Hibernation In Arctic Ground Squirrels Research Articles

Microbial gene expression during hibernation in arctic ground squirrels: greater differences across gut sections than in response to pre-hibernation dietary protein content.

Obligate seasonal hibernators fast for 5-9months depending on species yet resist muscle atrophy and emerge with little lean mass loss. The role of the gut microbiome in host nitrogen metabolism during hibernation is therefore of considerable interest, and recent studies support a role for urea nitrogen salvage (UNS) in host-protein conservation. We were interested in the effect of pre-hibernation diet on UNS and the microbial provision of essential amino acids (EAAs) during hibernation; therefore, we conducted a study whereby we fed arctic ground squirrels (Urocitellus parryii) pre-hibernation diets containing 9% vs. 18% protein and compared the expression of gut bacterial urease and amino acid (AA) metabolism genes in 4 gut sections (cecum mucosa, cecum lumen, small intestine [SI] mucosa, and SI lumen) during hibernation. We found that pre-hibernation dietary protein content did not affect expression of complete bacterial AA pathway genes during hibernation; however, several individual genes within EAA pathways were differentially expressed in squirrels fed 18% pre-hibernation dietary protein. Expression of genes associated with AA pathways was highest in the SI and lowest in the cecum mucosa. Additionally, the SI was the dominant expression site of AA and urease genes and was distinct from other sections in its overall microbial functional and taxonomic composition. Urease expression in the gut microbiome of hibernating squirrels significantly differed by gut section, but not by pre-hibernation dietary protein content. We identified two individual genes that are part of the urea cycle and involved in arginine biosynthesis, which were significantly more highly expressed in the cecum lumen and SI mucosa of squirrels fed a pre-hibernation diet containing 18% protein. Six bacterial genera were responsible for 99% of urease gene expression: Cupriavidus, Burkholderia, Laribacter, Bradhyrizobium, Helicobacter, and Yersinia. Although we did not find a strong effect of pre-hibernation dietary protein content on urease or AA metabolism gene expression during hibernation, our data do suggest the potential for pre-hibernation diet to modulate gut microbiota function during hibernation, and further investigations are warranted.

Effects of Spring Warming on Seasonal Neuroendocrinology and Activation of the Reproductive Axis in Hibernating Arctic Ground Squirrels.

Many animals adjust the timing of seasonal events, such as reproduction, molt, migration, and hibernation, in response to interannual variation and directional climate-driven changes in temperature. However, the mechanisms by which temperature influences seasonal timing are relatively under-explored. Seasonal timing involves retrograde signaling in which thyrotropin (TSH) in the pars tuberalis (PT) alters expression of thyroid hormone (TH) deiodinases (Dio2/Dio3) in tanycyte cells lining the third ventricle of the hypothalamus. This, in turn, affects the availability of triiodothyronine (T3) within the mediobasal hypothalamus-increased hypothalamic T3 restores a summer phenotype and activates the reproductive axis in long-day breeders. Recently, we showed that retrograde TH signaling is activated during late hibernation in arctic ground squirrels (Urocitellus parryii) held in constant darkness and constant ambient temperature. Sensitivity of seasonal pathways to nonphotic cues, such as temperature, is likely particularly important to hibernating species that are sequestered in hibernacula during spring. To address this issue, we exposed captive arctic ground squirrels of both sexes to an ecologically relevant increase in ambient temperature (from -6 to -1°C) late in hibernation and examined the effects of warming on the seasonal retrograde TSH/Dio/T3 signaling pathway, as well as downstream elements of the reproductive axis. We found that warmed males tended to have higher PT TSHβ expression and significantly heavier testis mass whereas the TSH/Dio/T3 signaling pathway was unaffected by warming in females, although warmed females exhibited a slight decrease in ovarian mass. Our findings suggest that temperature could have different effects on gonadal growth in male and female arctic ground squirrels, which could lead to mismatched timing in response to rapid climate change.

