Spermophilus Tridecemlineatus Research Articles

The complete mitochondrial genome of Ictidomys tridecemlineatus (Rodentia: Sciuridae)

The complete mitochondrial genome of the thirteen-lined ground squirrel, Ictidomys tridecemlineatus (Rodentia: Sciuridae) was sequenced to analyze the gene arrangement. It is a circular molecule of 16,458 bp in length including 37 genes typically found in other squirrels. The AT content of the overall base composition is 63.7% and the length of the control region is 1016 bp with 63.0% AT content. In BI and ML phylogenetic trees, I. tridecemlineatus is a sister clade to the genus Cynomys, and Tamias sibiricus is a sister clade to (Marmota himalayana + (I. tridecemlineatus + (C. leucurus + C. ludovicianus))). Ratufinae is well supported as the basal clade of Sciuridae. The monophyly of the family Sciuridae and its subfamilies Callosciurinae, Xerinae and Sciurinae are well supported.

Saponin-permeabilization is not a viable alternative to isolated mitochondria for assessing oxidative metabolism in hibernation.

ABSTRACTSaponin permeabilization of tissue slices is increasingly popular for characterizing mitochondrial function largely because it is fast, easy, requires little tissue and leaves much of the cell intact. This technique is well described for mammalian muscle and brain, but not for liver. We sought to evaluate how saponin permeabilization reflects aspects of liver energy metabolism typically assessed in isolated mitochondria. We studied the ground squirrel (Ictidomys tridecemlineatus Mitchell), a hibernating mammal that shows profound and acute whole-animal metabolic suppression in the transition from winter euthermia to torpor. This reversible metabolic suppression is also reflected in the metabolism of isolated liver mitochondria. In this study we compared euthermic and torpid animals using saponin permeabilized tissue and mitochondria isolated from the same livers. As previously demonstrated, isolated mitochondria have state 3 respiration rates, fueled by succinate, that are suppressed by 60-70% during torpor. This result holds whether respiration is standardized to mitochondrial protein, cytochrome a content or citrate synthase activity. In contrast, saponin-permeabilized liver tissue, show no such suppression in torpor. Neither citrate synthase activity nor VDAC content differ between torpor and euthermia, indicating that mitochondrial content remains constant in both permeabilized tissue and isolated mitochondria. In contrast succinate dehydrogenase activity is suppressed during torpor in isolated mitochondria, but not in permeabilized tissue. Mechanisms underlying metabolic suppression in torpor may have been reversed by the permeabilization process. As a result we cannot recommend saponin permeabilization for assessing liver mitochondrial function under conditions where acute changes in metabolism are known to occur.

Opposing activity changes in AMP deaminase and AMP-activated protein kinase in the hibernating ground squirrel.

Hibernating animals develop fatty liver when active in summertime and undergo a switch to a fat oxidation state in the winter. We hypothesized that this switch might be determined by AMP and the dominance of opposing effects: metabolism through AMP deaminase (AMPD2) (summer) and activation of AMP-activated protein kinase (AMPK) (winter). Liver samples were obtained from 13-lined ground squirrels at different times during the year, including summer and multiples stages of winter hibernation, and fat synthesis and β-fatty acid oxidation were evaluated. Changes in fat metabolism were correlated with changes in AMPD2 activity and intrahepatic uric acid (downstream product of AMPD2), as well as changes in AMPK and intrahepatic β-hydroxybutyrate (a marker of fat oxidation). Hepatic fat accumulation occurred during the summer with relatively increased enzymes associated with fat synthesis (FAS, ACL and ACC) and decreased enoyl CoA hydratase (ECH1) and carnitine palmitoyltransferase 1A (CPT1A), rate limiting enzymes of fat oxidation. In summer, AMPD2 activity and intrahepatic uric acid levels were high and hepatic AMPK activity was low. In contrast, the active phosphorylated form of AMPK and β-hydroxybutyrate both increased during winter hibernation. Therefore, changes in AMPD2 and AMPK activity were paralleled with changes in fat synthesis and fat oxidation rates during the summer-winter cycle. These data illuminate the opposing forces of metabolism of AMP by AMPD2 and its availability to activate AMPK as a switch that governs fat metabolism in the liver of hibernating ground squirrel.

