Various metabolic adaptations and regulatory controls are required to withstand metabolic rate depression, including reorganization of metabolic fuel and differential expression of genes/proteins . Most importantly, and of particular interest to this study, are the ways in which mammals coordinate their entry into arousal from torpor and sustain its metabolic function at low body temperature without causing hypothermia. Huge reserves of triglycerides act as a primary fuel for most organs during hibernation. Strict metabolic rate depression and allowing the body temperature to fall to near ambient temperatures during torpor bouts, allows hibernators to save huge amounts of energy as compared to euthermic animals (Ruf & Geiser, 2015). Richardson’s ground squirrel is additionally faced with oxidative stress during hibernation, particularly during arousals in which oxygen consumption increases drastically [9, 27]. In many instances of hypometabolism, an increase in antioxidant enzyme activity is observed to prepare for imminent oxidative stress upon arousal and preserve current cell integrity during periods of reduced repair [28–31]. NADP-IDH often contributes significantly to the NADPH pool required for reductive fatty acid biosynthesis. This enzyme provides NADPH for maintenance of proper oxidation-reduction balance and protection against oxidative damage .
NADP-IDH was purified to electrophoretic homogeneity from Richardson’s ground squirrel skeletal muscle through a combination of ion-exchange and affinity chromatography (Fig. 1). The apparent molecular weight (~ 46 kDa) determined by SDS-PAGE corresponds well with the molecular weights reported for the same enzyme through bioinformatics analysis. Kinetic analysis of NADP-IDH was assessed at high and low temperatures. Many significant changes in parameters were observed when comparing euthermic and hibernating samples. The Km values of isocitrate and NADP were both significantly reduced in hibernating squirrels at 22°C as compared to euthermic control squirrels (Table 2). A decrease in Km values is suggestive of an increased enzyme affinity and, in turn, enzyme activity in the hibernating state. Increased enzyme activity is indicative of elevated NADPH production in hibernating squirrels. This increased NADPH presence could contribute to enhanced antioxidant activity and overall oxidative stress defense in the organism while in the hypometabolic state. Previous studies done by Vucetic et al., 2013 , on the impact of hibernation on antioxidant defences in the European ground squirrel (Spermophilus citellus) showed increase in antioxidant defence enzyme protein expressions in the hibernating state. Similar study was also done by Allan & Storey on a molecular level, in which they analysed the expression downstream antioxidant target, NF-κB in skeletal muscle of hibernating ground squirrels (Ictidomys tridecemlineatus) . During hibernation, the expression of NF-κB was increased, particularly in early torpor and arousal suggesting increased antioxidant defense . Additionally, in this study, increased expression of manganese dependent superoxide dismutase in early torpor and increased expression of heme oxygenase 1 in early arousal was also noted . One more study suggested that there was a significant increase in MafK affirming an increase in relative Nrf2 and catalase levels seen in arousal. These results seen during arousal correspond to a surge in oxygen consumption, which causes increased reactive oxygen species production. Overall, this is also contributing to the idea that ground squirrels increase oxidative stress defense during hibernation to cope with increased oxygen fluctuations during periodic arousals and to preserve cells during reduced repair periods in hypometabolism.