Survival estimates of free-living arctic ground squirrels (Urocitellus parryii): effects of sex and biologging

Hibernation is associated with long lifespan: on average, hibernating mammals live 15% longer than nonhibernators of equivalent mass. We investigated how survival varies with sex, season, and the deployment of biologgers in arctic ground squirrels (Urocitellus parryii (Richardson, 1825)), a widely distributed northern hibernator. The duration of hibernation in arctic ground squirrels differs markedly by sex: females hibernate 30% longer each year than males, a behavioural trait that could positively affect female survival. Additionally, males engage in aggressive territorial and food cache defense in spring and fall, which may decrease survival in this sex. From 13 years of mark–recapture data, we estimated apparent survival of arctic ground squirrels in Arctic Alaska (USA) using Cormack–Jolly–Seber models in program MARK. We found that females had higher annual survival ([Formula: see text] (95% confidence interval (CI): 0.469, 0.913)) than males ([Formula: see text] (95% CI: 0.416, 0.670)), with a maximum observed lifespan (10 years) that exceeded that of males (6 years). We also show that biologger use and implantation did not significantly impact survival. Quantifying basic arctic ground squirrel demographics from this well-studied population illustrates how sex-specific hibernation parameters may influence lifespan differences in male and female arctic ground squirrels and provides support for the safety of biologging devices.

Seasonal decrease in thermogenesis and increase in vasoconstriction explain seasonal response to N6 -cyclohexyladenosine-induced hibernation in the Arctic ground squirrel (Urocitellus parryii).

Hibernation is a seasonal phenomenon characterized by a drop in metabolic rate and body temperature. Adenosine A1 receptor agonists promote hibernation in different mammalian species, and the understanding of the mechanism inducing hibernation will inform clinical strategies to manipulate metabolic demand that are fundamental to conditions such as obesity, metabolic syndrome, and therapeutic hypothermia. Adenosine A1 receptor agonist-induced hibernation in Arctic ground squirrels is regulated by an endogenous circannual (seasonal) rhythm. This study aims to identify the neuronal mechanism underlying the seasonal difference in response to the adenosine A1 receptor agonist. Arctic ground squirrels were implanted with body temperature transmitters and housed at constant ambient temperature (2°C) and light cycle (4L:20D). We administered CHA (N6 -cyclohexyladenosine), an adenosine A1 receptor agonist in euthermic-summer phenotype and euthermic-winter phenotype and used cFos and phenotypic immunoreactivity to identify cell groups affected by season and treatment. We observed lower core and subcutaneous temperature in winter animals and CHA produced a hibernation-like response in winter, but not in summer. cFos-ir was greater in the median preoptic nucleus and the raphe pallidus in summer after CHA. CHA administration also resulted in enhanced cFos-ir in the nucleus tractus solitarius and decreased cFos-ir in the tuberomammillary nucleus in both seasons. In winter, cFos-ir was greater in the supraoptic nucleus and lower in the raphe pallidus than in summer. The seasonal decrease in the thermogenic response to CHA and the seasonal increase in vasoconstriction, assessed by subcutaneous temperature, reflect the endogenous seasonal modulation of the thermoregulatory systems necessary for CHA-induced hibernation. Cover Image for this issue: doi: 10.1111/jnc.14528.

Effect of adenosine A1 receptor agonist induced‐hibernation on the arousal pathway in the Arctic Ground Squirrel