Investigating the Adaptive Role of Brain Mitochondria in a Mammalian Hibernator

During the hibernation season, thirteen‐lined ground squirrels (Ictidomys tridecemlineatus) regularly cycle between bouts of torpor and interbout arousal (IBA). This presents a unique seasonal change in energy requirements in the brain. Most of the brain is electrically quiescent during torpor, but regains activity upon arousal to IBA, resulting in extreme oscillations in energy demand during the hibernation season. We predicted that brain mitochondria undergo a seasonal change in function to accommodate the variable energy demands of hibernation. To address this hypothesis, we examined mitochondrial bioenergetics of brain in thirteen‐lined ground squirrels across four time points: Summer (SU), Fall (FA), Hibernation (HIB) and Spring (SP). Respiration rates of isolated brain mitochondria were measured using an oxygen electrode with three substrates: Glutamate/Malate, Succinate, and Glycerol‐3‐Phosphate. Membrane potential (mV) and proton leak were measured simultaneously using a TPP+ electrode. Glutamate/Malate‐fueled respiration was significantly higher in HIB than in both SU and FA (p<0.05). Succinate‐fueled respiration was significantly lower (p<0.05) in SP and SU than in HIB. We found no significant difference in mV between SP, SU and FA. These data imply that brain mitochondrial bioenergetics are not static across the year, and suggest that the brain requires more fuel and resources to function efficiently during hibernation. This study provides essential underpinnings to understanding the overall energy requirements of a hibernator's brain and provides clues to into the neuroprotective strategies employed by a mammalian hibernator.

Conserved microsatellite markers of high cross-species utility for flying, ground and tree squirrels

Many squirrel species around the world are threatened by forest loss and fragmentation. To facilitate studies of squirrel biodiversity, particularly of flying squirrels in Southeast Asia, we identified Hylopetes, Menetes, Glaucomys and Sciurus squirrel microsatellite sequences with homologs in a second squirrel species (Spermophilus tridecemlineatus), designed 40 consensus markers and tested three squirrel species. When tested in four individuals per species, 26 markers were variable in Hylopetes phayrei, 25 markers in H. lepidus and 25 markers in Menetes berdmorei. Eleven markers were selected from 14 that were polymorphic in all three species. Cross-species utility was confirmed for these 11 markers in seven additional squirrel species, including: the flying squirrels H. phayrei, H. lepidus, H. spadiceus and Petaurista petaurista; a ground squirrel, M. berdmorei; and the tree squirrels, Callosciurus caniceps and C. finlaysoni. The other markers that were variable in one or multiple species are also useful for those specific species.

Hypothalamic gene expression underlying pre-hibernation satiety.

Prior to hibernation, 13-lined ground squirrels (Ictidomys tridecemlineatus) enter a hypophagic period where food consumption drops by an average of 55% in 3 weeks. This occurs naturally, while the ground squirrels are in constant environmental conditions and have free access to food. Importantly, this transition occurs before exposure to hibernation conditions (5°C and constant darkness), so the ground squirrels are still maintaining a moderate level of activity. In this study, we used the Illumina HiSeq 2000 system to sequence the hypothalamic transcriptomes of ground squirrels before and after the autumn feeding transition to examine the genes underlying this extreme change in feeding behavior. The hypothalamus was chosen because it is known to play a role in the control and regulation of food intake and satiety. Overall, our analysis identified 143 genes that are significantly differentially expressed between the two groups. Specifically, we found five genes associated with feeding behavior and obesity (VGF, TRH, LEPR, ADIPOR2, IRS2) that are all upregulated during the hypophagic period, after the feeding transition has occurred. We also found that serum leptin significantly increases in the hypophagic group. Several of the genes associated with the natural autumnal feeding decline in 13-lined ground squirrels show parallels to signaling pathways known to be disrupted in human metabolic diseases, like obesity and diabetes. In addition, many other genes were identified that could be important for the control of food consumption in other animals, including humans.

The regulation of troponins I, C and ANP by GATA4 and Nkx2-5 in heart of hibernating thirteen-lined ground squirrels, Ictidomys tridecemlineatus.

