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Diffusibility and glycogen association of AMPK in rat skeletal muscle with and without in vitro stimulation

H. Xu,1 G.D. Lamb,2 N.T. Frankenberg,1 P.R. Gooley,3 D.I. Stapleton4 and R.M. Murphy,1 1Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC 3083, Australia, 2Department of Physiology, Anatomy and Microbiology, La Trobe University, Melbourne, VIC 3083, Australia, 3Department of Biochemistry & Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, VIC 3010, Australia and 4The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, VIC 3010, Australia.

The 5′-AMP-activated protein kinase (AMPK) functions as an intracellular fuel sensor that affects metabolism, and AMPK is activated in skeletal muscle in response to exercise and energy storage utilization (Jorgensen, Jensen & Richter, 2007). Mammalian AMPK is a heterotrimeric complex with a carbohydrate-binding module (CBM) in the β2-subunit (AMPKβ2) that shows high affinity for glycogen mimics (Koay et al., 2010). It has recently been demonstrated that glycogen-binding is blocked by a new AMPKβ phosphorylation site (Thr-148) within the CBM (Oligschlaeger et al., 2015) .

To investigate this finding in rat skeletal muscle, male Sprague-Dawley rats (6-8 mo old) were sacrificed using a lethal overdose of isoflurane in accordance with La Trobe University Ethics Committee. The extensor digitorium longus (EDL) muscle was excised and stimulated in vitro in order to activate AMPK as well as utilise glycogen storage. The stimulated and contralateral control muscles were then homogenized in a physiological K+ based solution with pCa >10 (Murphy et al., 2012).

Muscles were stimulated at 30 V with ten 50 Hz tetani for 0.5 s every two seconds, repeated every 2 min until peak force declined to < 20% of original (taking ∼1 h), which resulted in ∼33% increase in phosphorylated acetyl CoA carboxylase (p-ACC), a downstream product of activated AMPK. An enzymatic glycogen content assay showed ∼28% glycogen utilization during stimulation. Individual muscle fibres were isolated from control and stimulated muscles and AMPKβ2 content measured in the diffusible component (Murphy et al., 2012). Diffusibility of AMPKβ2 decreased ∼20% in stimulated compared to control muscles, indicating a pool of AMPKβ2 becomes bound in muscle as a consequence of stimulation. Amylase treatment, which is able to identify proteins associated with glycogen, indicated that the bound AMPKβ2 was not associated with glycogen. A phospho-specific AMPKβ-Thr-148 antibody was used in an immunoprecipitation (IP) assay to detect the AMPKβ phosphorylation, and it was found that the entire pool of AMPKβ2 was phosphorylated in both control and stimulated muscles. This finding further confirmed that skeletal muscle AMPKβ2 is not associated with glycogen in vivo, and that activation of AMPK by muscle contraction does not dephosphorylate AMPKβ2. These findings confirm that when AMPKβ2 is phosphorylated at Thr-148, AMPK does not associate with glycogen.

Jorgensen SB, Jensen TE, & Richter EA. (2007). Role of AMPK in skeletal muscle gene adaptation in relation to exercise. Appl Physiol Nutr Metab 32, 904-11.

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Oligschlaeger Y, Miglianico M, Chanda D, Scholz R, Thali RF, Tuerk R, Stapleton DI, Gooley PR, Neumann D. (2015). The recruitment of AMP-activated protein kinase to glycogen is regulated by autophosphorylation. J Biol Chem 290, 11715-28.

Murphy RM, Xu H, Latchman H, Larkins NT, Gooley PR, Stapleton DI. (2012). Single fibre analyses of glycogen-related proteins reveal their differential association with glycogen in rat skeletal muscle. Am J Physiol Cell Physiol 303, C1146-55.