Regulation of pyruvate dehydrogenase (PDH) in the hibernating ground squirrel, (Ictidomys tridecemlineatus)

Author’s Accepted Manuscript Regulation of pyruvate dehydrogenase (PDH) in the hibernating ground squirrel, (Ictidomys tridecemlineatus) Sanoji Wijenayake, Shannon N. Tessier, Kenneth B. Storey www.elsevier.com/locate/jtherbio

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S0306-4565(17)30136-5 http://dx.doi.org/10.1016/j.jtherbio.2017.07.010 TB1962

To appear in: Journal of Thermal Biology Received date: 10 April 2017 Revised date: 19 July 2017 Accepted date: 20 July 2017 Cite this article as: Sanoji Wijenayake, Shannon N. Tessier and Kenneth B. Storey, Regulation of pyruvate dehydrogenase (PDH) in the hibernating ground squirrel, (Ictidomys tridecemlineatus) , Journal of Thermal Biology, http://dx.doi.org/10.1016/j.jtherbio.2017.07.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Regulation of pyruvate dehydrogenase (PDH) in the hibernating ground squirrel, (Ictidomys tridecemlineatus). Sanoji Wijenayake, Shannon N. Tessier1, Kenneth B. Storey* Institute of Biochemistry, Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, ON, K1S 5B6, Canada.

*Corresponding author: Kenneth B. Storey, Professor of Biochemistry , Canada Research Chair in Molecular Physiology , Institute of Biochemistry, Department of Biology, Department of Chemistry, 1125 Colonel By Drive, Ottawa, ON. Canada. K1S 5B. , Tel: 1-613-520-3678; Fax: 1-613-520-3749, E-mail address: [email protected]

Abstract Pyruvate dehydrogenase (PDH) is a vital regulatory enzyme that catalyzes the conversion of pyruvate into acetyl-CoA and connects anaerobic glycolysis to aerobic TCA cycle. Post-translational inhibition of PDH activity via three serine phosphorylation sites (pS232, pS293, and pS300) regulate the metabolic flux through the TCA cycle, decrease glucose utilization, and facilitate lipid metabolism during times of nutrient deprivation. As metabolic readjustment is necessary to survive hibernation, the purpose of this study was to explore the post-translational regulation of pyruvate dehydrogenase and the expression levels of four mitochondrial serine/threonine kinases (PDHKs), during torpor-arousal cycles in liver, heart, and skeletal muscle of 13-lined ground squirrels. A combination of Luminex multiplex technology and western immunoblotting were used to measure the protein expression levels of total PDH, three phosphorylation sites, S232, 293, 300, and the expression levels of the corresponding PDH kinases (PDHK1-4) during

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Present Address: Department of Surgery and Center for Engineering in Medicine, Massachusetts General Hospital & Harvard Medical School, 114 16th Street, Charlestown, MA. USA, 02129.

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euthermic control, entrance, late torpor, and interbout arousal. Liver and heart showed strong inhibitory PDH regulation, indicating a possible decrease in glucose utilization and a possible preference for β-oxidation of fatty acids during periods of low temperature and starvation. On the contrary, skeletal muscle showed limited PDH regulation via phosphorylation, possibly due to alternate controls. Phosphorylation of PDH may play an important role in regulating aerobic and anaerobic metabolic responses during hibernation in the 13-lined ground squirrel. Abbreviations PDH, Pyruvate dehydrogenase; PDHK, Pyruvate dehydrogenase kinases; PDP, Pyruvate dehydrogenase phosphatases; MRD, Metabolic rate depression; RPP, Reversible protein phosphorylation; TCA, Tricarboxylic acid cycle; F-2,6-P2, Fructose 2,6-bisphosphate; PFK, Phosphofructokinase; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase.

Keywords: PDH, hibernation, metabolism, phosphorylation, fat metabolism, glycolysis.