Hibernation is an adaptive strategy used by various mammals to survive the winter under situations of low ambient temperatures and limited or no food availability. The heart of hibernating thirteen-lined ground squirrels (Ictidomys tridecemlineatus) has the remarkable ability to descend to low, near 0°C temperatures without falling into cardiac arrest. We hypothesized that the transcription factors GATA4 and Nkx2-5 may play a role in cardioprotection by facilitating the expression of key downstream targets such as troponin I, troponin C, and ANP (atrial natriuretic peptide). This study measured relative changes in transcript levels, protein levels, protein post-translational modifications, and transcription factor binding over six stages: euthermic control (EC), entrance into torpor (EN), early torpor (ET), late torpor (LT), early arousal (EA), and interbout arousal (IA). We found differential regulation of GATA4 whereby transcript/protein expression, post-translational modification (phosphorylation of serine 261), and DNA binding were enhanced during the transitory phases (entrance and arousal) of hibernation. Activation of GATA4 was paired with increases in cardiac troponin I, troponin C and ANP protein levels during entrance, while increases in p-GATA4 DNA binding during early arousal was paired with decreases in troponin I and no changes in troponin C and ANP protein levels. Unlike its binding partner, the relative mRNA/protein expression and DNA binding of Nkx2-5 did not change during hibernation. This suggests that either Nkx2-5 does not play a substantial role or other regulatory mechanisms not presently studied (e.g. posttranslational modifications) are important during hibernation. The data suggest a significant role for GATA4-mediated gene transcription in the differential regulation of genes which aid cardiac-specific challenges associated with torpor-arousal.

Neuronal UCP1 expression suggests a mechanism for local thermogenesis during hibernation

Hibernating mammals possess a unique ability to reduce their body temperature to ambient levels, which can be as low as -2.9 °C, by active down-regulation of metabolism. Despite such a depressed physiologic phenotype, hibernators still maintain activity in their nervous systems, as evidenced by their continued sensitivity to auditory, tactile, and thermal stimulation. The molecular mechanisms that underlie this adaptation remain unknown. We report, using differential transcriptomics alongside immunohistologic and biochemical analyses, that neurons from thirteen-lined ground squirrels (Ictidomys tridecemlineatus) express mitochondrial uncoupling protein 1 (UCP1). The expression changes seasonally, with higher expression during hibernation compared with the summer active state. Functional and pharmacologic analyses show that squirrel UCP1 acts as the typical thermogenic protein in vitro. Accordingly, we found that mitochondria isolated from torpid squirrel brain show a high level of palmitate-induced uncoupling. Furthermore, torpid squirrels during the hibernation season keep their brain temperature significantly elevated above ambient temperature and that of the rest of the body, including brown adipose tissue. Together, our findings suggest that UCP1 contributes to local thermogenesis in the squirrel brain, and thus supports nervous tissue function at low body temperature during hibernation.

Gene expression changes controlling distinct adaptations in the heart and skeletal muscle of a hibernating mammal.

Throughout the hibernation season, the thirteen-lined ground squirrel (Ictidomys tridecemlineatus) experiences extreme fluctuations in heart rate, metabolism, oxygen consumption, and body temperature, along with prolonged fasting and immobility. These conditions necessitate different functional requirements for the heart, which maintains contractile function throughout hibernation, and the skeletal muscle, which remains largely inactive. The adaptations used to maintain these contractile organs under such variable conditions serves as a natural model to study a variety of medically relevant conditions including heart failure and disuse atrophy. To better understand how two different muscle tissues maintain function throughout the extreme fluctuations of hibernation we performed Illumina HiSeq 2000 sequencing of cDNAs to compare the transcriptome of heart and skeletal muscle across the circannual cycle. This analysis resulted in the identification of 1,076 and 1,466 differentially expressed genes in heart and skeletal muscle, respectively. In both heart and skeletal muscle we identified a distinct cold-tolerant mechanism utilizing peroxisomal metabolism to make use of elevated levels of unsaturated depot fats. The skeletal muscle transcriptome also shows an early increase in oxidative capacity necessary for the altered fuel utilization and increased oxygen demand of shivering. Expression of the fetal gene expression profile is used to maintain cardiac tissue, either through increasing myocyte size or proliferation of resident cardiomyocytes, while skeletal muscle function and mass are protected through transcriptional regulation of pathways involved in protein turnover. This study provides insight into how two functionally distinct muscles maintain function under the extreme conditions of mammalian hibernation.

