Energetics of arousal episodes in hibernating arctic ground squirrels

Arctic ground squirrels overwintering in northern Alaska experience average soil temperature of −10°C. To examine energetic costs of arousing from hibernation under arctic compared to temperate conditions, captive ground squirrels were maintained in ambient temperatures (T _a) of 2, −5 and −12°C. Rates of oxygen consumption and carbon dioxide production were used to estimate metabolic rate and fuel use during the three phases of arousal episodes: rewarming, euthermia, and recooling. Respiratory quotient comparisons suggest exclusive use of lipid during rewarming and mixed fuel use during euthermia. Animals rewarming from torpor at T _a −12°C took longer, consumed more oxygen, and attained higher peak rates of oxygen consumption when compared to 2°C. T _a had no significant effect on cost or duration of the euthermic phase. Animals recooled faster at −12°C than at 2°C, but total oxygen consumption was not different. T _a had no significant effect on the total cost of arousal episodes when all three phases are included. Arousal episodes account for 86% of estimated costs of a complete hibernation cycle including torpor when at 2°C and only 23% at −12°C. Thus, due to the higher costs of steady-state metabolism during torpor, proportional metabolic costs of arousal episodes at T _a characteristic of the Arctic are diminished compared to relative costs of arousals in more temperate conditions.     

Tissue-specific depression of mitochondrial proton leak and substrate oxidation in hibernating arctic ground squirrels

A significant proportion of standard metabolic rate is devoted to driving mitochondrial proton leak, and this futile cycle may be a site of metabolic control during hibernation. To determine if the proton leak pathway is decreased during metabolic depression related to hibernation, mitochondria were isolated from liver and skeletal muscle of nonhibernating (active) and hibernating arctic ground squirrels (Spermophilus parryii). At an assay temperature of 37 degrees C, state 3 and state 4 respiration rates and state 4 membrane potential were significantly depressed in liver mitochondria isolated from hibernators. In contrast, state 3 and state 4 respiration rates and membrane potentials were unchanged during hibernation in skeletal muscle mitochondria. The decrease in oxygen consumption of liver mitochondria was achieved by reduced activity of the set of reactions generating the proton gradient but not by a lowered proton permeability. These results suggest that mitochondrial proton conductance is unchanged during hibernation and that the reduced metabolism in hibernators is a partial consequence of tissue-specific depression of substrate oxidation.     

Inhibition of NMDA-type glutamate receptors induces arousal from torpor in hibernating arctic ground squirrels (Urocitellus parryii)

J. Neurochem. (2012) 122, 934-940. ABSTRACT: Hibernation is an adaptation to overcome periods of resource limitation often associated with extreme climatic conditions. The hibernation season consists of prolonged bouts of torpor that are interrupted by brief interbout arousals. Physiological mechanisms regulating spontaneous arousals are poorly understood, but may be related to a need for gluconeogenesis or elimination of metabolic wastes. Glutamate is derived from glutamine through the glutamate-glutamine cycle and from glucose via the pyruvate carboxylase pathway when nitrogen balance favors formation of glutamine. This study tests the hypothesis that activation of NMDA-type glutamate receptors (NMDAR) maintains torpor in arctic ground squirrel (arctic ground squirrel (AGS); Urocitellus parryii). Administration of NMDAR antagonists MK-801 (5?mg/kg, i.p.) that crosses the blood-brain barrier and AP5 (5?mg/kg, i.p.) that does not cross the blood-brain barrier induced arousal in AGS. Central administration of MK-801 (0.2, 2, 20 or 200?μg; icv) to hibernating AGS failed to induce arousal. Results suggest that activation of NMDAR at a peripheral or circumventricular site is necessary to maintain prolonged torpor and that a decrease in glutamate at these sites may contribute to spontaneous arousal in AGS.     