1.1 Introduction: Hibernation is used as a survival strategy by selected animals to survive extended periods of starvation and exposure to cold temperatures. This method of survival has representatives documented in monotreams, marsupials, lemurs, shrews, rodents, bats, bears, and birds (McNab, 1978);(Wang et al., 2011);(Storey and Storey, 2010);(Storey, 2015). In the obligate hibernator, 13-lined ground squirrels (Ictidomys tridecemlineatus), the core body temperature (Tb) decrease to approximately 5 ˚C from 35-38 ˚C, heart rate reduces from 350-400 beats/min to approximately 5-10 beats/min, organ perfusion rate drops to <10% of control conditions, and respiratory rate drops from >40 breaths/min to less than one breath/min during hibernation (Storey, 2010). However, the global suppression of metabolic rate is considered to be the most prominent characteristic of hibernation and the 13-lined ground squirrel is capable of reducing the metabolic rate

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>90% during hibernation compared to the euthermic control conditions (Storey, 2010);(Storey and Storey, 2010). Two of the central components of metabolic rate depression (MRD) in hibernators is the reduction of metabolic flux through glycolysis and a swift transition in metabolic fuel usage from carbohydrates to fatty acids (Buck and Barnes, 2000);(Storey and Storey, 2004). In this capacity, Brooks and Storey (1992) showed glycolytic rate significantly decreased in response to hibernation in the liver, heart, kidney, and muscle of the ground squirrel, Spermophilus lateralis. Furthermore, hibernating mammals have been documented to have a RQ (respiratory quotient) of ~0.7, a value indicative of a near complete reliance on β-oxidation of fatty acids to generate ATP (Dark, 2005). RQ is a unit-less value, which represents the moles of CO2, respired per moles of O2 consumed (Burlington and Klain, 1967; Cortes et al., 2009; Lyman and Chatfield, 1955). Furthermore, obligate hibernators such as yellow-bellied marmots (Marmota flaviventris) and 13-lined ground squirrels have shown to accumulate extensive fat stores primarily in the white adipocytes (WAT) rather than storing high levels of glycogen in the liver during hyperphagia (a period of over-eating that prelude the winter season). For instance, ~ 2-fold increase in total lipids, triacylglycerol, cholesterol, and fatty acid levels were documented in the hibernating 13-lined ground squirrel (Serkova et al., 2007). In addition, during mass gain prior to hibernation in marmots, lipogenesis is favored over lipolysis in which the expression levels of lipolytic enzymes such as HSL (hormonesensitive lipase) was seen to decrease and lipogenic enzymes such as LPL (lipoprotein lipase) was shown to increase (Wilson et al., 1992). These examples may indicate that hibernating mammals preferentially store and use fatty acids as the primary source of fuel

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to generate ATP during times of low energy availability. However, this study is the first to explore reversible protein phosphorylation (RPP) of pyruvate dehydrogenase (PDH) enzyme and its possible role in facilitating fatty acid oxidation to generate ATP during hibernation in 13-lined ground squirrels liver, heart, and skeletal muscle. PDH catalyzes the irreversible conversion of pyruvate into acetyl-CoA and links anaerobic glycolysis to aerobic TCA (tricarboxylic acid) cycle. The function of PDC (pyruvate dehydrogenase complex) is tightly regulated to maintain a stable glucose homeostasis during fed and fasted states through RPP of PDH at the E1α subunit (Bowker-Kinley et al., 1998a; Patel and Korotchkina, 2003). Mammalian PDH enzymatic activity is regulated through three phosphorylation sites, S232, S293, and S300 depending on ratios of NADH/NAD+, acetyl-CoA/CoA, and plasma glucose levels during fed and fasted states (Kolobova et al., 2001; Korotchkina and Patel, 2001; Patel and Roche, 1990). Phosphorylation at any one of these residues by PDH kinase 1-4 (PDHK) renders the entire PDH inactive (Korotchkina and Patel, 2001; Yeaman et al., 1978). On the other hand, PDP 1-2 (pyruvate dehydrogenase phosphatases) dephosphorylates PDH and renders it active (Patel and Korotchkina, 2006). Therefore, depending on the energy demands of the cell, PDH enzymatic activity could swiftly be inhibited through phosphorylation by the PDHKs or activated through dephosphorylation by the PDPs and/or PDHK inhibition in an energy efficient manner. As such, PDH is considered to be a vital enzymatic hub that can function as a metabolic switch between glycolysis and βoxidation of fatty acids during extended periods of starvation. The current study investigates the RPP of PDH and the relative expression levels of PDHKs during torpor-arousal cycles in the well-established natural hibernator model,

