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Mitochondria are the main organelles for mammalian energy metabolism and have been implicated in the regulation of germ cell maintenance and spermatogenesis. However, little is known about the changes in the mitochondria of the testis of seasonal breeders. Here, we characterized the seasonal changes in the mitochondria in the testis of the wild ground squirrels. Increased testicle weight, seminiferous tubule diameter, and sperm count were observed in the wild ground squirrels at the breeding season. RNA-seq analysis of the wild ground squirrel testes revealed that mitochondrial-related genes were expressed differentially between the breeding and nonbreeding seasons. Immunohistochemical staining showed that key mitophagy factors including PINK1, MFN2, and PARKIN were highly expressed in various cell types of testis during the breeding season. In addition, the abundance and enzymatic activities of mitochondrial-localized antioxidative enzymes superoxide dismutase 2 (SOD2) and Catalase were decreased in the testis during the breeding season, suggesting a tightly controlled redox balance at least partially facilitated by mitophagy during the seasonal breeding. Taken together, our study reveals that mitochondrial autophagy and oxidative stress may be implicated in the seasonal reproductive recrudescence of the wild ground squirrels, which deepens our understanding of the mitochondrial regulation of seasonal reproductivity in wildlife and provides new insights into the development of potential therapeutic interventions of male infertility.

It is well established that hibernating mammals rely predominantly on lipid stores to fuel metabolism throughout the hibernation season. However, it is unclear if other endogenous fuels contribute to the rapid, ~400-fold increase in metabolic rate during the early phase of arousal from torpor. To investigate this issue, we used cavity ring-down spectroscopy, a technique that provides a real-time indication of fuel use by measuring the ratio of 13 C to 12 C in the exhaled CO 2 of arousing 13-lined ground squirrels ( Ictidomys tridecemlineatus ). We used infrared thermography to simultaneously measure ventilation and surface temperature change in various body regions, and we interpreted these data in light of changing plasma metabolite abundances at multiple stages of arousal from torpor. We found that hibernating squirrels use a combination of lipids and, likely, carbohydrates to fuel the initial ~60 min of arousal before switching to predominantly lipid oxidation. This fuel switch coincided with times of maximal rates of ventilation and rewarming of different body surface regions, including brown adipose tissue. Infrared thermography revealed zonal rewarming, whereby the brown adipose tissue region was the first to warm, followed by the thoracic and head regions and, finally, the posterior half of the body. Consistent with the results from cavity ring-down spectroscopy, plasma metabolite dynamics during early arousal suggested a large reliance on fatty acids, with a contribution from carbohydrates and glycerol. Because of their high oxidative flux rates and efficient O 2 use, carbohydrates might be an advantageous metabolic fuel during the early phase of arousal, when metabolic demands are high but ventilation rates and, thus, O 2 supply are relatively low.