Hibernation is a seasonal phenomenon and in the arctic ground squirrel (AGS), the effect of seasonal changes is very evident. In late fall AGS decrease their metabolic demand followed by the entry into hibernation in winter. Previously in the lab it has been shown that N6‐cyclohexyladenosine (CHA), an A1 adenosine receptor agonist, induces hibernation in AGS in a seasonally dependent manner. In winter AGS treated with CHA experience a decrease in metabolic demand and temperature as seen during natural hibernation. However, in summer CHA triggers a brief and transient metabolic suppression that does not lead to hibernation onset.As adenosine is known to promote sleep, we decided to investigate if the sleep‐arousal pathways were affecting the seasonal difference in response to CHA. The tuberomammillary nucleus (TMN) is a nucleus of the ascending arousal pathway characterized by fast firing rates to promote wakefulness. Previously we showed a decrease in neuronal activation in the TMN in AGS treated with CHA in both summer and winter seasons.Here we test the hypothesis that seasonal response to CHA is associated with difference in activation in another nucleus of the ascending arousal pathway, the locus coeruleus (LC).AGS implanted with body temperature (Tb) transmitter, were treated with CHA or vehicle (0.5mg/kg, IP). AGS were perfused with 4% paraformaldehyde and brains were dissected and cryoprotected in 5%, 10%, 15%, 20%, 30% sucrose. We performed free‐floating immunohistochemistry on 40μm brain slices in four experimental groups: winter CHA, winter control, summer CHA and summer control. We sectioned brains from eight AGS per group for a total of 32 animals and identified active nuclei with cFos immunoreactivity (mouse anti‐cFos 1:20,000, Santa Cruz) and active tyrosine hydroxylase neurons (rabbit anti‐TH). Blinded analysis of cFos immunoreactive neurons was done using bright field microscopy and Metamorph software. Statistical analysis was performed on R.CHA produced a hibernation‐like response measured by a decrease in Tb in winter AGS but not in the summer. We did not observe any significant difference in neuronal activation in the LC, thus CHA is associated with a decreased neuronal activity in the TMN, resulting in the TMN being the major wakefulness center affected by CHA but not by seasons.Support or Funding InformationResearch was supported by NIH R03 NS081637, NSF IOS‐1258179, RL5GM118990 and Alaska INBRE P20GM103395. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Tissue-specific telomere dynamics in hibernating arctic ground squirrels (Urocitellus parryii).

Hibernation is used by a variety of mammals to survive seasonal periods of resource scarcity. Reactive oxygen species (ROS) released during periodic rewarming throughout hibernation, however, may induce oxidative damage in some tissues. Telomeres, which are the terminal sequences of linear chromosomes, may shorten in the presence of ROS, and thus the telomere length of an individual reflects the degree of accrued oxidative damage. This study quantified telomere length dynamics throughout hibernation in arctic ground squirrels (Urocitellus parryii). We hypothesized that telomere dynamics are tissue specific and predicted that telomere shortening would be most pronounced in brown adipose tissue (BAT), the organ that directly supports non-shivering thermogenesis during arousals. We used qPCR to determine relative telomere length (RTL) in DNA extracted from liver, heart, skeletal muscle (SM) and BAT of 45 juvenile and adult animals sampled either at mid- or late hibernation. Age did not have a significant effect on RTL in any tissue. At mid-hibernation, RTL of juvenile females was longer in BAT and SM than in liver and heart. In juvenile females, RTL in BAT and SM, but not in liver and heart, was shorter at late hibernation than at mid-hibernation. At late hibernation, juvenile males had longer RTL in BAT than did juvenile females, perhaps due to the naturally shorter hibernation duration of male arctic ground squirrels. Finally, BAT RTL at late hibernation negatively correlated with arousal frequency. Overall, our results suggest that, in a hibernating mammal, telomere shortening is tissue specific and that metabolically active tissues might incur higher levels of molecular damage.

Skeletal muscle non‐shivering thermogenesis in hibernating Arctic Ground Squirrels