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13-lined ground squirrels. Western immunoblotting and Luminex multiplex technology were used to measure the relative changes in total PDH, and phosphorylation levels of PDH at S232, S293, and S300 as well as the relative protein expression levels of PDHK1-4 in the liver, heart, and skeletal muscle during euthermic control (EC), entrance (EN), late torpor (LT), and interbout arousal (IA) (n = 4/experimental condition). Overall, the results indicate a strong repression of PDH in the liver and cardiac muscle during hibernation, suggesting a possible shift from carbohydrate catabolism to breakdown of lipids in the 13-lined ground squirrel.

1.2 Materials and Methods 1.21 Animal Protocol 13-lined ground squirrels (I. tridecemlineatus), weighing approximately 150-300 g, were captured in the wild by United States Department of Agriculture-licensed trappers (TLS Research, Bloomingdate, IL). The animals were transported to the Animal Hibernation Facility, National Institute of Neurological Disorders and Stroke (NINDS) (NIH, Bethesda, MD). The hibernation experiments were done in the laboratory of Dr. J.M. Hallenbeck, as reported in (McMullen and Hallenbeck, 2010). The animals were injected with a sensor chip (Bio Medic Data Systems; IPTT-300) subcutaneously while under 5% isoflurane anesthesia soon after arrival. The 13-lined ground squirrels were individually housed in shoebox cages in a holding room at 21 °C and fed standard rodent diet and water ad libitum until they have gained enough mass to enter hibernation. Subsequently, the animals were transferred to constant darkness in an environmental chamber at 5 °C and allowed to transition naturally into torpor. Body temperature (Tb), time, and respiratory rates were monitored

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and used to determine the stage of torpor-arousal cycles. All animals, including control animals, had been through bouts of torpor-arousal prior to sampling. All animals were sacrificed and sampled according to (McMullen and Hallenbeck, 2010). All hibernation experiments and animal housing were approved by the NINDS animal care and use committee (ACUC). The sampled tissues were flash frozen in liquid nitrogen immediately upon excision and shipped to Carleton University on dry ice. Upon arrival the tissues were stored at -80 °C until further use. The experimental conditions used in this experiment are as follows; Euthermic control (EC) denotes euthermic animals (Tb = ~ 37 °C) that were capable of entering torpor but had not done so in the past 72 hrs. Entrance into torpor (EN) denotes animals that were in the entrance phase of the hibernation cycle (Tb= 18-31 °C). Late torpor (LT) denotes animals that were in deep torpor for at least 5 consecutive days (Tb=5-8 °C). Interbout arousal (IA) represent animals that have aroused after multiday torpor period with core body temperature returned back to 37 °C for ~18 hrs. 1.22 Protein extraction Soluble protein extraction of frozen tissue samples were done as per manufactures instructions (EMD Millipore; PDHMAG13K). Approximately 50 mg of frozen liver, heart, and skeletal muscle of I. tridecemlineatus were weighed and homogenized using 1X lysis buffer (EMD Millipore; 43-045) with a 1:5 (w/v) ratio using a Dounce homogenizer. Prior to use, 1X lysis buffer (EMD Millipore; 43-040) was pre-chilled and combined with 1mM Na3VO4, 10mM NaF, 10mM β-Glycerophosphate (Bioshop; GYP001.50), of phosphatase inhibitors and 10 µL/mL of protease inhibitor cocktail containing 104 mM AEBSF, 80 μM aprotinin, 4 mM bestatin, 1.4 mM E-64, 2 mM leupeptin, 1.5 mM pepstatin A (Bioshop; PIC001.1). Homogenates were subsequently

