Mammalian Hibernation Research Articles

Apneic uptake of atmospheric O2 by deeply hypothermic nestlings of the white-footed mouse (Peromyscus leucopus): circulation and lungs.

Nestling white-footed mice (Peromyscus leucopus) are born in the earliest days of spring in cold climates. If the nestlings are by accident exposed to ambient temperatures near freezing (0-7°C) at early ages (2-10 days old), they may experience body temperatures (Tbs) equally low. During such hypothermia, although their heart keeps beating, they become apneic (cease inhaling and exhaling). However, they have an exceptional ability (e.g., compared to Mus musculus) to tolerate these conditions for at least several hours, after which they revive if rewarmed by parents. This paper addresses the physiology of the apneic period. We show that apneic, hypothermic nestlings undergo physiologically important exchanges of gases with the atmosphere. These gas exchanges do not occur across the skin. Instead they occur via the trachea and lungs even though the animals are apneic. Most significantly, when hypothermic neonates are in apnea in ordinary air, they take up O2 steadily from the atmosphere throughout the apneic period, and the evidence available indicates that this O2 uptake is essential for the nestlings' survival. At Tbs of 2-7°C, the nestlings' rate of O2 consumption varies quasi-exponentially with Tb and averages 0.04 mL O2 g- 1 h- 1, closely similar to the rate expressed by adult mammalian hibernators in hibernation at similar Tbs. Morphometric analysis indicates that, at all focal ages, O2 transport along the full length of the trachea can take place by diffusion at rates adequate to meet the measured rates of metabolic O2 consumption.

Identification of genes supporting cold resistance of mammalian cells: lessons from a hibernator

Susceptibility of human cells to cold stress restricts the use of therapeutic hypothermia and long-term preservation of organs at low temperatures. In contrast, cells of mammalian hibernators possess remarkable cold resistance, but little is known about the molecular mechanisms underlying this phenomenon. In this study, we conducted a gain-of-function screening of genes that confer cold resistance to cold-vulnerable human cells using a cDNA library constructed from the Syrian hamster, a mammalian hibernator, and identified Gpx4 as a potent suppressor of cold-induced cell death. Additionally, genetic deletion of or pharmacological inhibition of Gpx4 revealed that Gpx4 is necessary for suppressing lipid peroxidation specifically under cold in hamster cell lines. Genetic disruption of other ferroptosis-suppressing pathways, namely biopterin synthesis and mitochondrial or plasma membrane CoQ reduction pathways, also accelerated cold-induced cell death under Gpx4 dysfunction. Collectively, ferroptosis-suppressing pathways protect the cells of a mammalian hibernator from cold-induced cell death and the augmentation of these pathways renders cold resistance to cells of non-hibernators, including humans.

Hypothalamic hormone deficiency enables physiological anorexia in ground squirrels during hibernation

Mammalian hibernators survive prolonged periods of cold and resource scarcity by temporarily modulating normal physiological functions, but the mechanisms underlying these adaptations are poorly understood. The hibernation cycle of thirteen-lined ground squirrels (Ictidomys tridecemlineatus) lasts for 5–7 months and comprises weeks of hypometabolic, hypothermic torpor interspersed with 24–48-h periods of an active-like interbout arousal (IBA) state. We show that ground squirrels, who endure the entire hibernation season without food, have negligible hunger during IBAs. These squirrels exhibit reversible inhibition of the hypothalamic feeding center, such that hypothalamic arcuate nucleus neurons exhibit reduced sensitivity to the orexigenic and anorexigenic effects of ghrelin and leptin, respectively. However, hypothalamic infusion of thyroid hormone during an IBA is sufficient to rescue hibernation anorexia. Our results reveal that thyroid hormone deficiency underlies hibernation anorexia and demonstrate the functional flexibility of the hypothalamic feeding center.

Frequency-modulated timer regulates torpor–arousal cycles during hibernation in distinct small mammalian hibernators