Previous hibernation studies demonstrated that such a natural model of skeletal muscle disuse causes limited muscle atrophy and a significant fast-to-slow fiber type shift. However, the underlying mechanism as defined in a large-scale analysis remains unclarified. Isobaric tags for relative and absolute quantification (iTRAQ) based quantitative analysis were used to examine proteomic changes in the fast extensor digitorum longus muscles (EDL) of Daurian ground squirrels (Spermophilus dauricus). Although the wet weights and fiber cross-sectional area of the EDL muscle showed no significant decrease, the percentage of slow type fiber was 61% greater (P < 0.01) in the hibernation group. Proteomics analysis identified 264 proteins that were significantly changed (ratio < 0.83 or >1.2-fold and P < 0.05) in the hibernation group, of which 23 proteins were categorized into energy production and conversion and translation and 22 proteins were categorized into ribosomal structure and biogenesis. Along with the validation by western blot, MAPKAP kinase 2, ATP5D, ACADSB, calcineurin, CSTB and EIF2S were up-regulated in the hibernation group, whereas PDK4, COX II and EIF3C were down-regulated in the hibernation group. MAPKAP kinase 2 and PDK4 were associated with glycolysis, COX II and ATP5D were associated with oxidative phosphorylation, ACADSB was associated with fatty acid metabolism, calcineurin and CSTB were associated with catabolism, and EIF2S and EIF3C were associated with anabolism. Moreover, the total proteolysis rate of EDL in the hibernation group was significantly inhibited compared with that in the pre-hibernation group. These distinct energy and protein metabolism characteristics may be involved in myofiber type conversion and resistance to atrophy in the EDL of hibernating Daurian ground squirrels., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Hibernation is a seasonally adaptive strategy that allows hibernators to live through extremely cold conditions. Despite the profound reduction of blood flow to the retinas, hibernation causes no lasting retinal injury. Instead, hibernators show an increased tolerance to ischemic insults during the hibernation period. To understand the molecular changes of the retinas in response to hibernation, we applied an integrative transcriptome and metabolome analysis to explore changes in gene expression and metabolites of 13-lined ground squirrel retinas during hibernation. Metabolomic analysis showed a global decrease of ATP synthesis in hibernating retinas. Decreased glucose and galactose, increased beta-oxidation of carnitine and decreased storage of some amino acids in hibernating retinas indicated a shift of fuel use from carbohydrates to lipids and alternative usage of amino acids. Transcriptomic analysis revealed that the down-regulated genes were enriched in DNA-templated transcription and immune-related functions, while the up-regulated genes were enriched in mitochondrial inner membrane and DNA packaging-related functions. We further showed that a subset of genes underwent active alternative splicing events in response to hibernation. Finally, integrative analysis of the transcriptome and metabolome confirmed the shift of fuel use in the hibernating retina by the regulation of catabolism of amino acids and lipids. Through transcriptomic and metabolomic data, our analysis revealed the altered state of mitochondrial oxidative phosphorylation and the shift of energy source in the hibernating retina, advancing our understanding of the molecular mechanisms employed by hibernators. The data will also serve as a useful resource for the ocular and hibernation research communities., (© 2017 Wiley Periodicals, Inc.)

Stable carbon isotope ratios (δ 13 C) in breath show promise as an indicator of immediate metabolic fuel utilization in animals because tissue lipids have a lower δ 13 C value than carbohydrates and proteins. Metabolic fuel consumption is often estimated using the respiratory exchange ratio (RER), which has lipid and carbohydrate boundaries, but does not differentiate between protein and mixed fuel catabolism at intermediate values. Because lipids have relatively low δ 13 C values, measurements of stable carbon isotopes in breath may help distinguish between catabolism of protein and mixed fuel that includes lipid. We measured breath δ 13 C and RER concurrently in arctic ground squirrels (Urocitellus parryii) during steady-state torpor at ambient temperatures from -2 to -26°C. As predicted, we found a correlation between RER and breath δ 13 C values; however, the range of RER in this study did not reach intermediate levels to allow further resolution of metabolic substrate use with the addition of breath δ 13 C measurements. These data suggest that breath δ 13 C values are 1.1‰ lower than lipid tissue during pure lipid metabolism. From RER, we determined that arctic ground squirrels rely on nonlipid fuel sources for a significant portion of energy during torpor (up to 37%). The shift toward nonlipid fuel sources may be influenced by adiposity of the animals in addition to thermal challenge., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Studies demonstrate the importance of metabolic resources in the regulation of reproduction and immune functions in seasonal breeders. In this regard, the restricted energy availability can be considered as an environmental variable that may act as a seasonal stressor and can lead to compromised immune functions. The present study explored the effect of photoperiodic variation in the regulation of immune function under metabolic stress condition. The T-cell-dependent immune response in a tropical seasonal breeder Funambulus pennanti was studied following the inhibition of cellular glucose utilization with 2-deoxy-d-glucose (2-DG). 2-DG treatment resulted in the suppression of general (e.g., proliferative response of lymphocytes) and antigen-specific [anti-keyhole limpet hemocyanin IgG titer and delayed-type hypersensitivity response] T-cell responses with an activation of the hypothalamic-pituitary-adrenal axis, which was evident from the increased levels of plasma corticosterone. 2-DG administration increased the production of inflammatory cytokines [interleukin (IL)-1β and tumor necrosis factor (TNF)-α] and decreased the autocrine T-cell growth factor IL-2. The immunocompromising effect of 2-DG administration was retarded in animals exposed to short photoperiods compared with the control and long photoperiod-exposed groups. This finding suggested that short photoperiodic conditions enhanced the resilience of the immune system, possibly by diverting metabolic resources from the reproductive organs toward the immune system. In addition, melatonin may have facilitated the energy "trade-off" between reproductive and immune mechanisms, thereby providing an advantage to the seasonal breeders for their survival during stressful environmental conditions., (Copyright © 2017. Published by Elsevier Inc.)