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kept on ice for 30 min with intermittent vortexing (every 10 min) at RT. Following incubation, the samples were centrifuged at 14,000 x g for 20 min at 4 °C. The supernatants were collected and total soluble protein concentrations were measured using the Bradford assay (Bio-Rad; 500-0006) with bovine serum albumin (BSA) as the standard. All samples were adjusted to a final concentration of 10 µg/µL using the 1X lysis buffer with added protease and phosphatase inhibitors (described above). 100 µL aliquots of the extracted total protein samples were then taken to conduct the multiplex analysis. These homogenates were stored at -80 °C until use. A second round of Bradford assay was performed on the samples to confirm the final concentration of 10 µg/µL just before use. The remaining sample volume were mixed 1:1 v:v with 2X SDS loading buffer (100 mM Tris-base, 4% w:v SDS, 20% v:v glycerol, 0.2% w:v bromophenol blue, 10% v:v 2-mercaptoethanol) and boiled for 15 min to further denature the proteins. The samples were then stored at -80 °C for further use. 1.23 Western Immunoblotting Western immunoblotting was done as previously described by (Wijenayake and Storey, 2016). In brief, 15-25 µg of total soluble protein extracts from EC, EN, LT, and IA conditions were loaded on to 8-10% SDS-polyacrylamide gels according to the molecular weight of the target protein. The gels were resolved using a Mini-Protean 3 Electrophoresis Module (Biorad; 165-3301) for 45-90 min at 180 V in 1X Tris-glycine running buffer (0.25 M of Tris-base, 2.45 M glycine, 0.035 M SDS, and ddH2O to add up to 2.5 L). 4 µL of pre-stained PiNK Plus protein ladder (Froggabio; PM005-0500) were run alongside the samples for molecular weight reference. Subsequently, the samples

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were electroblotted onto 0.45 µm PVDF membranes (Millipore; IPVH00010) at 160 mA for 100-120 min using 1X transfer buffer (25mM Tris-base, 192 mM glycine, 10% v/v methanol, 16 L ddH2O. pH 8.5). The membranes were blocked with 2.5-5% w/v skim milk in 0.5X TBST for 30-45 min and incubated with primary antibody (1:100 dilution with 0.5X TBST for PDHK1-4) at 4 °C overnight. The antibodies used are as follows; PDHK1 (Genetex; GTX107405), PDHK2 (Genetex; GTX105251), PDHK3 (Genetex; GTX104286), PDHK4 (Genetex; GTX105667). Following primary incubation, the membranes were incubated with HRPconjugated anti-rabbit IgG secondary antibody (1:8000 v:v 0.5X TBST) (Bioshop; APA007P) for 30-40 min on a plate rocker at RT. Finally, the membranes were visualized using enhanced chemiluminescence and a ChemiGenius Bio-Imaging System (Syngene, Frederick, MD) using H2O2 and Luminol. 1.24 Multiplex Analysis Multi-species pyruvate dehydrogenase (PDH) complex magnetic bead panel (EMD Millipore; PDHMAG-13K) was used to investigate the relative expression of PDH (total), and relative phosphorylation state of PDH at S232, S293, and S300 residues in the liver, skeletal muscle, and heart over torpor-arousal cycle of 13-lined ground squirrels (n=4/experimental condition). The PDH assay was performed as per manufacturer’s instructions. In detail, aliquots of 10 µg/µL of protein samples were diluted with Milliplex MAP Assay Buffer (Millipore; 43-010), to a final concentration of 0.5 µg/µL and 12.5 µg of protein in total were added per well. HepG2 cell lysate: unstimulated (Millipore; 47-234) were used as the positive control, and HepG2 cell lysate treated with dichloroacetate (DCA) (Millipore; 47-232) was used as an additional control for the phospho-PDH assay specificity. 25 µL of assay buffer was added to the blank wells, 25

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µL of sample lysates were added to the test wells, and 25 µL of HepG2 cell lysate and HepG2 lysate with DCA were added to the control wells. Subsequently, 25 µL of premixed beads were added to each well and the plate was incubated with agitation on a plate shaker for 2 hr at RT (protected from light). Following incubation, 50 µL of detection antibody was added to each well and incubated on a plate shaker for 1 h at RT. Thereafter, 50 µL of SAPE (StreptavidinPhycoerythrin) (Millipore; L-SAPE5) was added to each well and incubated with agitation on a plate shaker for 30 min at RT. Finally, the beads were re-suspended in 100 µL of Sheath Fluid on a plate shaker for 5 min at RT. The data acquisition was performed on a Luminex 200 instrument (Luminex, Austin, TX) using Milliplex Analyst software (Millipore, Billerica, MA). The equipment settings were; 50 events per bead, 50 µL sample size, 8,000 – 15, 000 gate settings, Default (low PMT) reporter gains, 60 sec time out, and bead number 64 for PDH (Total), 28 for p-PDH (S232), 34 for p-PDH (S293), and 43 for p-PDH (S300). It should be noted that in order to ensure the cross-reactivity of the antibodies that were used in the Luminex PDH bead panel, protein sequence alignments comparing human, mouse, rat, and 13-lined ground squirrel were generated using CLUSTAL OMEGA online bioinformatics tool. All three phosphorylation sites showed 100% conservation amongst human, mouse, rat, and 13-lined ground squirrel.

