Characterizing the regulation of pyruvate kinase in response to hibernation in ground squirrel liver (Urocitellus richardsonii)

https://doi.org/10.1016/j.cbpb.2020.110466Get rights and content

Highlights

  • Purification to homogeneity of Richardson's ground squirrel liver pyruvate kinase

  • Lower maximal liver pyruvate kinase activity in hibernation compared to euthermia.

  • Serine/threonine phosphorylation increased in pyruvate kinase during hibernation.

  • Decreased sensitivity to FBP activation in hibernating liver pyruvate kinase.

Abstract

The Richardson's ground squirrel (Urocitellus richardsonii) undergoes numerous changes to its core physiological and metabolic processes over the months it spends hibernating during the winter. Winter torpor is characterized by an overall reduction in metabolic rate, a lowering of core body temperature, and a switch to preferential consumption of lipids instead of carbohydrates. The alterations in central metabolic pathways are often accomplished by the regulation of key enzymes within the glycolytic pathway. The regulation of one such enzyme, pyruvate kinase (PK), was characterized in the present study in the liver of torpid ground squirrels. PK was purified from liver tissue of euthermic and hibernating U. richardsonii and subsequently assayed to determine the kinetic parameters of the enzyme at 22° and 5 °C. Additional studies assessed the relative degree of post-translational modifications in PK from control and hibernating ground squirrels. The results from this study demonstrated significantly lowered maximal activity in the hibernating form of the enzyme and decreased sensitivity to the activator FBP when compared to the control. Immunoblotting demonstrated increased relative serine and threonine phosphorylation (~3 fold) in the hibernating PK. Taken together these results suggest that phosphorylation of liver PK is an important step in inhibiting glycolytic activity in the liver of the Richardson's ground squirrel during torpor.

Introduction

Seasonal fluctuations of temperature and resource availability present a significant challenge to animals living in northern environments. The arrival of extreme winter conditions requires that animals adopt one of several strategies: caching food supplies to consume over the winter, undergoing a seasonal migration to more forgiving climates, or seeking shelter and entering a dormant state until more favorable conditions return. Mammalian hibernation is an excellent example of this third strategy and is characterized by an animal entering into a dormant state, known as torpor, which is interspersed by brief periods of arousal, dubbed inter-bout arousals (van Breukelen and Martin, 2015). Periods of torpor may last anywhere between a few days to 5 weeks while the interbout arousals typically last between 12 and 24 h (Carey et al., 2003). Hibernation is also characterized by major physiological and biochemical changes, as animals enter a hypometabolic state with metabolic rates decreasing by as much as 95% (Geiser, 2004). Indeed, hibernator heart rates have been observed to drop to just 5% that of the resting euthermic rate during torpor, while breathing rates can drop from >40 to <1 breaths per minute (McArthur and Milsom, 1991; Zatzman, 1984). At the molecular level, energetically expensive processes like transcription and translation are suppressed during entry into torpor by preferential expression of less active forms of RNA polymerase II, and inhibition of translational machinery function via protein phosphorylation (Chen et al., 2001; Frerichs et al., 1998; Hittel and Storey, 2002; Morin and Storey, 2006). Studying the cycling between periods of metabolic depression and normal activity could provide novel insight into mammalian metabolic regulation.

The Richardson's ground squirrel (Urocitellus richardsonii) is a well-studied hibernator model and is found across the prairies of southern Canada and northern United States. Hibernating for 7–9 months between September and May (Michener, 1983), these animals conserve up to 87.8% of their energy stores over the course of the entire hibernation season by entering a state of torpor (Wang, 1979). Furthermore, differential regulation of metabolic enzymes in this animal suggests that metabolic remodeling to optimize energy consumption efficiency occurs during hibernation (Bell and Storey, 2018; Ruberto et al., 2016; Thatcher and Storey, 2001).

