Assessment of required conditions for detecting glycogenin and other glycogen related proteins in rat skeletal muscle

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Master of Science to the School of Life Sciences, Faculty of Science, Technology and Engineering, La Trobe University, Bundoora.

Glycogen is an important energy source within contracting skeletal muscle. It has a simple but strictly ordered structure comprising α-1,4-glycosidic bonds with α-1,6-glycosidic bonds at branch points. Glycogen granules are generated on the core protein, glycogenin. Glycogenin is a 37 kDa protein which is a self-glycosylating protein that regulates the formation of glycogen. Glycogen debranching enzyme (GDE) and glycogen phosphorylase (GP) are necessary for the enzymatic steps of glycogen breakdown. Previous studies have typically assessed glycogenin using fractionated muscle, where some of the sample was discarded. This study aimed to determine the optimal conditions for detecting glycogenin as well as other glycogen related proteins (GDE and GP) in whole skeletal muscle. Additionally, time lapse and electrical stimulation were used in this study to show the different glycogen degradation patterns in order to speculate the location of glycogenin and other glycogen related proteins. It was hypothesised that glycogenin and GDE would require amylase treatment for their optimal detection, but not GP. It was also hypothesised that other glycogen utilising processes (eg. stimulation of muscle or leaving muscle at room temperature) would result in a greater amount of the glycogenin being detected. Male Long-Evans hooded rats (6-8 months old) were used and the extensor digitorum longus (EDL) muscles and Soleus (SOL) muscles were excised. Skeletal muscles were either homogenized or fibres were dissected from muscle and mechanically-skinned. Western blotting was used to examine the amount of glycogenin, GDE and GP proteins following incubation with or without various conditions of amylase treatment. Overall, it was found that amylase was required to detect glycogenin but not GDE or GP, however, some glycogenin was revealed in muscle left at room temperature for 3 hours without amylase. This was likely due to glycogen degradation, which was determined to be greater in the older muscle. Furthermore, the optimal temperature for amylase activity to reveal glycogenin was determined. Interestingly, there was a fibre type dependence on the optimal temperature. We also used stimulation of EDL muscle to utilize cellular glycogen and found that the diffusibility of GDE was higher in fibres from stimulated muscle compared with control fibres.