Interestingly, when observing the same Km kinetic parameters at a reduced temperature (5°C), the increase in enzyme affinity in the hibernating state is no longer observed. In fact, both isocitrate and Mg2+ Km values were increased in the hibernating state when compared to their respective euthermic counterparts (Table 2). Such results are more indicative of a decrease in enzyme affinity in the hibernating state at the cooler temperature. Perhaps, reduced NADP-IDH activity in the hibernating state would allow energy conservation in ground squirrels during hypometabolism. Reduced metabolic rate and the suppression of unnecessary processes during stages of reduced oxygen, food, and water consumption has been well studied in the past [8, 35, 36]. In hibernators, evidence has been presented that global suppression of ATP expensive processes such as transcription, translation, and growth stimulated by the Akt transcription factor takes place in order to facilitate energy conservation during hibernation [37–39]. Therefore, decreased activity of NADP-IDH in ground squirrel skeletal muscle at reduced temperatures could be the result of an assortment of energy conservation mechanisms. The conflicting results of Km values of NAP-IDH at 22°C and 5°C are indicative of more complex regulation of this enzyme. During hibernation, the ground squirrel reduces its core body temperature to near ambient temperatures and can remain at approximately 5°C for prolonged bouts of torpor . However, the squirrels do not always remain in a state of torpor throughout hibernation and instead cycle back to normal body temperature and metabolic rate periodically through interbout arousal periods [4, 9]. During torpor at colder temperatures, there is a decrease in oxidative stress. When moving to periods of arousal, the immediate phase of high oxygen consumption puts the hibernating squirrel at risk of increased mitochondrial reactive oxygen species (ROS) production [27, 40]. Thus, enzyme activity is likely being controlled in Richardson’s ground squirrel based on antioxidant need by regulating NADP-IDH not only according to euthermic or hibernating state, but also according to temperature. Altogether, this study explored the idea that at low temperatures, when oxidative stress is low and energy conservation needs are high, NADP-IDH can be downregulated. Yet, at increased temperatures and increased ROS threat during interbout arousal periods, NADPH production becomes more essential to cell survivability and is thus upregulated. Previous studies of enzymes from ground squirrels have also documented significant changes in the kinetic properties of enzymes in hibernating versus euthermic states and in high versus low assay temperatures. These include creatine kinase , hexokinase , pyruvate kinase  and citrate synthase  (all well-known regulatory enzymes) from skeletal muscle of Richardson’s ground squirrels. A significant increase in I50 value of KCl at room temperature was observed in hibernating as compared to euthermic squirrel (Table 2). Increased KCl tolerance could mean that the hibernating form of NADP-IDH has increased chemical stability in Richardson’s ground squirrels. A similar phenomenon was observed in the SERCA (sarcoendoplasmic reticulum Ca2+-ATPase) enzyme of turtles which undergoes anoxia, another form of hypometabolic stress .
The differences observed in the kinetic properties of NADP-IDH purified from euthermic and hibernating skeletal muscles may be explained by structural differences. Phosphorylation levels of serine, threonine, and tyrosine residues were therefore assessed by western blotting. There was a significant increase in phosphorylation on serine and threonine residues of NADP-IDH in the hibernating ground squirrels (Fig. 2). Reversible protein phosphorylation has been known to control the activity of key metabolic enzymes during hibernation in Richardson’s ground squirrel in earlier studies. A study done by Bell & Storey showed skeletal muscle pyruvate kinase of hibernating ground squirrel, U. richardsonii was regulated post-translationally . They indicated that skeletal muscle pyruvate kinase from the hibernating animal was significantly more phosphorylated . In another study, immunoblotting showed that hibernator muscle glycerol-3-phosphate dehydrogenase had a higher phosphoserine content than in euthermic controls [3, 46]. Protein phosphorylation is an important post-translational modification that can quickly and reversibly alter the properties of metabolic enzymes . Phosphorylation of NADP-IDH during hibernation could thus also be responsible for the kinetic differences observed in the present study.
To complement the experimental studies on U. richardsonii NADP-IDH, bioinformatics tools were used to analyze the NADP-IDH protein sequence from the thirteen-lined ground squirrel, a closely related hibernator with a sequenced genome (Fig. 3). This analysis was aimed at identifying sites of post-translational modifications that may play a role in stabilizing enzyme structure and enhancing enzyme functionality in euthermic versus hibernating states. Bioinformatics results further confirmed the presence of potential threonine and serine residues which can likely be phosphorylated in hibernating NADP-IDH enzyme. (Fig. 3). The addition of phosphate groups to these serine or threonine amino acid residues could play a role in modifying ground squirrel skeletal muscle functionality at high (euthermic) versus low (hibernating) body temperatures.