All mammals depend on heat producing mechanisms to maintain homeostasis. Skeletal muscle shivering and brown adipose tissue (BAT) uncoupling through the expression of uncoupling protein 1 (UCP‐1) are the identified major sources of heat production, though recently, skeletal muscle has also been highlighted as a thermogenic source. Skeletal muscle nonshivering heat production occurs via sarcolipin (SLN) uncoupling of sarcoendoplasmic reticulum (SR) calcium ATPase (SERCA)‐mediated ATP hydrolysis. SLN may play a significant role in hibernating animals due to the wide temperature range that requires regulation. A thermoregulatory mechanism needs to be in place to suppress heat production during hibernation and also initiate rapid thermogenesis during arousal. We hypothesized that skeletal muscle uncoupling driven by SLN activity is a significant source of thermogenesis and inhibiting this pathway can alter the metabolic rate during hibernation in Arctic ground squirrels (AGS). Specifically, this work aimed to elucidate the role of skeletal muscle SLN uncoupling and its connection with BAT heat production in thermoregulation and metabolic control during temperature fluctuations. AGS were treated with inhibitors of BAT‐uncoupling or skeletal muscle‐uncoupling during hibernation to assess skeletal muscle and BAT ability to aid in heat production. Rate of temperature increase and changes in VO2 were compared to internal controls during inter‐bout arousals to quantify the effect of suppressing BAT or skeletal muscle uncoupling throughout hibernation. Results show that suppressing skeletal muscle uncoupling by SLN causes a significant reduction in metabolic rate during AGS rewarming. Interestingly, inhibition of BAT thermogenesis did not reduce metabolic rate. Inhibition of thermogenic sources highlighted the importance of SLN uncoupling and metabolism in thermogenesis and systemic thermoregulation. Thermogenesis control is directly linked to metabolic rate and energy loss regulation, and thus influencing thermoregulation processes has the potential for translation to innovative treatments for obesity and other diseases and conditions relevant to metabolism.Support or Funding InformationResearch reported in this publication was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103395. The content is solely the responsibility of the authors and does not necessarily reflect the official views of the NIH.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Shifting Nitrogen Balance Induces Arousal from Hibernation in Arctic Ground Squirrels

Hibernators, such as arctic ground squirrels (AGS), undergo brief periods of euthermy (termed interbout arousals) where they radically change their physiological and metabolic state. These interbout arousals rebalance metabolic and nitrogen homeostasis. A key metabolic characteristic of a hibernation bout is a progressive increase in glutamine in liver, representing an increase in nitrogen load, which is resolved over the interbout arousal. We hypothesized that increasing the nitrogen load over a bout of torpor would induce arousal. We tested the impact of increased nitrogen load on timing of arousal and metabolic profile by blinded infusion of ammonium acetate at 6 and 30 mg/kg/hr for a 12 hour period early in a torpor bout. Sodium acetate (6 and 30 mg/kg/hr) and saline served as controls. Plasma was sampled at 0, 6, 12, 30 hour from infusion start and at points defined as mid and late torpor. We monitored the core body temperature and rate of oxygen consumption to assess the hibernation state of the animal. The metabolic impact of exogenous nitrogen load was tracked over time in plasma with ultra high‐performance liquid chromatography mass spectrometry (UHPLC‐MS) metabolomics.Infusion with the highest concentration of ammonium acetate resulted in a shorter bout length than infusion with saline (Table 1). The effect of infusions on metabolic profiles is pending analysis. In conclusion, results support the hypothesis that nitrogen load limits torpor bout length. Further work is needed to determine if shifting nitrogen balance in hibernation mimics endogenous mechanisms which naturally induce arousal.This work was performed in conformance with the FASEB Statement of Principles for animals used in research.Support or Funding InformationThis work was supported by the NSF award number 1258179 and NIH 20GM103395.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Seasonal loss and resumption of circadian rhythms in hibernating arctic ground squirrels.