Thyroid hormones (THs), key regulators of lipid and carbohydrate metabolism, are likely modulators of energy allocation within and among animal life history stages. Despite their role in modulating metabolism, few studies have investigated whether THs vary among life history stages in free-living animals or if they exhibit stage-specific relationships to total energy expenditure and activity levels. We measured plasma total triiodothyronine (tT3) and thyroxine (tT4) at four, discrete life history stages of female arctic ground squirrels from two different populations in northern Alaska to test whether plasma THs correlate with life history stage-specific changes in metabolic rate and energy demand. We also tested whether THs explained individual variation in aboveground activity levels within life history stages. T3 peaked during lactation and was lowest during pre-hibernation fattening, consistent with known changes in basal metabolism and core body temperature. In contrast, T4 was elevated shortly after terminating hibernation but remained low and stable across other life-history stages in the active season. THs were consistently higher in the population that spent more time above-ground but the relationship between THs and activity varied among life history stages. T3 was positively correlated with activity only during lactation (r(2)=0.50) whereas T4 was positively correlated with activity immediately following lactation (r(2)=0.48) and during fattening (r(2)=0.53). Our results support the hypothesis that THs are an important modulator of basal metabolism but also suggest that the relationship between THs and activity varies among life history stages., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The hearts of mammalian hibernators maintain contractile function in the face of severe environmental stresses during winter heterothermy. To enable survival in torpor, hibernators regulate the expression of numerous genes involved in excitation-contraction coupling, metabolism, and stress response pathways. Understanding the basis of this transition may provide new insights into treatment of human cardiac disease. Few studies have investigated hibernator heart performance during both summer active and winter torpid states, and seasonal comparisons of whole heart function are generally lacking. We investigated the force-frequency relationship and the response to ex vivo ischemia-reperfusion in intact isolated hearts from 13-lined ground squirrels (Ictidomys tridecemlineatus) in the summer (active, July) and winter (torpid, January). In standard euthermic conditions, we found that winter hearts relaxed more rapidly than summer hearts at low to moderate pacing frequencies, even though systolic function was similar in both seasons. Proteome data support the hypothesis that enhanced Ca(2+) handling in winter torpid hearts underlies the increased relaxation rate. Additionally, winter hearts developed significantly less rigor contracture during ischemia than summer hearts, while recovery during reperfusion was similar in hearts between seasons. Winter torpid hearts have an increased glycogen content, which likely reduces development of rigor contracture during the ischemic event due to anaerobic ATP production. These cardioprotective mechanisms are important for the hibernation phenotype and highlight the resistance to hypoxic stress in the hibernator., (Copyright © 2015 the American Physiological Society.)