Social conditioned place preference in the captive ground squirrel (Ictidomys tridecemlineatus): Social reward as a natural phenotype.

Social behaviors of wild animals are often considered within an ultimate framework of adaptive benefits versus survival risks. By contrast, studies of laboratory animals more typically focus on affective aspects of behavioral decisions, whether a rodent derives a rewarding experience from social encounter, and how this experience might be initiated and maintained by neural circuits. Artificial selection and inbreeding have rendered laboratory animals more affiliative and less aggressive than their wild conspecifics, leaving open the possibility that social reward is an artifact of domestication. We compared social behaviors of wild and captive population of juvenile 13-lined ground squirrels (Ictidomys tridecemlineatus), the latter being 2nd- and 3rd-generation descendants of wild individuals. At an age corresponding to emergence from the burrow, postnatal day (PD) 38, captive squirrels engaged in vigorous social approach and play and these juvenile behaviors declined significantly by PD 56. Similarly, young wild squirrels expressed social proximity and play; affiliative interactions declined with summer's progression and were replaced by agonistic chasing behaviors. Social conditioned place preference testing (conditioned PDs 40-50) indicated that adolescent squirrels derived a rewarding experience from social reunion. Our results support the contention that undomesticated rodents have the capacity for social reward and more generally suggest the possibility that positive affective experiences may support group cohesion, social cooperation, and altruism in the wild.

Dynamic changes in global and gene-specific DNA methylation during hibernation in adult thirteen-lined ground squirrels, Ictidomys tridecemlineatus.

Hibernating mammals conserve energy in the winter by undergoing prolonged bouts of torpor, interspersed with brief arousals back to euthermia. These bouts are accompanied by a suite of reversible physiological and biochemical changes; however, much remains to be discovered about the molecular mechanisms involved. Given the seasonal nature of hibernation, it stands to reason that underlying plastic epigenetic mechanisms should exist. One such form of epigenomic regulation involves the reversible modification of cytosine bases in DNA by methylation. DNA methylation is well known to be a mechanism that confers upon DNA its cellular identity during differentiation in response to innate developmental cues. However, it has recently been hypothesized that DNA methylation also acts as a mechanism for adapting genome function to changing external environmental and experiential signals over different time scales, including during adulthood. Here, we tested the hypothesis that DNA methylation is altered during hibernation in adult wild animals. This study evaluated global changes in DNA methylation in response to hibernation in the liver and skeletal muscle of thirteen-lined ground squirrels along with changes in expression of DNA methyltransferases (DNMT1/3B) and methyl binding domain proteins (MBDs). A reduction in global DNA methylation occurred in muscle during torpor phases whereas significant changes in DNMTs and MBDs were seen in both tissues. We also report dynamic changes in DNA methylation in the promoter of the myocyte enhancer factor 2C (mef2c) gene, a candidate regulator of metabolism in skeletal muscle. Taken together, these data show that genomic DNA methylation is dynamic across torpor-arousal bouts during winter hibernation, consistent with a role for this regulatory mechanism in contributing to the hibernation phenotype.

Transcriptional Activation of p53 during Cold Induced Torpor in the 13-Lined Ground Squirrel Ictidomys tridecemlineatus.

The transcription factor p53 is located at the centre of multiple pathways relating the cellular response to stress. Commonly known as a tumor suppressor, it is responsible for initiating diverse actions to protect the integrity of the genome, ranging from cell cycle arrest to apoptosis. This study investigated the regulation of p53 protein in hibernating 13-lined ground squirrel Ictidomys tridecemlineatus during multiple stages of the torpor-arousal cycle. Transcript and protein levels of p53 were both elevated in the skeletal muscle during early and late torpor stages of the hibernation cycle. Nuclear localization of p53 was also increased during late torpor, and this is associated with an increase in its DNA binding activity and expression of p53 transcriptional targets p21CIP, gadd45α, and 14-3-3σ. The increase in p53 transcriptional activity appears to be independent of its phosphorylation at Ser-15, Ser-46, and Ser-392, consistent with an absence of checkpoint kinase activation during torpor. Sequence analysis revealed unique amino acid substitutions in the ground squirrel p53 protein, which may contribute to an increase in protein stability compared to nonhibernators. Overall, the study results provided evidences for a potential role of p53 in the protection of the skeletal muscle during torpor.