Global analysis of circulating metabolites in hibernating ground squirrels

Hibernation in mammals involves major alterations in nutrition and metabolism that would be expected to affect levels of circulating molecules. To gain insight into these changes we conducted a non-targeted LC–MS based metabolomic analysis of plasma using hibernating ground squirrels in late torpor (LT, T_b ~ 5 °C) or during an interbout arousal period (IBA, T_b ~ 5 °C) and non-hibernating squirrels in spring (T_b ~ 37 °C). Several metabolites varied and allowed differentiation between hibernators and spring squirrels, and between torpid and euthermic squirrels. Methionine and the short-chain carnitine esters of propionate and butyryate/isobutyrate were reduced in LT compared with the euthermic groups. Pantothenic acid and several lysophosphatidylcholines were elevated in LT relative to the euthermic groups, whereas lysophosphatidylethanolamines were elevated during IBA compared to LT and spring animals. Two regulatory lipids varied among the groups: sphingosine 1-phosphate was lower in LT vs. euthermic groups, whereas cholesterol sulfate was elevated in IBA compared to spring squirrels. Levels of long-chain fatty acids (LCFA) and total NEFA tended to be elevated in hibernators relative to spring squirrels. Three long-chain acylcarnitines were reduced in LT relative to IBA; free carnitine was also lower in LT vs. IBA. Our results identified several biochemical changes not previously observed in the seasonal hibernation cycle, including some that may provide insight into the metabolic limitations of mammalian torpor.     

Contrasting stress response of male arctic ground squirrels and red squirrels

A hormonal-challenge protocol was used to compare the stress response of males of Arctic ground squirrels and red squirrels during the breeding season (May). These squirrels live in the same boreal forest of the Yukon, but have very different life histories and utilize the forest in markedly different ways. Red squirrels had levels of total cortisol, maximum corticosteroid-binding capacity, and free cortisol that were 5, 7, and 2 times, respectively, those of Arctic ground squirrels. Red squirrels were resistant to suppression by an artificial glucocorticoid, dexamethasone (DEX); Arctic ground squirrels were not. Cortisol levels in red squirrels responded slowly but continuously to the ACTH injection; Arctic ground squirrels responded rapidly and then stabilized. Testosterone levels in red squirrels were extremely sensitive to the challenge, being suppressed by both DEX and ACTH; levels in Arctic ground squirrels were resistant to the challenge, being modestly suppressed by DEX and stimulated by ACTH. Energy mobilization, as measured by glucose and free fatty acid responses, was not affected. Red squirrels had four times the levels of white blood cells and higher proportions of lymphocytes and lower proportions of eosinophils than Arctic ground squirrels, indicating that the latter were in worse condition immunologically. Our evidence suggests that the functions associated with the hypothalamic-pituitary-adrenal axis are compromised in breeding male Arctic ground squirrels, but not in red squirrels. We propose that in male red squirrels this axis has evolved in the context of a stable social system based on long-lived animals with individual territories which are needed to deal with unpredictable winter food supplies. In contrast, Arctic ground squirrels escape the rigors of winter by hibernation and this hormonal axis has evolved in short-lived males in the context of intense intra-sexual competition in a social system based on female kin groups and regular male dispersal to avoid inbreeding. J. Exp. Zool. 286:390-404, 2000.     

A new hepadnavirus endemic in arctic ground squirrels in Alaska.

We present evidence for a novel member of the hepadnavirus family that is endemic in wild arctic ground squirrels (Spermophylus parryi kennicotti) in Alaska. This virus, designated arctic squirrel hepatitis virus (ASHV), was initially detected in the livers of animals bearing large hepatic nodules by nucleic acid hybridization with hepadnavirus probes and in plasma by cross-reactivity with antibodies to hepadnavirus surface and core antigens. The complete nucleotide sequence of the 3,302-bp-long ASHV genome was determined and compared with those of ground squirrel hepatitis virus (GSHV) and woodchuck hepatitis virus (WHV); all sequences were organized into four open reading frames, designated pre-C/C, pre-S/S, pol, and X. Despite roughly equivalent variability among the three rodent hepadnaviruses (around 16% base and 19% amino acid exchanges), ASHV appeared to be more closely related to GSHV than to WHV in phylogenetic analysis. Accordingly, preliminary studies of the pathology of ASHV infection suggested that ASHV may be a less efficient oncogenic agent than WHV. About one-third of aged animals maintained in captivity, including virus-infected as well as uninfected squirrels, developed large liver nodules, consisting of hepatocellular adenomas or carcinomas or nonmalignant lesions characterized by drastic microvesicular steatosis. ASHV-infected arctic ground squirrels may serve as a new model with which to analyze the contribution of hepadnavirus- and host-specific determinants to liver pathology and tumorigenesis.     