Mammalian hibernators undergo significant physiological and biochemical changes when confronted with cold temperatures. Metabolic depression and translational repression are two examples of the various processes impacted during a torpor bout. MicroRNAs (miRNAs), non-coding transcripts that bind to mRNAs, are known regulators of mRNA translation and a growing number of these molecules have been found to be differentially expressed during hibernation. We hypothesized that a group of six miRNAs, with targets involved in various metabolic cascades, is modulated in selected tissues of the hibernating thirteen-lined ground squirrel Ictidomys tridecemlineatus. Expression levels of these miRNAs were assessed in the liver, heart, and skeletal muscle ground squirrel tissues using qRT-PCR. miR-29a, miR-152, miR-195, miR-223, and miR-486 were shown to be up-regulated in the hibernating liver, while miR-378 was shown to be down-regulated in hibernating skeletal muscle tissue samples. Interestingly, fatty acid synthase (FAS), an enzyme involved in fatty acid biosynthesis and a miR-195 target, was shown to be down-regulated in hibernating squirrel liver. This data add to the growing signature of differentially expressed miRNAs during hibernation and puts the light on the potential regulation of fatty acid homeostasis by a miRNA in torpid animals.

Small-bodied hibernators partition the year between active homeothermy and hibernating heterothermy accompanied by fasting. To define molecular events underlying hibernation that are both dependent and independent of fasting, we analyzed the liver proteome among two active and four hibernation states in 13-lined ground squirrels. We also examined fall animals transitioning between fed homeothermy and fasting heterothermy. Significantly enriched pathways differing between activity and hibernation were biased toward metabolic enzymes, concordant with the fuel shifts accompanying fasting physiology. Although metabolic reprogramming to support fasting dominated these data, arousing (rewarming) animals had the most distinct proteome among the hibernation states. Instead of a dominant metabolic enzyme signature, torpor-arousal cycles featured differences in plasma proteins and intracellular membrane traffic and its regulation. Phosphorylated NSFL1C, a membrane regulator, exhibited this torpor-arousal cycle pattern; its role in autophagosome formation may promote utilization of local substrates upon metabolic reactivation in arousal. Fall animals transitioning to hibernation lagged in their proteomic adjustment, indicating that the liver is more responsive than preparatory to the metabolic reprogramming of hibernation. Specifically, torpor use had little impact on the fall liver proteome, consistent with a dominant role of nutritional status. In contrast to our prediction of reprogramming the transition between activity and hibernation by gene expression and then within-hibernation transitions by posttranslational modification (PTM), we found extremely limited evidence of reversible PTMs within torpor-arousal cycles. Rather, acetylation contributed to seasonal differences, being highest in winter (specifically in torpor), consistent with fasting physiology and decreased abundance of the mitochondrial deacetylase, SIRT3., (Copyright © 2014 the American Physiological Society.)

Hibernating arctic ground squirrels (Urocitellus parryii), overwintering in frozen soils, maintain large gradients between ambient temperature (T(a)) and body temperature (T(b)) by substantially increasing metabolic rate during torpor while maintaining a subzero T(b). We used quantitative reverse-transcription PCR (qRT-PCR) to determine how the expression of 56 metabolic genes was affected by season (active in summer vs hibernating), metabolic load during torpor (imposed by differences in T(a): +2 vs -10°C) and hibernation state (torpid vs after arousal). Compared with active ground squirrels sampled in summer, liver from hibernators showed increased expression of genes associated with fatty acid catabolism (CPT1A, FABP1 and ACAT1), ketogenesis (HMGCS2) and gluconeogenesis (PCK1) and decreased expression of genes associated with fatty acid synthesis (ACACB, SCD and ELOVL6), amino acid metabolism, the urea cycle (PAH, BCKDHA and OTC), glycolysis (PDK1 and PFKM) and lipid metabolism (ACAT2). Stage of hibernation (torpid vs aroused) had a much smaller effect, with only one gene associated with glycogen synthesis (GSY1) in liver showing consistent differences in expression levels between temperature treatments. Despite the more than eightfold increase in energetic demand associated with defending T(b) during torpor at a T(a) of -10 vs +2°C, transcript levels in liver and brown adipose tissue differed little. Our results are inconsistent with a hypothesized switch to use of non-lipid fuels when ambient temperatures drop below freezing.