1.25 Statistical Analysis The multiplex assays measuring relative protein and phosphorylation levels were measured over torpor-arousal in liver, heart, and skeletal muscle using median fluorescent intensity (MFI). The band densities on the immunoblots were visualized using a Chemic Genius Bioimaging system (Syngene, Frederick, MD) and quantified using

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GeneTools software. Immunoblot band intensities were normalized against the summed intensity of coomassie stained protein bands in the same lane (without including the target band) to normalize against protein loading irregularities. The molecular weight of the target immunoblot band was identified with the use of FroggaBio protein molecular weight ladder (Froggabio; PM005-0500). All numerical data are expressed as mean ±SEM (n = 4) normalized to EC values. Statistical analysis was done using Sigmaplot 12 software (Systat Software Inc., San Jose, CA). One-way ANOVA with a Tukey post-hoc test (p<0.05) was used for the statistical analysis as well as for generating the figures.

1.3 Results: PDH catalyze the irreversible conversion of pyruvate into acetyl-CoA and is considered to be one of the main enzymatic hubs that regulate the teeter-totter between glycolysis and β-oxidation of fats. The total levels of PDH, changes in phosphorylation levels at three serine residues (S232, S293, and S300), along with the relative protein expression levels of four PDHK isozymes (PDHK1-4) that are known to phosphorylate the three serine residues were measured in the liver, heart, and skeletal muscle of I. tridecemlineatus over four stages of the torpor-arousal cycle. Overall, liver exhibited the most robust increase in phosphorylation levels at S300 with a 15-fold increase during EN and 30-fold increase during LT compared to EC. The phosphorylation at S300 returned to control levels during IA. The expression levels of PDH (total) and phosphorylation levels at S293 remained unchanged in response to LT, and the phosphorylation at S232 increased significantly during LT (Figure 1). In correlation, the protein levels of PDHK1 increased by 2-2.5-fold during EN and LT,

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respectively. In addition, PDHK4 levels significantly increased by 1.5-fold in response to LT, whereas PDHK2 levels significantly decreased in response to LT and PDHK3 levels significantly decreased in response to EN and LT (Figure 2) and returned to EC levels during IA. In the heart (Figure 3) of hibernating I. tridecemlineatus, phosphorylation levels at S293 and S300 significantly increased by 1.5-2-fold in response to LT, whereas phosphorylation at S232 and 300 significantly decreased during EN. Similarly to liver, total levels of PDH did not change during the torpor-arousal cycle. Furthermore, PDHK13 levels remained unchanged in the heart. However, the protein expression levels of PDHK4, significantly increased by 2-fold in response to LT, but remained unchanged during EN and IA compared to EC (Figure 4). Contrary to liver and heart, skeletal muscle showed limited changes in phosphorylation levels during hibernation at all three serine residues with a slight decrease in phosphorylation levels during EN and LT at S293. Total PDH levels also slightly decreased during EN compared to EC (Figure 5). Overall, the expression levels of PDHK2 and PDHK3 significantly decreased in response to EN and IA, yet remained unchanged during LT compared to EC. Furthermore, PDHK1 and PDHK4 levels increased by 1-1.5 fold during IA, while the rest of the sampling points remained unchanged (Figure 6).