Regulation of gene expression by NFAT transcription factors in hibernating ground squirrels is dependent on the cellular environment

Calcineurin is a calmodulin-stimulated phosphatase that regulates the nuclear translocation of nuclear factor of activated T cell (NFAT) c1-4 through dephosphorylation. We believe that this mechanism plays various roles in the remodeling and maintenance of Ictidomys tridecemlineatus skeletal muscle. During hibernation, bouts of torpor and arousal take place, and squirrels do not lose muscle mass despite being inactive. Protein expression of Ca²⁺ signaling proteins were studied using immunoblotting. A DNA-protein interaction ELISA technique was created to test the binding of NFATs in the nucleus to DNA probes containing the NFAT response element under environmental conditions reflective of those during hibernation. Calcineurin protein levels increased by 3.08-fold during torpor (compared to euthermic control), whereas calpain1 levels also rose by 3.66-fold during torpor. Calmodulin levels were elevated upon entering torpor. NFATc4 binding to DNA showed a 1.4-fold increase during torpor, and we found that this binding was further enhanced when 600 nM of Ca²⁺ was supplemented. We also found that decreasing the temperature of ELISAs resulted in progressive decreases in the binding of NFATs c1, c3, and c4 to DNA. In summary, calmodulin and calpain1 appear to activate calcineurin and NFATc4 during torpor. NFAT binding to target promoters is affected by intranuclear [Ca²⁺] and environmental temperatures. Therefore, Ca²⁺ signaling and temperature changes play key roles in regulation of the NFAT-calcineurin pathway in skeletal muscle of hibernating 13-lined ground squirrels over the torpor-arousal cycle, and they may contribute to the avoidance of disuse-induced muscle atrophy that occurs naturally in these animals.     

Territoriality and male reproductive success in arctic ground squirrels

Although territorial defense is a common form of reproductivecompetition among male vertebrates, the exact reproductiveconsequences of this behavior are often poorly understood.To explore relationships between territoriality and reproductivesuccess in a nongroup-living mammal, we quantified patterns of space use, mating success, and fertilization success formales in a free-living population of arctic ground squirrels(Spermophilus parryii plesius). Because litters of this speciesare sired almost exclusively by a female's first mate, we predictedthat territory ownership would be associated with first accessto estrous females. During the 2-week period when mating occurred,each male in the study population attempted to defend a distinctportion of the habitat, although the success of this defensevaried among individuals. Twenty-six of 28 females monitoredmated with the male on whose territory they resided. However,the majority of females observed throughout estrus (65%; n= 20) also mated with at least one other male, indicating thatterritory ownership was not associated with exclusive accessto females. In contrast, territory ownership was significantly associated with first access to estrous females; 20 (71.4%)of 28 females mated first with the male on whose territorythey resided. In this regard, the behavior of S. parryii plesiusparallels that of socially monogamous birds in which territorialdefense by males functions to deter extrapair copulations byfemales. Although territorial defense represents an important component of male reproductive success in arctic ground squirrels,other aspects of male behavior (e.g., the ability to dominateagonistic interactions on the day of a female's estrus) arealso critical. We suggest that future studies of vertebratemating systems will benefit by viewing such defense as onlyone of multiple axes along which conspecific males compete foraccess to females.     

Morphometric and metabolic indices of disuse in muscles of hibernating ground squirrels.

1. Morphological, biochemical and metabolic characteristics of hindlimb muscles from summer-active (SA), winter-active (WA) and hibernating (H) golden-mantled ground squirrels (Spermophilus lateralis) were examined to identify alterations resulting from seasonal periods of inactivity. 2. Cross-sectional areas of fibers from the soleus were reduced in both WA and H, although only significantly (P less than 0.05) in WA. Fibers in the EDL exhibited significant reductions in cross-sectional areas in both H and WA groups. Muscle fiber and capillary densities were altered in quantitative agreement with changes in cross-sectional areas. 3. Protein content was reduced 20% (P less than 0.05) in EDL from H and WA groups, but reductions (10%) in the soleus were not statistically significant. RNA content in WA and H groups was significantly decreased in soleus (20%) and EDL (35%) compared with SA, but DNA content was unchanged. 4. In the plantaris, triglyceride content was unchanged, but citrate synthase activity in H (210 +/- 13 mumol min-1 g-1) was significantly greater than in SA (177 +/- 10). In contrast, LDH activity in H was reduced by 25% (P less than 0.05) compared with SA. 5. These results demonstrate atrophic effects associated with seasonal inactivity in hibernating ground squirrels, but suggest the existence of natural mechanisms which limit the response.     

