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

Abstract

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 α - and β -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, ~ 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 ~ 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, how- ever, had no effect on the proportion of total β2 -AMPK that was phosphorylated in whole muscle homogenates measured by immuno- precipitation. These findings suggest that, in rat skeletal muscle, β2 -AMPK is not associated with glycogen and that activation of AMPK by muscle contraction does not dephosphorylate β2 -AMPK. These findings question the physiological relevance of the carbohy- drate-binding function of β2 -AMPK in skeletal muscle.