Hibernation allows mammals to endure harsh seasons by reducing their basal metabolism and body temperature (Tb) to minimize energy expenditure. During hibernation in small animals such as Syrian hamsters and 13-lined ground squirrels, Tb decreases to an ambient level ( < 5 °C) and remains constant for days to weeks in a physiological condition termed deep torpor. Torpor is interrupted by periods of arousal, during which Tb recovers to a euthermic level (approximately 37 °C), and these torpor–arousal cycles are repeated multiple times during hibernation. However, little is known about the mechanisms governing Tb fluctuations during hibernation. In this study, we employed an unbiased model selection approach to Tb data and revealed that a model incorporating frequency modulation quantitatively reproduced Tb fluctuation during hibernation in Syrian hamsters. We found that an unexpectedly long period of 120–430 days modulates a shorter period of several days. In addition, the aforementioned model reproduced Tb fluctuation in 13-lined ground squirrels, which can undergo repeated hibernation according to intrinsic circannual rhythms in constant laboratory conditions. This is the first quantitative study to demonstrate the concerted action of two endogenous periods, one lasting a few days and the other lasting a year, in the torpor–arousal cycles of distinct mammalian hibernators. We anticipate that our theoretical analysis of Tb fluctuation will be a starting point for quantitative comparisons of hibernation patterns across various hibernating species. Furthermore, quantification of Tb data using models will foster our understanding of the molecular mechanisms of hibernation by revealing the biological processes operating within these periods.

Conserved Noncoding Cis-Elements Associated with Hibernation Modulate Metabolic and Behavioral Adaptations in Mice.

Our study elucidates functional roles for conserved cis-elements associated with the evolution of mammalian hibernation. Genomic analyses found topologically associated domains (TADs) that disproportionately accumulated convergent genomic changes in hibernators, including the TAD for the Fat Mass & Obesity (Fto) locus. Some hibernation-linked cis-elements in this TAD form regulatory contacts with multiple neighboring genes. Knockout mice for these cis-elements exhibit Fto, Irx3, and Irx5 gene expression changes, impacting hundreds of genes downstream. Profiles of pre-torpor, torpor, and post-torpor phenotypes found distinct roles for each cis-element in metabolic control, while a high caloric diet uncovered different obesogenic effects. One cis-element promoting a lean phenotype influences foraging behaviors throughout life, affecting specific behavioral sequences. Thus, convergent evolution in hibernators pinpoints functional genetic mechanisms of mammalian metabolic control.

Convergent accelerated evolution of mammal-specific conserved non-coding elements in hibernators

Mammals maintain their body temperature, yet hibernators can temporarily lower their metabolic rate as an energy-saving strategy. It has been proposed that hibernators evolved independently from homeotherms, and it is possible that the convergent evolution of hibernation involved common genomic changes among hibernator-lineages. Since hibernation is a seasonal trait, the evolution of gene regulatory regions in response to changes in season may have been important for the acquisition of hibernation traits. High-frequency accumulation of mutations in conserved non-coding elements (CNEs) could, in principle, alter the expression of neighboring genes and thereby contribute to the acquisition of new traits. To address this possibility, we performed a comparative genomic analysis of mammals to identify accelerated CNEs commonly associated with hibernation. We found that accelerated CNEs are common to hibernator-lineages and could be involved with hibernation. We also found that common factors of genes that located near accelerated CNEs and are differentially expressed between normal and hibernation periods related to gene regulation and cell-fate determination. It suggests that the molecular mechanisms controlling hibernation have undergone convergent evolution. These results help broaden our understanding of the genetic adaptations that facilitated hibernation in mammals and may offer insights pertaining to stress responses and energy conservation.

Torpor in the golden-mantled ground squirrel: investigating transcriptomic dynamics across body temperature

The golden-mantled ground squirrel ( Callospermophilus lateralis) is considered an elite hibernator. Seasonal torpor in the face of environmental extremes and food scarcity can elicit a decrease in body temperature to near freezing temperatures and oxygen consumption rates 1/100th of normothermic rates. Not all hibernators are capable of body temperature and metabolic depression to this degree; instead, mammalian hibernators use diverse strategies related to body size and environment and conduct torpor at body temperatures ranging from cold (rodents) to hot (bears). Tropical hibernators (tenrecs, lemurs) conduct torpor at more intermediate, warm body temperatures, and exhibit large variation in strategies. One strategy that likely enables cold-temperature torpor is control of mRNA and protein synthesis; in ground squirrels this occurs at body temperatures below 18°C. We studied the ground squirrel model as a hibernator that is capable of executing torpor over a range of temperatures, aiming to evaluate physiological and molecular strategies that vary across hot, warm, and cold torpor. We analyzed the transcriptomes of heart, brain, liver, and kidney collected from ground squirrels during the early phase of torpor bouts across a range of ambient temperatures (4, 12, 20, 25, 30°C). Temperature-responsive differential gene expression was compared between euthermic animals (37°C) and torpid animals at each experimental temperature using DESeq2. In the brain, we detected the highest number of differentially expressed genes between the non-hibernating squirrels and those torpid at 25ºC (3425 DE genes; pFDR&lt;0.01). The highest number of differentially expressed genes for heart (2742 genes), liver (3586 genes), and kidney (2233 genes) occurred between non-hibernating animals and those torpid at 20ºC. These results suggest warm torpid temperatures around 20-25ºC induce the largest molecular response, which may indicate cellular dysregulation. Using WGCNA, we produced a consensus network of genes exhibiting correlated differential expression in all four tissues and across all pairwise comparisons between the normothermic control animals and each temperature of torpor; this network contained 488 genes. Pathway analysis within the consensus network demonstrates enrichment of key pathways related to ubiquitination, gene silencing, RNA binding, DNA repair, and alternative splicing. These pathways clearly are central to molecular responses to torpor at any temperature. Elucidating the temperature-specific differences occurring in conjunction with consensus responses will tease apart molecular strategies employed by mammals across wide-ranging hibernation phenotypes. Funded by the Translational Research Institute for Space Health. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Hibernating Golden-Mantled Ground Squirrels (Spermophilus [Callospermophilus] lateralis) have High Inter-individual Variability in Oxygen Consumption Rates with Warmer Ambient Temperatures