Expression Profiling and Structural Characterization of MicroRNAs in Adipose Tissues of Hibernating Ground Squirrels

MicroRNAs (miRNAs) are small non-coding RNAs that are important in regulating metabolic stress. In this study, we determined the expression and structural characteristics of 20 miRNAs in brown (BAT) and white adipose tissue (WAT) during torpor in thirteen-lined ground squirrels. Using a modified stem-loop technique, we found that during torpor, expression of six miRNAs including let-7a, let-7b, miR-107, miR-150, miR-222 and miR-31 was significantly downregulated in WAT (P<0.05), which was 16%–54% of euthermic non-torpid control squirrels, whereas expression of three miRNAs including miR-143, miR-200a and miR-519d was found to be upregulated by 1.32–2.34-fold. Similarly, expression of more miRNAs was downregulated in BAT during torpor. We detected reduced expression of 6 miRNAs including miR-103a, miR-107, miR-125b, miR-21, miR-221 and miR-31 (48%–70% of control), while only expression of miR-138 was significantly upregulated (2.91±0.8-fold of the control, P<0.05). Interestingly, miRNAs found to be downregulated in WAT during torpor were similar to those dysregulated in obese humans for increased adipogenesis, whereas miRNAs with altered expression in BAT during torpor were linked to mitochondrial β-oxidation. miRPath target prediction analysis showed that miRNAs downregulated in both WAT and BAT were associated with the regulation of mitogen-activated protein kinase (MAPK) signaling, while the miRNAs upregulated in WAT were linked to transforming growth factor β (TGFβ) signaling. Compared to mouse sequences, no unique nucleotide substitutions within the stem-loop region were discovered for the associated pre-miRNAs for the miRNAs used in this study, suggesting no structure-influenced changes in pre-miRNA processing efficiency in the squirrel. As well, the expression of miRNA processing enzyme Dicer remained unchanged in both tissues during torpor. Overall, our findings suggest that changes of miRNA expression in adipose tissues may be linked to distinct biological roles in WAT and BAT during hibernation and may involve the regulation of signaling cascades.

Purification and properties of glyceraldehyde-3-phosphate dehydrogenase from the skeletal muscle of the hibernating ground squirrel, Ictidomys tridecemlineatus.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the skeletal muscle of euthermic and torpid Ictidomys tridecemlineatus was purified to electrophoretic homogeneity using a novel method involving Blue-agarose and Phenyl-agarose chromatography. Kinetic analysis of the enzymes isolated from the two conditions suggested the existence of two structurally distinct proteins, with GAPDH V max being 40–60% less for the enzyme from the torpid condition (in both glycolytic and gluconeogenic directions) as compared to the euthermic enzyme form. Thermal denaturation, in part determined by differential scanning fluorimetry, revealed that purified GAPDH from the torpid animals was significantly more stable that the enzyme from the euthermic condition. Mass spectrometry combined with Western blot analyses of purified GAPDH indicate that the cellular GAPDH population is extensively modified, with posttranslational phosphorylation, acetylation and methylation being detected. Global reduction in GAPDH tyrosine phosphorylation during torpor as well as site specific alterations in methylation sites suggests that that the stable changes observed in kinetic and structural GAPDH properties may be due to posttranslational modification of this enzyme during torpor. Taken together, these results suggest a stable suppression of GAPDH (possibly by some reversible posttranslational modification) during ground squirrel torpor, which likely contributes to the overall reduction in carbohydrate metabolism when these animals switch to lipid fuels during dormancy.

Global DNA modifications suppress transcription in brown adipose tissue during hibernation

