Single fiber analyses of glycogen-related proteins reveal their differential association with glycogen in rat skeletal muscle

Murphy RM, Xu H, Latchman H, Larkins NT, Gooley PR, Stapleton DI. Single fiber analyses of glycogen-related proteins reveal their differential association with glycogen in rat skeletal muscle. Am J Physiol Cell Physiol 303: C1146-C1155, 2012. First published September 26, 2012; doi:10.1152/ajpcell.00252.2012.-To understand how glycogen affects skeletal muscle physiology, we examined enzymes essential for muscle glycogen synthesis and degradation using single fibers from quiescent and stimulated rat skeletal muscle. Presenting a shift in paradigm, we show these proteins are differentially associated with glycogen granules. Protein diffusibility and/or abundance of glycogenin, glycogen branching enzyme (GBE), debranching enzyme (GDE), phosphorylase (GP), and synthase (GS) were examined in fibers isolated from rat fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscle. GDE and GP proteins were more abundant (similar to 10-to 100-fold) in fibers from EDL compared with SOL muscle. GS and glycogenin proteins were similar between muscles while GBE had an approximately fourfold greater abundance in SOL muscle. Mechanically skinned fibers exposed to physiological buffer for 10 min showed similar to 70% total pools of GBE and GP were diffusible (nonbound), whereas GDE and GS were considerably less diffusible. Intense in vitro stimulation, sufficient to elicit a similar to 50% decrease in intracellular glycogen, increased diffusibility of GDE, GP, and GS (similar to 15-60%) and decreased GBE diffusibility (similar to 20%). Amylase treatment, which breaks alpha-1,4 linkages of glycogen, indicated differential diffusibilities and hence glycogen associations of GDE and GS. Membrane solubilization (1% Triton-X-100) allowed a small additional amount of GDE and GS to diffuse from fibers, suggesting the majority of nonglycogen-associated GDE/GS is associated with myofibrillar/contractile network of muscle rather than membranes. Given differences in enzymes required for glycogen metabolism, the current findings suggest glycogen particles have fiber-type-dependent structures. The greater catabolic potential of glycogen breakdown in fast-twitch fibers may account for different contraction induced rates of glycogen utilization.