Gliding allows mammals to exploit canopy habitats of old-growth forests possibly as a means to save energy. To assess costs of quadrupedal locomotion for a gliding arboreal mammal, we used open-flow respirometry and a variable-speed treadmill to measure oxygen consumption and to calculate cost of transport, excess exercise oxygen consumption, and excess post-exercise oxygen consumption for nine northern flying squirrels (Glaucomys sabrinus) and four fox squirrels (Sciurus niger). Our results indicate that oxygen consumption during exercise by flying squirrels was 1.26-1.65 times higher than predicted based on body mass, and exponentially increased with velocity (from 0.84 ± 0.03 ml O(2) kg(-1) s(-1) at 0.40 m s(-1) to 1.55 ± 0.03 ml O(2) kg(-1) s(-1) at 0.67 m s(-1)). Also, cost of transport in flying squirrels increased with velocity, although excess exercise oxygen consumption and excess post-exercise oxygen consumption did not. In contrast, oxygen consumption during exercise for fox squirrels was similar to predicted, varying from 0.51 (±0.02) ml O(2) kg(-1) s(-1) at 0.63 m s(-1) to 0.54 (±0.03) ml O(2) kg(-1) s(-1) at 1.25 m s(-1). In addition, the cost of transport for fox squirrels decreased with velocity, while excess exercise oxygen consumption and excess post-exercise oxygen consumption did not. Collectively, these observations suggest that unlike fox squirrels, flying squirrels are poorly adapted to prolonged bouts of quadrupedal locomotion. The evolution of skeletal adaptations to climbing, leaping, and landing and the development of a gliding membrane likely has increased the cost of quadrupedal locomotion by >50% while resulting in energy savings during gliding and reduction in travel time between foraging patches.

During the torpor phase of mammalian hibernation when core body temperature is near 4 degrees C, the autonomic system continues to maintain respiration, blood pressure and heartbeat despite drastic reductions in brain activity. In addition, the hibernator's neuronal tissues enter into a protected state in which the potential for ischemia-reperfusion injury is markedly minimized. Evolutionary adaptations for continued function and neuroprotection throughout cycles of torpor and euthermia in winter are predicted to manifest themselves partly in changes in the brainstem proteome. Here, we compare the soluble brainstem protein complement from six summer active ground squirrels and six in the early torpor (ET) phase of hibernation. Thirteen percent of the approximately 1,500 quantifiable 2D gel spots alter significantly from summer to ET; the proteins identified in these differing spots are known to play roles in energy homeostasis via the tricarboxylic acid cycle (8 proteins), cytoarchitecture and cell motility (14 proteins), anabolic protein processes (13 proteins), redox control (11 proteins) and numerous other categories including protein catabolism, oxidative phosphorylation, signal transduction, glycolysis, intracellular protein trafficking and antiapoptotic function. These protein changes represent, at least in part, the molecular bases for restructuring of cells in the brainstem, a shift away from glucose as the primary fuel source for brain in the winter, and the generation of a streamlined mechanism capable of efficient and rapid energy production and utilization during the torpor and arousal cycles of hibernation.

Hibernating mammals use reduced metabolism, hypothermia, and stored fat to survive up to 5 or 6 mo without feeding. We found serum levels of the fat-derived ketone, D-beta-hydroxybutyrate (BHB), are highest during deep torpor and exist in a reciprocal relationship with glucose throughout the hibernation season in the thirteen-lined ground squirrel (Spermophilus tridecemlineatus). Ketone transporter monocarboxylic acid transporter 1 (MCT1) is upregulated at the blood-brain barrier, as animals enter hibernation. Uptake and metabolism of 13C-labeled BHB and glucose were measured by high-resolution NMR in both brain and heart at several different body temperatures ranging from 7 to 38 degrees C. We show that BHB and glucose enter the heart and brain under conditions of depressed body temperature and heart rate but that their utilization as a fuel is highly selective. During arousal from torpor, glucose enters the brain over a wide range of body temperatures, but metabolism is minimal, as only low levels of labeled metabolites are detected. This is in contrast to BHB, which not only enters the brain but is also metabolized via the tricarboxylic acid (TCA) cycle. A similar situation is seen in the heart as both glucose and BHB are transported into the organ, but only 13C from BHB enters the TCA cycle. This finding suggests that fuel selection is controlled at the level of individual metabolic pathways and that seasonally induced adaptive mechanisms give rise to the strategic utilization of BHB during hibernation.