Circadian clocks are near universal among organisms and play a key role in coordinating physiological and metabolic functions to anticipate or coincide with predictable daily changes in the physical and social environment. However, whether circadian rhythms persist and are functionally important during hibernation in all mammals is currently unclear. We examined whether circadian rhythms of body temperature (T b) persist during multi-day, steady-state torpor and investigated the association between timing of animal emergence, exposure to light, and resumption of activity and T b rhythms in free-living and captive male arctic ground squirrels. High-resolution (0.02 °C) temperature loggers revealed that circadian rhythms of T b were not present during deep torpor in free-living arctic ground squirrels. Significant circadian rhythms of T b resumed, however, following the resumption of euthermia, but prior to emergence, though rhythms became much more robust coincident with aboveground emergence. Additionally, squirrels maintained in captivity under conditions of constant darkness spontaneously developed significant circadian rhythms of T b and activity soon after ending torpor. Exposing animals to a 5-s pulse of light within a week when they ended torpor increased the strength of rhythms. Our results are consistent with the hypothesis that circadian clock function is inhibited during hibernation in arctic ground squirrels, and we postulate that exposure to external stimuli, such as light in free-living animals, and meals or acute disturbance for captive squirrels, may enhance T b rhythmicity by synchronizing loosely coupled circadian oscillators within the suprachiasmatic nucleus.

Shotgun Proteomics Analysis of Hibernating Arctic Ground Squirrels

Mammalian hibernation involves complex mechanisms of metabolic reprogramming and tissue protection. Previous gene expression studies of hibernation have mainly focused on changes at the mRNA level. Large scale proteomics studies on hibernation have lagged behind largely because of the lack of an adequate protein database specific for hibernating species. We constructed a ground squirrel protein database for protein identification and used a label-free shotgun proteomics approach to analyze protein expression throughout the torpor-arousal cycle during hibernation in arctic ground squirrels (Urocitellus parryii). We identified more than 3,000 unique proteins from livers of arctic ground squirrels. Among them, 517 proteins showed significant differential expression comparing animals sampled after at least 8 days of continuous torpor (late torpid), within 5 h of a spontaneous arousal episode (early aroused), and 1-2 months after hibernation had ended (non-hibernating). Consistent with changes at the mRNA level shown in a previous study on the same tissue samples, proteins involved in glycolysis and fatty acid synthesis were significantly underexpressed at the protein level in both late torpid and early aroused animals compared with non-hibernating animals, whereas proteins involved in fatty acid catabolism were significantly overexpressed. On the other hand, when we compared late torpid and early aroused animals, there were discrepancies between mRNA and protein levels for a large number of genes. Proteins involved in protein translation and degradation, mRNA processing, and oxidative phosphorylation were significantly overexpressed in early aroused animals compared with late torpid animals, whereas no significant changes at the mRNA levels between these stages had been observed. Our results suggest that there is substantial post-transcriptional regulation of proteins during torpor-arousal cycles of hibernation.

Physiological oxidative stress after arousal from hibernation in Arctic ground squirrel

Hibernation in Arctic ground squirrels (AGS), Spermophilus parryii, is characterized by a profound decrease in oxygen consumption and metabolic demand during torpor that is punctuated by periodic rewarming episodes, during which oxygen consumption increases dramatically. The extreme physiology of torpor or the surge in oxygen consumption during arousal may increase production of reactive oxygen species, making hibernation an injurious process for AGS. To determine if AGS tissues experience cellular stress during rewarming, we measured carbonyl proteins, lipid peroxide end products and percent oxidized glutathione in brown adipose tissue (BAT) and liver of torpid, hibernating (hAGS), late arousal (laAGS), and cold-adapted, euthermic AGS (eAGS). In BAT carbonyl proteins and lipid peroxide end products were higher in eAGS and laAGS than in hAGS. By contrast, in liver, no significant difference in carbonyl proteins was observed. In another group of animals, comparison of carbonyl proteins and percent oxidized glutathione in frontal cortex, liver, and BAT of eAGS and hAGS showed no evidence of oxidative stress associated with torpor. These results indicate that increased thermogenesis associated with arousal AGS results in tissue specific oxidative stress in BAT but not in liver. Moreover, torpor per se is largely devoid of oxidative stress, likely due to suppression of oxidative metabolism.