Skeletal muscle is protected from disuse in hibernating dauria ground squirrels

The purpose of this study is to test the hypothesis that muscle fibers are protected from undue atrophy in hibernating dauria ground squirrels (Spermophilus dauricus, Brandt). Muscle mass, fiber cross sectional area (CSA, video analysis) and fiber type distribution (m-ATPase staining) were determined in extensor digitorum longus (EDL) muscle from non-hibernating control animals (Pre-H), from animals who hibernated for one (H1) or two (H2) months, and from animals 2-4 days after arousal (Post-H; N = 8 each). Muscle wet weight decreased less than body weight in hibernating animals, resulting in a steady increase in muscle-to-body mass ratio (+ 37% in Post-H compared to Pre-H, p < 0.001). In the Pre-H group, Type I (6.3 ± 2.0%) and II (93.7 ± 2.0%) fiber CSAs were 1719 ± 201 and 2261 ± 287 μm², respectively. There was a tendency (n.s.) of larger CSA of type I in hibernators compared to pre-H. In the Post-H group, fiber CSA and type distribution were not different from Pre-H. We are the first to report data on EDL fiber type distribution and confirm a protective effect that prevents muscle atrophy in spite of prolonged disuse during hibernation in dauria ground squirrels.     

Myosin isoform expression and MAFbx mRNA levels in hibernating golden-mantled ground squirrels (Spermophilus lateralis)

Hibernating mammals present many unexplored opportunities for the study of muscle biology. The hindlimb muscles of a small rodent hibernator (Spermophilus lateralis) atrophy slightly during months of torpor, representing a reduction in the disuse atrophy commonly seen in other mammalian models. How torpor affects contractile protein expression is unclear; therefore, we examined the myosin heavy-chain (MHC) isoform profile of ground squirrel skeletal muscle before and after hibernation. Immunoblotting was performed first to identify the MHC isoforms expressed in this species. Relative percentages of MHC isoforms in individual muscles were then measured using SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The soleus and diaphragm did not display differences in isoforms following hibernation, but we found minor fast-to-slow isoform shifts in MHC protein in the gastrocnemius and plantaris. These subtle changes are contrary to those predicted by other models of inactivity but may reflect the requirement for shivering thermogenesis during arousals from torpor. We also measured mRNA expression of the Muscle Atrophy F-box (MAFbx), a ubiquitin ligase important in proteasome-mediated proteolysis. Expression was elevated in the hibernating gastrocnemius and the plantaris but was not associated with atrophy. Skeletal muscle from hibernators displays unusual plasticity, which may be a combined result of the intense activity during arousals and the reduction of metabolism during torpor.     

Ubiquitin conjugate dynamics in the gut and liver of hibernating ground squirrels

Protein synthesis is severely depressed in hibernating mammals. In the absence of significant protein synthesis, the continued turnover of proteins as a function of normal cellular activity would result in the net depletion of protein pools. We measured levels of ubiquitylated proteins in the gut of thirteen-lined ground squirrels ( Spermophilus tridecemlineatus) and liver of golden-mantled ground squirrels ( Spermophilus lateralis). In both tissues, ubiquitin conjugate concentrations increased during entrance into torpor and were elevated 2-3 fold by late torpor compared with levels in active animals. The data are consistent with a depression of proteolysis with a resultant high level of ubiquitylated proteins during the natural hypothermia of torpor. The periodic returns to euthermy during the hibernation season allow for degradation of these conjugated proteins and may serve to restore protein pools.     

