Regulation of citric acid cycle enzymes and related pathways in the skeletal muscle of hibernating Richardson’s ground squirrels, Urocitellus Richardsonii
Richardson’s ground squirrels (Urocitellus richardsonii) are small rodents inhabiting western Canada that spend a large portion of their life in hibernation. Hibernation is accompanied by a profound drop in body temperature to a minimum of 2-3 °C and a notable shift from carbohydrate to lipid consumption that involves large-scale rearrangements of central metabolic processes. This thesis investigated the regulation of key enzymatic checkpoints in the citric acid cycle (CAC) as well as enzymes that shuttle substrates into the CAC in skeletal muscle of ground squirrels during hibernation. Initial work investigated regulation of the pyruvate dehydrogenase complex (PDC) that bridges glycolysis and the CAC. Muscle PDC showed few changes in properties in terms of activity and inhibitory phosphorylation of the enzyme. This was in stark contrast to liver where strong suppression of PDC activity occurred during hibernation correlated with increased inhibitory phosphorylation on serine-300. This then led to investigation of two crucial irreversible regulatory steps of the CAC in the muscle: citrate synthase (CS) and the α-ketoglutarate dehydrogenase complex (KGDC). CS activity decreased significantly during hibernation. This correlated with decreased lysine succinylation of CS that reflected increased SIRT5 levels, the enzyme responsible for desuccinylase activity in mitochondria. KGDC also showed decreased affinity for coenzyme A in hibernating squirrels and marked differences in posttranslational modifications including increased tyrosine phosphorylation on all three enzyme subunits and increased serine phosphorylation on E2 subunit. Stimulating the action of endogenous protein kinases demonstrated decreased affinity for coenzyme A. Finally, regulation of muscle glutamate dehydrogenase (GDH) was analyzed to ascertain how GDH regulation mediated the flow of α-ketoglutarate into the CAC from amino acid catabolism. Most GDH kinetic parameters were unaffected between hibernating and euthermic states, except that glutamate affinity was substantially lower at 8 °C (a physiologically relevant temperature) for the enzyme from hibernating squirrels. GDH from hibernating animals also exhibited significantly higher ADP-ribosylation, suggesting a regulatory mechanism for modulating GDH. Taken together these findings suggest that enzymatic regulation in Richardson’s ground squirrel muscle is actively mediated by a variety of posttranslational mechanisms of the CAC and related enzymes to coordinate metabolic suppression during hibernation.