Torpor is a reversible reduction in endotherm body temperature and metabolic rate. Because torpid endotherms can attain lower body temperatures in colder environments, minimum torpor metabolism generally increases with rising air temperature whereas euthermic metabolism generally declines with rising air temperature. As a result, the fundamental metabolic niche of endotherms that express torpor should be driven by climate and should be broadest in colder environments. On the other hand, if torpor serves primarily as an energy conservation strategy and its expression is influenced by energy availability, then the realized metabolic niche should be defined by resources. To evaluate the influence of resource and climate on torpor use and metabolism of hibernating mammals, we monitored the torpor expression of free-ranging eastern chipmunks (Tamias striatus) over two winters of varying resource abundance. In the low-food year, soil temperature constrained maximum torpor expression but was too invariant across small spatial scales to explain individual variation in torpor expression. In the high-food year, torpor was drastically reduced, and local density of seed-producing trees predicted fine-scale spatial variation in torpor expression. Thus, the fundamental metabolic niche of hibernating chipmunks in cold environments is broad and constrained by climate, whereas the realized metabolic niche is highly variable among individuals and years and is determined primarily by local resource abundance.

Parasites have been suggested to influence many aspects of host behaviour. Some of these effects may be mediated via their impact on host energy budgets. This impact may include effects on both energy intake and absorption as well as components of expenditure, including resting metabolic rate (RMR) and activity (e.g. grooming). Despite their potential importance, the energy costs of parasitism have seldom been directly quantified in a field setting. Here we pharmacologically treated female Cape ground squirrels (Xerus inauris) with anti-parasite drugs and measured the change in body composition, the daily energy expenditure (DEE) using doubly labelled water, the RMR by respirometry and the proportions of time spent looking for food, feeding, moving and grooming. Post-treatment animals gained an average 19g of fat or approximately 25kJd-1. DEE averaged 382kJd-1 prior to and 375kJd-1 post treatment (p>0.05). RMR averaged 174kJd-1 prior to and 217kJd-1 post treatment (p<0.009). Post-treatment animals spent less time looking for food and grooming, but more time on feeding. A primary impact of infection by parasites could be suppression of feeding behaviour and, hence, total available energy resources. The significant elevation of RMR after treatment was unexpected. One explanation might be that parasites produce metabolic by-products that suppress RMR. Overall, these findings suggest that impacts of parasites on host energy budgets are complex and are not easily explained by simple effects such as stimulation of a costly immune response. There is currently no broadly generalizable framework available for predicting the energetic consequences of parasitic infection.

Glycolysis, glucose oxidation, palmitate oxidation, and cardiac function were measured in isolated working hearts from ground squirrels and rats subjected to a hypothermia-rewarming protocol. Hearts were perfused initially for 30 min at 37 degrees C, followed by 2 h of hypothermic perfusion at 15 degrees C, after which hearts were rewarmed to 37 degrees C and further perfused for 30 min. Functional recovery in ground squirrel hearts was greater than in rat hearts after rewarming. Hypothermia-rewarming had a similar general effect on the various metabolic pathways in both species. Despite these similarities, total energy substrate metabolic rates were greater in rat than ground squirrel hearts during hypothermia despite a lower level of work being performed by the rat hearts, indicating that rat hearts are less efficient than ground squirrel hearts during hypothermia. After rewarming, energy substrate metabolism recovered completely in both species, although cardiac work remained depressed in rat hearts. The difference in functional recovery between rat and ground squirrel hearts after rewarming cannot be explained by general differences in energy substrate metabolism during hypothermia or after rewarming.