Energy consumption in mammalian hibernators transitions from primarily carbohydrate sources in the summer and fall to mostly lipid sources during hibernation in the winter (Lang-Ouellette et al., 2014). In fact, the respiratory quotient (RQ) of many hibernators shifts from 1 to 0.7–0.8 during torpor, suggesting a significant shift towards lipid metabolism occurs during this period (Buck and Barnes, 2000). Preferential consumption of lipids is observed in almost all organs, including the liver, and is primarily brought about by torpor-induced changes in the levels of circulating hormones. High levels of circulating glucagon during hibernation stimulates the release of free fatty acids from white adipose tissue (WAT) where they are stored in preparation for winter. Meanwhile, insulin levels decrease during torpor due to a lack of feeding and in some instances insulin resistance can develop (Bauman et al., 1987; Martin, 2008). The decreased level of insulin has two main effects: first, it is thought to decrease rates of lipogenesis and glycogen synthesis through decreased PI3K/Akt signaling (Cai et al., 2004; Eddy and Storey, 2003). Secondly, decreased insulin levels result in reduced uptake of glucose by cells, ultimately decreasing glucose availability and therefore carbohydrate metabolism. This effect is amplified further through enzymatic regulation via reversible protein phosphorylation (RPP). In the liver of the Djungarian hamster (Phodopus sungorus), for example, an inactivation of pyruvate dehydrogenase (PDH) was observed in response to daily torpor, and this change in activity was consistent with regulation by RPP (Heldmaier et al., 1999). This pattern also held true in a seasonal hibernator, the 13-lined ground squirrel (Ictidomys tridecemlineatus) where increased levels of key site-specific inhibitory phosphorylated residues were observed in liver PDH (Wijenayake et al., 2017). Similar results were observed in the hibernating mouse Zapus hudsonius which exhibited stress-responsive inactivation of glycogen phosphorylase (GP), phosphofructokinase 1 (PFK1) and PK consistent with RPP-regulatory mechanisms (Storey, 1987). Inactivation of these enzymes limits glycolytic activity and the entrance of pyruvate into the TCA cycle, ultimately reducing the rate of carbohydrate metabolism and energy production in general. The research performed to date has clearly demonstrated that carbohydrate metabolism is highly regulated in mammalian liver tissue such that it is suppressed during hibernation.

PK is the terminal enzyme of aerobic glycolysis and catalyzes the transfer of a phosphate group from phosphoenol pyruvate (PEP) to ADP. It is located at a locus of ATP production and both its substrate (PEP) and product (pyruvate) have multiple potential metabolic fates. For these reasons, PK is commonly targeted via RPP in mammalian liver tissue to effect enzymatic regulation. Indeed, in response to glucagon signaling, mammalian PK is typically inactivated via phosphorylation such that gluconeogenesis is prioritized (Blair et al., 1976). Furthermore, PK is highly regulated by multiple allosteric effectors, including fructose bisphosphate (FBP), ATP and alanine (Jurica et al., 1998; Tanaka et al., 1967; Van Veelen et al., 1979). The regulation of PK in animals undergoing hypometabolic states has been explored. For example, in the freeze-tolerant wood frog (Rana sylvatica), reduced affinity for PEP was associated with the frozen and dehydrated states in the muscle tissue (Smolinski et al., 2017). Stress-induced regulation of PK has also been observed in hibernators. In skeletal muscle of the ground squirrel U. richardsonii PK displayed decreased levels of phosphorylation in tissues obtained from torpid animals which correlated with a three-fold increase in the Km PEP, suggesting inhibition of glycolytic processes in the muscle (Bell and Storey, 2018). Given the complex nature of PK regulation and the precedence established by these studies, it is possible that PK is also regulated in the liver to reduce the rate of glycolysis. However, the nature of this regulation is still unknown in mammalian hibernators. This study therefore explored PK in the liver of a model hibernating mammal, Richardson's ground squirrel (Urocitellus richardsonii), specifically investigating changes in its kinetic and structural properties following the animal's entry into torpor.

Section snippets

Animal treatments

The protocols used for the care and handling of the Richardson's ground squirrels used in these experiments were as previously reported (Thatcher and Storey, 2001). Briefly, animals were captured in late summer near Calgary, Alberta. All animals were individually housed in rat cages with free access to food and water at 22 °C and on an autumn photoperiod (10 h light, 14 h dark). After 8 weeks under this regime, half of the animals were maintained under these control (euthermic) conditions. The

Purification of PK

PK was successfully purified from euthermic and hibernating U. richardsonii liver using a combination of ion-exchange and affinity column chromatography (Table 1, Fig. 1). PK was eluted from the first column by increasing the pH of the elution buffer from pH 6.0 to pH 6.5. This step resulted in 6.6-fold purification with a 30% yield. The second step, a 0–1 M KCl gradient elution from a Cibacron blue column, resulted in a 10-fold purification with a 24% yield. The final purification step was a

Discussion

Mammalian hibernation during winter is characterized by long periods of torpor where metabolic rates can drop to 5% of their normal value, interspersed with brief periods of interbout arousal where metabolic rates return to normal. It is thought that the interbout arousal periods play a role in restoring levels of glucose through liver gluconeogenesis needed to sustain the brain and other carbohydrate-consuming organs upon re-entry into torpor (Carey et al., 2003). Regulation of glycolysis and

Acknowledgments and funding

Special thanks to JM Storey for her work in editorial review of this manuscript.KB Storey holds the Canada Research Chair in Molecular Physiology and SR Green was supported by an NSERC Canada Graduate Scholarship at the Doctoral level. This research was funded through a Discovery Grant (grant number 6793) through the Natural Sciences and Engineering Research Council of Canada (NSERC).

Declaration of Competing Interests

The authors declare no conflict of interest.

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