1.4 Discussion: PDH is considered to be the rate-limiting enzyme in the irreversible decarboxylation of pyruvate (the end product of glycolysis) into acetyl-CoA, and thereby links anaerobic glycolysis to oxidative TCA cycle (Kelley et al., 1993; Patel and

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Korotchkina, 2006; Patel and Roche, 1990). In addition, PDH is the main enzymatic switch that regulates fuel selection from glucose to fatty acids (Patel and Korotchkina, 2006; Rardin et al., 2009), and regulates the metabolic flux during extended periods of starvation. PDH activity is regulated by a phospho-dephospho mechanisms at three serine residues (S232, S293, S300) depending on intra mitochondrial concentrations of pyruvate, CoA/acetyl CoA, FADH/FADH2, NAD+/NADH (Kolobova et al., 2001; Patel and Korotchkina, 2003) and changes in phosphorylation state of PDH is shown to correlate with changes in expression levels of PDHKs (Bowker-Kinley et al., 1998a). Accumulation of pyruvate, NAD+, and CoA is an indication of low energy state in the cell and leads to the activation of PDH, whereas large amounts of acetyl-CoA and NADH accumulation repress PDH activity. Site specificity for PDH phosphorylation by various PDHKs is thought to contribute to tissue-specific regulation (Korotchkina and Patel, 2001) and the interaction of PDH with coenzymes can alter the rate and stoichiometries of these phosphorylation events at indvidual sites (Kolobova et al., 2001). This complexity of regulation allows for critical metabolic hubs such as PDH to be very finely regulated based on multiple competing factors and sensing information across several molecular pathways. Evidence of a possible metabolic switch from glucose utilization to fatty acids during starvation have been reported in other hibernators such as mice, bears, bats, and lemurs (Buck et al., 2002; Gehnrich and Aprille, 1988; Storey, 1989, 2010; Tessier et al., 2015). Consequently, PDH/p-PDH interplay may also play an important role in MRD and regulate the conversion of pyruvate into acetyl-CoA in the champion hibernator 13-lined ground squirrel. As such, the present study investigates posttranslational regulation of PDH at three phosphorylation sites and corresponding

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expression levels of PDHK isozymes (PDHK1-4) in liver, heart, and skeletal muscle of 13-lined ground squirrels. We chose to study multiple phosphorylation sites and kinase isoforms in order gain a more complete understanding of the role of competing phosphorylation sites on PDH activity. Overall, the results indicate a tissue-specific RPP of PDH as well as PDHK expression in response to torpor-arousal. In particular, liver showed a robust increase in PDH phosphorylation during entrance and late torpor (Figure 1). In correlation, PDHK1 and PDHK4 levels also increased in expression when compared to euthermic control (Figure 2). As phosphorylation of any one of the three serine residues by PDHK’s render PDH inactive (Korotchkina and Patel, 2001), phosphorylation at S300 could indicate a strong reduction in the decarboxylation of pyruvate into acetyl-CoA, perhaps contributing to a metabolic shift towards β-oxidation of fats (Buck and Barnes, 2000; Storey and Storey, 2004) in hibernating ground squirrels. Liver is a large, proliferative, and critical metabolic tissue in mammals (Serkova et al., 2007). It is the primary site of glycogen storage and is vital for regulating biochemical processes such as ketogenesis, detoxification of chemicals, synthesis of glucose (gluconeogenesis) from amino acids, as well as break down of glycogen (glycogenolyis) into glucose (Hadj-Moussa et al., 2016). Therefore, to spare and ensure enough glycogen storage is available to meet the energetic demands of the arousal phase, hibernating ground squirrels may use RPP to inhibit the activity of PDH and several upstream glycolytic enzymes, both of which would contribute to reduced glucose utilization. Glucose sparing effects through glycogenolysis inhibition has also been shown in the liver of hibernating mouse, Zapus hudsonius (Storey, 1987) and hibernating ground squirrel, Spermophilus lateralis (Brooks and