Antioxidant defense in hibernation: cloning and expression of peroxiredoxins from hibernating ground squirrels, Spermophilus tridecemlineatus

Mammalian hibernation is characterized by prolonged torpor bouts interspersed by brief arousal periods. Adequate antioxidant defenses are needed both to sustain cell viability over weeks of deep torpor and to defend against high rates of oxyradical formation associated with massive oxygen-based thermogenesis during arousal. The present study shows that up-regulation of peroxiredoxins contributes to antioxidant defense during torpor in thirteen-lined ground squirrels, Spermophilus tridecemlineatus. Expression levels of three isozymes of the 2-Cys peroxiredoxin (Prdx) family were quantified by Western blotting, the results showing 4.0- and 12.9-fold increases in Prdx1 protein in brown adipose tissue (BAT) and heart, respectively, during hibernation compared with euthermia. Comparable increases in Prdx2 were 2.4- and 3.7-fold whereas Prdx3 rose by 3.1-fold in heart of torpid animals. Total 2-Cys peroxiredoxin enzymatic activity also rose during hibernation by 1.5-fold in heart and 3.5-fold in BAT. Furthermore, RT-PCR showed that prdx2 mRNA levels increased by 1.7- and 3.7-fold in BAT and heart, respectively, during hibernation. A partial nucleotide sequence of prdx2 from ground squirrels was obtained by PCR amplification, the deduced amino acid sequence showing 96-97% identity with Prdx2 from other mammals. Some unique amino acid substitutions were identified that might contribute to stabilizing Prdx2 conformation at the near 0 degrees C body temperatures during torpor.     

Ubiquitylation of proteins in livers of hibernating golden-mantled ground squirrels, Spermophilus lateralis

Rodent hibernators experience low core body temperature (as low as −2 °C) and reduced metabolic rates during hibernation. Concordant with energetic constraints, protein synthesis is negligible during torpor. To maintain pools of key regulatory proteins, proteolysis must be depressed as well. Ubiquitin-dependent proteolysis consists of two major steps: (1) ubiquitylation or tagging of a protein substrate by ubiquitin and (2) the protein substrate’s subsequent degradation by the 26S proteasome. Earlier, we demonstrated that the low temperatures typical of torpor virtually arrest proteolytic processing. Here, we demonstrate that in vitro ubiquitylation still continues at greater than 30% of maximal rates at temperatures as low as 0 °C. Continued ubiquitylation in the presence of severely depressed proteolysis may explain the previously observed 2- to 3-fold increase of ubiquitin conjugates during torpor. We determined if there is a qualitative change in the type of ubiquitylation e.g., monoubiquitylation vs polyubiquitylation that occurs during torpor. We found no bias for monoubiquitylation in any state of the torpor cycle. We further determined that substrate limitation of free ubiquitin is not limiting ubiquitylation during torpor. We conclude that while the cold temperatures of torpor may limit proteolysis in accordance with metabolic demands, continued ubiquitylation may result in increased ubiquitin conjugate concentrations that must be processed upon arousal.     

Ubiquitylation of proteins in livers of hibernating golden-mantled ground squirrels,Spermophilus lateralis

Rodent hibernators experience low core body temperature (as low as −2 °C) and reduced metabolic rates during hibernation. Concordant with energetic constraints, protein synthesis is negligible during torpor. To maintain pools of key regulatory proteins, proteolysis must be depressed as well. Ubiquitin-dependent proteolysis consists of two major steps: (1) ubiquitylation or tagging of a protein substrate by ubiquitin and (2) the protein substrate’s subsequent degradation by the 26S proteasome. Earlier, we demonstrated that the low temperatures typical of torpor virtually arrest proteolytic processing. Here, we demonstrate that in vitro ubiquitylation still continues at greater than 30% of maximal rates at temperatures as low as 0 °C. Continued ubiquitylation in the presence of severely depressed proteolysis may explain the previously observed 2- to 3-fold increase of ubiquitin conjugates during torpor. We determined if there is a qualitative change in the type of ubiquitylation e.g., monoubiquitylation vs polyubiquitylation that occurs during torpor. We found no bias for monoubiquitylation in any state of the torpor cycle. We further determined that substrate limitation of free ubiquitin is not limiting ubiquitylation during torpor. We conclude that while the cold temperatures of torpor may limit proteolysis in accordance with metabolic demands, continued ubiquitylation may result in increased ubiquitin conjugate concentrations that must be processed upon arousal.