Physiological regulation of tau phosphorylation during hibernation

The microtubule-associated protein tau is abnormally hyperphosphorylated in the brains of individuals with Alzheimer disease and other tauopathies, and is believed to play a critical role in the pathogenesis of these diseases. While the mechanisms leading to abnormal tau phosphorylation remain elusive, the recent demonstration of reversible tau phosphorylation during hibernation provides an ideal physiological model to study this critical process in vivo. In this study, Arctic ground squirrels (AGS) during hibernation were used to study mechanisms related to tau hyperphosphorylation. Our data demonstrate that tau is hyperphosphorylated at all six sites (S199, T205, S214, S262, S396, and S404) examined in hibernating AGS. Interestingly, only three of these sites (S199, S262, and S404) are dephosphorylated in aroused animals, suggesting a reversible phosphorylation at selective sites. Summer-active AGS demonstrated the lowest tau phosphorylation at all these sites. To explore the mechanisms underlying increased tau phosphorylation during hibernation, the expression level and enzyme activity of various potential tau kinases and protein phosphatases were examined. The kinetic analysis of enzyme activity at different temperatures revealed differential changes in enzyme activity with temperature decline. Specifically, increased protein kinase A activity, decreased protein phosphatase 2A activity, as well as substantial contribution from glycogen synthase kinase-3beta, likely play a key role in increased tau phosphorylation during hibernation in AGS.

Absence of cellular stress in brain after hypoxia induced by arousal from hibernation in Arctic ground squirrels

Although hypoxia tolerance in heterothermic mammals is well established, it is unclear whether the adaptive significance stems from hypoxia or other cellular challenge associated with euthermy, hibernation, or arousal. In the present study, blood gases, hemoglobin O2 saturation (S(O2), and indexes of cellular and physiological stress were measured during hibernation and euthermy and after arousal thermogenesis. Results show that arterial O2 tension (Pa(O2)) and S(O2) are severely diminished during arousal and that hypoxia-inducible factor (HIF)-1alpha accumulates in brain. Despite evidence of hypoxia, neither cellular nor oxidative stress, as indicated by inducible nitric oxide synthase (iNOS) levels and oxidative modification of biomolecules, was observed during late arousal from hibernation. Compared with rats, hibernating Arctic ground squirrels (Spermophilus parryii) are well oxygenated with no evidence of cellular stress, inflammatory response, neuronal pathology, or oxidative modification following the period of high metabolic demand necessary for arousal. In contrast, euthermic Arctic ground squirrels experience mild, chronic hypoxia with low S(O2) and accumulation of HIF-1alpha and iNOS and demonstrate the greatest degree of cellular stress in brain. These results suggest that Arctic ground squirrels experience and tolerate endogenous hypoxia during euthermy and arousal.

Ascorbate dynamics and oxygen consumption during arousal from hibernation in Arctic ground squirrels.

During hibernation in Arctic ground squirrels (Spermophilus parryii), O(2) consumption and plasma leukocyte counts decrease by >90%, whereas plasma concentrations of the antioxidant ascorbate increase fourfold. During rewarming, O(2) consumption increases profoundly and plasma ascorbate and leukocyte counts return to normal. Here we investigated the dynamic interrelationships among these changes. Plasma ascorbate and uric acid (urate) concentrations were determined by HPLC from blood samples collected at approximately 15-min intervals via arterial catheter; leukocyte count and hematocrit were also determined. Body temperature, O(2) consumption, and electromyographic activity were recorded continuously. Ascorbate, urate, and glutathione contents in body and brain samples were determined during hibernation and after arousal. During rewarming, the maximum rate of plasma ascorbate decrease occurred at the time of peak O(2) consumption and peak plasma urate production. The ascorbate decrease did not correlate with mouth or abdominal temperature; uptake into leukocytes could account for only a small percentage. By contrast, liver and spleen ascorbate levels increased significantly after arousal, which could more than account for ascorbate clearance from plasma. Brain ascorbate levels remained constant. These data suggest that elevated concentrations of ascorbate [(Asc)] in plasma [(Asc)(p)] provide an antioxidant source that is redistributed to tissues during the metabolic stress that accompanies arousal.