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Storey, 1992), in which glycogen phosphorylase (which catalyzes the rate-limiting step of glycogenolysis) activity was depressed during both short-term and long-term hibernation. Furthermore, a profound decrease in overall glycolytic rate was seen in bat liver (Borgmann and Moon, 1976), Arctic ground squirrels (Tashima et al., 1970), as well as S. lateralis during hibernation (Brooks and Storey, 1992). Heart showed a robust increase in phosphorylation at S293 and S300 in response to LT and no change in expression of total PDH over torpor-arousal (Figure 3). In correlation, PDHK4 increased in expression (Figure 4). PDHK4 isoform, although expressed in several other tissues such as skeletal muscle, liver, kidney, mammary glands, and adipose tissues (Bowker-Kinley et al., 1998b; Jeoung, 2015), have cardiacrelated functions and increased in expression by several-folds during starvation in rat hearts (Wu et al., 1998), suggesting a potential link between starvation and the regulatory properties of PDHK4. Similar conclusions to liver can be made regarding the regulation of PDH in the heart, in which the inactivation of PDH complex via PDHK4 may indicate a preference for β-oxidation of fatty acids during hibernation. Similarly, (Brooks and Storey, 1992) reported a significant reduction in cardiac PDH enzymatic activity during hibernation in S. lateralis. Cardiac response to hibernation is a fascinating phenomenon, as heart is known to be functional during low temperature. Although, heart rate is reduced to 3-5 beats/min from euthermic values of 200-300 beats/min, the strength of the contractions are significantly enhanced in order to pump viscous blood through the body at 4 °C (Storey and Storey, 2004; Tessier and Storey, 2012; Wickler et al., 1991). As such, heart experience increased levels of mechanical as well as biochemical stress during times of hibernation. Additionally, although a global reduction in metabolic rate

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has been reported in the heart of hibernating ground squirrels, various cardiac-specific proteins such as myocyte enhancer factor-2 (MEF2) (Tessier and Storey, 2012), Foxo1, Foxo3a, MyoG, MAFbx, and MuRF1(Zhang et al., 2016) have shown increased expression in response to late torpor in 13-lined ground squirrels. p38 and MK2 activity were also elevated in the heart during torpor (MacDonald and Storey, 2005) and one of the most energy requiring cellular process, protein translation was largely maintained in the heart during hibernation (Wu and Storey, 2012). Therefore, a sufficient ATP supply is needed to maintain a reduced yet active cardiac output during hibernation and fatty acid oxidation could be the main source of ATP production in the heart during this time period. Skeletal muscle is remarkable in its ability to remodel and adapt to low energy conditions. As such, during hibernation, skeletal muscle remains largely inactive and hibernators are known to experience prolonged periods of muscle disuse yet experience little to no muscle atrophy (Storey and Storey, 2010; Tessier and Storey, 2012). Others have reported a state of translational repression via mTOR signaling cascade in the skeletal muscle during late torpor in 13-lined ground squirrels (Wu and Storey, 2012). Skeletal muscle in hibernators is dominantly used during the arousal phase to induce shivering thermogenesis, a thermoregulatory mechanism that is used to return the body temperature from 4 °C back to 37 °C (Storey and Storey, 2010). Despite these important balances between energy expenditure and availability during hibernation, skeletal muscle showed limited regulation in PDH activity through phospho/dephospho mechanisms during hibernation (Figure 5,6). This could suggest that RPP of PDH by PDHKs may not be the primary mode of metabolic regulation in the skeletal muscle. This data is

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comparable to a previously published study by (Brooks and Storey, 1992), in which no PDH enzymatic activity was detected in the skeletal muscle during hibernation in S. lateralis. Consequently, it is possible that glycolysis is regulated upstream in the skeletal muscle during hibernation. Fructose 2,6-bisphosphate (F-2,6-P2) concentration (nM/g of wet weight) has been shown to significantly decrease in skeletal muscle of hibernating S. lateralis. In general, a low F-2,6-P2 levels inhibit glycolysis by directly influencing the activity of phosphofructokinase (PFK), a rate-limiting enzyme of glycolysis (Brooks and Storey, 1992). Reduced glyceraldehyde 3-phosphate dehydrogenase (GAPDH) activity was also reported in the skeletal muscle of hibernating Jerbova. Reduced GAPDH activity should result in a decrease in glycolytic flux during periods of starvation (Soukri et al., 1996). Furthermore, (Gerhart-Hines et al., 2007) showed that fasting induce PGC1α deacetylation in skeletal muscle in rodents. Deacetylation of PGC-1α by SIRT1 in required for the activation of mitochondrial fatty acid oxidation genes. 1.5 Conclusions In summary, the current study assessed the role of RPP in regulating the activity of PDH and the expression levels of four PDHKs in liver, heart, and skeletal muscle in response to torpor-arousal in the champion hibernator I. tridecemlineatus. Liver and heart showed a robust increase in phosphorylation of PDH in response to hibernation. This may suggest an overall decrease in PDH activity and a reliance on fatty acid oxidation over glycolysis to generate ATP in these two tissues. Similar to other known hibernators, the inhibition of PDH activity through phosphorylation during extended periods of low temperature and starvation could be a notable characteristic of MRD in hibernating 13lined ground squirrels.

