protein kinase (AMPK), a heterotrimeric complex that functions as an intracellular fuel sensor that affects metabolism, is activated in skeletal muscle in response to exercise and utilization of stored energy. The diffusibility properties of alpha-and beta-AMPK were examined in isolated skeletal muscle fiber segments dissected from rat fast-twitch extensor digitorum longus and oxidative soleus muscles from which the surface membranes were removed by mechanical dissection. After the muscle segments were washed for 1 and 10 min, similar to 60% and 75%, respectively, of the total AMPK pools were found in the diffusible fraction. After in vitro stimulation of the muscle, which resulted in an similar to 80% decline in maximal force, 20% of the diffusible pool became bound in the fiber. This bound pool was not associated with glycogen, as determined by addition of a wash step containing amylase. Stimulation of extensor digitorum longus muscles resulted in 28% glycogen utilization and a 40% increase in phosphorylation of the downstream AMPK target acetyl carboxylase-CoA. This, however, had no effect on the proportion of total beta(2)-AMPK that was phosphorylated in whole muscle homogenates measured by immuno-precipitation. These findings suggest that, in rat skeletal muscle, beta(2)-AMPK is not associated with glycogen and that activation of AMPK by muscle contraction does not dephosphorylate beta(2)-AMPK. These findings question the physiological relevance of the carbohydrate-binding function of beta(2)-AMPK in skeletal muscle.