Hibernation is crucial to winter survival for many small mammals and is characterized by prolonged periods of torpor during which strong global controls are applied to suppress energy-expensive cellular processes. We hypothesized that one strategy of energy conservation is a global reduction in gene transcription imparted by reversible modifications to DNA and to proteins involved in chromatin packing. Transcriptional regulation during hibernation was examined over euthermic control groups and five stages of the torpor/arousal cycle in brown adipose tissue of thirteen-lined ground squirrels (Ictidomys tridecemlineatus). Brown adipose is crucial to hibernation success because it is responsible for the non-shivering thermogenesis that rewarms animals during arousal. A direct modification of DNA during torpor was revealed by a 1.7-fold increase in global DNA methylation during long term torpor as compared with euthermic controls. Acetylation of histone H3 (on Lys23) was reduced by about 50% when squirrels entered torpor, which would result in increased chromatin packing (and transcriptional repression). This was accompanied by strong increases in histone deacetylase protein levels during torpor; e.g. HDAC1 and HDAC4 levels rose by 1.5- and 6-fold, respectively. Protein levels of two co-repressors of transcription, MBD1 and HP1, also increased by 1.9- and 1.5-fold, respectively, in long-term torpor and remained high during early arousal. MBD1, HP1 and HDACs all returned to near control values during interbout indicating a reversal of their inhibitory actions. Overall, the data presents strong evidence for a global suppression of transcription during torpor via the action of epigenetic regulatory mechanisms in brown adipose tissue of hibernating thirteen-lined ground squirrels.

Hibernation alters the diversity and composition of mucosa-associated bacteria while enhancing antimicrobial defence in the gut of 13-lined ground squirrels.

The gut microbiota plays important roles in animal nutrition and health. This relationship is particularly dynamic in hibernating mammals where fasting drives the gut community to rely on host-derived nutrients instead of exogenous substrates. We used 16S rRNA pyrosequencing and caecal tissue protein analysis to investigate the effects of hibernation on the mucosa-associated bacterial microbiota and host responses in 13-lined ground squirrels. The mucosal microbiota was less diverse in winter hibernators than in actively feeding spring and summer squirrels. UniFrac analysis revealed distinct summer and late winter microbiota clusters, while spring and early winter clusters overlapped slightly, consistent with their transitional structures. Communities in all seasons were dominated by Firmicutes and Bacteroidetes, with lesser contributions from Proteobacteria, Verrucomicrobia, Tenericutes and Actinobacteria. Hibernators had lower relative abundances of Firmicutes, which include genera that prefer plant polysaccharides, and higher abundances of Bacteroidetes and Verrucomicrobia, some of which can survive solely on host-derived mucins. A core mucosal assemblage of nine operational taxonomic units shared among all individuals was identified with an average total sequence abundance of 60.2%. This core community, together with moderate shifts in specific taxa, indicates that the mucosal microbiota remains relatively stable over the annual cycle yet responds to substrate changes while potentially serving as a pool for 'seeding' the microbiota once exogenous substrates return in spring. Relative to summer, hibernation reduced caecal crypt length and increased MUC2 expression in early winter and spring. Hibernation also decreased caecal TLR4 and increased TLR5 expression, suggesting a protective response that minimizes inflammation.

Inhibition of Borna disease virus replication by an endogenous bornavirus-like element in the ground squirrel genome.

Animal genomes contain endogenous viral sequences, such as endogenous retroviruses and retrotransposons. Recently, we and others discovered that nonretroviral viruses also have been endogenized in many vertebrate genomes. Bornaviruses belong to the Mononegavirales and have left endogenous fragments, called "endogenous bornavirus-like elements" (EBLs), in the genomes of many mammals. The striking features of EBLs are that they contain relatively long ORFs which have high sequence homology to the extant bornavirus proteins. Furthermore, some EBLs derived from bornavirus nucleoprotein (EBLNs) have been shown to be transcribed as mRNA and probably are translated into proteins. These features lead us to speculate that EBLs may function as cellular coopted genes. An EBLN element in the genome of the thirteen-lined ground squirrel (Ictidomys tridecemlineatus), itEBLN, encodes an ORF with 77% amino acid sequence identity to the current bornavirus nucleoprotein. In this study, we cloned itEBLN from the ground squirrel genome and investigated its involvement in Borna disease virus (BDV) replication. Interestingly, itEBLN, but not a human EBLN, colocalized with the viral factory in the nucleus and appeared to affect BDV polymerase activity by being incorporated into the viral ribonucleoprotein. Our data show that, as do certain endogenous retroviruses, itEBLN potentially may inhibit infection by related exogenous viruses in vivo.