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Additional Information and Declaration Funding This work was supported by a discovery grant (Funding #: 6793) awarded to Dr. KB Storey from the Natural Sciences and Engineering Research Council (NSERC) Canada. S Wijenayake holds a postgraduate Queen Elizabeth II Graduate Scholarship in Science and Technology, SN Tessier holds a NSERC postdoctoral fellowship, and KB Storey holds the Canada Research Chair for Molecular Physiology. Grant Disclosures NSERC Discovery Grant Canada Research Chair NSERC Post-doctoral Fellowship Queen Elizabeth II Graduate Scholarship in Science and Technology Author Contributions Sanoji Wijenayake conceived, designed, and performed the experiments, analyzed the data, and wrote the paper. Shannon N. Tessier conceived, designed, and performed some of the experiments. Kenneth B. Storey contributed reagents, materials, and wrote the paper.

Acknowledgements We thank JM Storey for editorial review of this manuscript. We thank Dr. J.M. Hallenbeck and Dr. D.C. McMullen (NINDS, NIH, Bethesda) for providing the tissue samples for this study.

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Figure 1a/b. Post-translational regulation of pyruvate dehydrogenase (PDH) in the liver of hibernating I. tridecemlineatus. The relative changes in the protein level of PDH (total), and the phosphorylation levels of p-PDH (S232), p-PDH (S293), and p-PDH (S300) were measured using Luminex multiplex technology. The relative Median Fluorescent Intensity (MFI) for a given protein target in a sample was calculated by normalizing all samples to EC. Data are mean ± SEM (n = 3–4 independent protein isolations from different animals). Data were analyzed using a one-way ANOVA with a post-hoc Tukey test (p<0.05). a significantly different from the EC, b significantly different from EN, c significantly different from LT, and d significantly different from IA.

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Figure 2. Relative protein expression levels of PDHK 1-4 (pyruvate dehydrogenase kinase) in the liver of hibernating I. tridecemlineatus as determined by western immunoblotting. Data are mean ± SEM (n = 3–4 independent protein isolations from different animals). Data were analyzed using a one-way ANOVA with a post-hoc Tukey test (p<0.05). a significantly different from the EC, b significantly different from EN, c significantly different from LT, and d significantly different from IA. Figure 3. Post-translational regulation of Pyruvate dehydrogenase (PDH) in the heart of hibernating I. tridecemlineatus. Other information as in Figure 1. Figure 4. Relative protein expression levels of PDHK 1-4 (Pyruvate dehydrogenase kinase) in the heart of hibernating I. tridecemlineatus as determined by western immunoblotting. Other information as in Figure 2. Figure 5. Post-translational regulation of Pyruvate dehydrogenase (PDH) in the skeletal muscle of hibernating I. tridecemlineatus. Other information as in Figure 1. Figure 6. Relative protein expression levels of PDHK 1-4 (Pyruvate dehydrogenase kinase) in the skeletal muscle of hibernating I. tridecemlineatus as determined by western immunoblotting. Other information as in Figure 2.

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Highlights  PDH activity can be regulated through reversible protein phosphorylation at three serine residues.  A strong increase in PDH phosphorylation is evident during hibernation in Ictidomys tridecemlineatus.  Liver and heart may prefer -oxidation of fats to generate ATP during prolonged starvation compared to glycolysis.  A glucose sparing effect thorough glycolytic inhibition is evident in liver and heart.  PDH may be a regulatory hub that controls mitochondrial respiration during hibernation.

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Results







1.6

1.2 1.0 0.8

a

a

a

0.6 0.4 0.2

Euthermic Control Entrance Late Torpor Interbout Arousal

0) PD H

(p S

30

3) 29 PD H

(p S

23 (p S PD H

(T o

ta l

)

2)

0.0

PD H

Relative MFI for Muscle

1.4