P06 : Posttranslational modification of glyceraldehyde-3-phosphate dehydrogenase from a hibernating mammal: Insight into cold-adaptation and structural diversity of a housekeeping enzyme

Thirteen-lined ground squirrels, Spermophilus tridecemlineatus, endure cold winters by entering into hibernation; a period that entails extended bouts of aerobic torpor where body temperature drops to near freezing and metabolic rate falls to ∼5% of normal levels. The drastic depression in metabolic rate during torpor is brought about through dramatic changes to cellular biochemistry. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous enzyme that is critical, not only to glucose metabolism, but to a variety of cellular processes including oxidative stress response, autophagy and gene regulation. Its functional diversity makes it a potentially important protein during ground squirrel torpor. Analysis of purified skeletal muscle GAPDH from control and hibernating ground squirrels revealed two structurally diverse proteins. GAPDH from both conditions were extensively modified posttranslationally, with significantly decreased levels of tyrosine phosphorylation, acetylation, and ubiquitination being identified for GAPDH from the torpid condition as compared to control. Furthermore, analysis through mass spectrometry revealed novel acetylation, methylation, dimethylation, oxidation and deamidation sites within the GAPDH protein. The structural diversity of GAPDH matches its functional diversity in the cell and this study begins to correlate these structural differences with changes in GAPDH stability and kinetics during torpor.

P04 : Role of antiapoptotic signaling in mammalian hibernation

In the context of normal cell turnover, apoptosis is an essential natural phenomenon. Apoptosis depends on signals that promote cell death – proapoptotic pathways “and those that neutralize these signals” anti-apoptotic pathways. We proposed that changes in anti-apoptotic proteins would counteract stresses associated with hibernation (e.g. cold body temperatures, changes in metabolic requirements, ischemia–reperfusion) in thirteen-lined ground squirrels ( Spermophilus tridecemlineatus ). Immunoblotting was used to analyze expression of proteins in three tissues. In brain, phospho-Bcl-2 (T56), phospho-Bcl-2(S70), Bcl-3, xIAP, Mcl-1, and BI-1 increased significantly during torpor (compared with euthermia) whereas cIAP was unaltered. In liver, all proteins were unchanged except for cIAP and Bcl-3 that decreased and BI-1 which increased during torpor. In kidney, the majority of proteins did not change except for a decrease in phospho-Bcl-2(T56) and an increase in BI-1 during torpor. The data show that anti-apoptotic pathways have organ-specific responses in hibernators with a prominent potential role in brain.

A05 Session 1: Translational Hibernation : Lessons from mammalian hibernators: Surviving the adverse effects of hypothermia and ischemia–reperfusion

The ability to induce a hypometabolic state holds immense clinical promise since it serves as a means of lowering tissue energy needs and reducing ischemia damage; however, is also associated with many adverse effects in humans and most mammals. Nonetheless, hypometabolic states produced by drug administration or chilling have been applied for treatment of stroke, heart attack, multiple organ failure, as well as brain and spinal cord injury. Moreover, the ability to induce and maintain a dormant state could improve chances of survival for injured patients during transport to medical facilities, delay the onset of age-related diseases, or extend the viability of donor organs removed for transplantation. Naturally occurring hypometabolic states occur in species from seven orders of mammals, suggesting that the torpor phenotype arises from a genotype present in all mammals, including humans. Consequently, mammalian hibernators are of particular interest because they endure hypothermia, ischemia–reperfusion, restricted nutritional resources and yet transition seamlessly to and from the torpid state. To investigate the mechanisms that promote cycles of torpor-arousal without injurious effects, we surveyed targets across a range of cellular processes including metabolism, signal transduction and cellular survival, and epigenetics in 13-lined ground squirrels (Ictidomys tridecemlineatus). As a result, we identified key targets that showed differential expression during torpor with involvement in: (1) fuel utilization and balancing cellular energetics, (2) mitogen-activated protein kinase (MAPK) signal transduction and MAPK-responsive transcription factors and downstream genes, and (3) epigenetic changes including histone modifications and methylation. The data show that central to achieving a hypometabolic state are global molecular controls to inhibit ATP-expensive cellular processes as well as selective implementation of cytoprotective strategies. Furthermore, these data collectively isolate a set of molecular signatures or “fingerprints” in the areas of metabolic rate depression, stress resistance, and epigenetics which define suspended animation. Ultimately, scientific exploration of all forms of hibernation/torpor which differ in length, depth, and body temperature will enable the possibility of exploiting the properties of hypometabolism for the benefit of disease research and human health.