Ambient temperature (Ta) is a prominent part of mammalian hibernation. An excellent example of this can be found in the ground squirrels, which hibernate relatively well in cold Tas of &lt;4°C, where extremely low body temperatures (Tb) of -2.9°C and profound metabolic depression (1% of active oxygen consumption rates [VȮ2]) have been reported. These characteristics highlight the importance of initiating and maintaining a decreased Tb to effectively shut down key homeostatic processes. Resumption of such processes occurs during interbout arousals (IBAs), where VȮ2 and Tb return to active (‘euthermic’) values. An important question then is whether these torpor characteristics would continue at warmer Tas, and if so, up to which Ta? Previous research demonstrated an acute uncoupling of translational initiation from elongation precisely at Tb = 18°C in squirrels entering torpor. Given that protein synthesis often accounts for ~30% of VȮ2, one might expect a large change in VȮ2 at ~18-20°C. Further, we hypothesized that squirrels hibernating at warmer temperatures would be more variable as their ability to depress energetically expensive processes was compromised. We hibernated golden-mantled ground squirrels ( Spermophilus [Callospermophilus] lateralis) at Tas of 4, 8, 12, 16, 20, 24, 28, and 30°C and measured VȮ2, carbon dioxide production (V̇CO2), and core Tb. We found hibernation at Ta&gt;20°C caused VȮ2 to become less predictable and more variable. Further, animals had a larger difference between Tb and Ta at higher Tas. We interpret the results to suggest an inability to effectively depress homeostatic processes. These data have profound implications for the engineering of torpor use in humans as it relates to spaceflight. The data suggests that humans will either 1) have to be very cold wherein homeostatic processes are depressed and the person is arrested in a quasi-homeostatic state or 2) be at a higher temperature that provides for adequate homeostasis. NASA-Translational Research Institute for Space Health (TRISH) grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Depressing Ubiquitin-Dependent Proteolysis in a Hibernator Whose Body Temperature Is Normally Variable: The Common Tenrec

In a canonical mammalian hibernator like a ground squirrel, the cold body temperatures (Tb) of the torpid squirrel depresses protein degradation at the level of the 26S proteasome. Ubiquitylation per se is less temperature sensitive as ubiquitylation rates of colder temperatures continue at 30% to 60% of the maximal rates of warmer temperatures. This leads to an accumulation of ubiquitin-conjugated proteins of 2-3 fold in torpid squirrels as compared to active squirrels. The common tenrec is a bizarre Afrotherian placental mammal with many plesiomorphic traits such as a cloaca, internal testes, indeterminate growth, and the smallest brain of all extant mammals with very little neocortex. Interestingly, these tenrecs may be fully active with a Tb of 12°C or hibernating at a Tb of 28°C. Moreover, oxygen consumption rates may vary 25 fold in active tenrecs. We found expected decreases of 26S proteolytic activity at low temperatures. Capacitance for proteolysis varied by state and there was no evidence that torpor per se would arrest protein degradation. Indeed, in sharp contrast to the squirrel, ubiquitylation rates were essentially unaffected by temperature and ranged at colder temperatures from 80% to 90% of the maximal ubiquitylation rates. In other words, the only in vitro evidence for the control of ubiquitin-dependent proteolysis that we found would only be available to very cold tenrecs independent of if they were active or torpid. However, ubiquitin-conjugate concentrations appear low but unaffected by state. All in all, we hypothesize a ‘Life in the Slow Lane’ model for tenrecs wherein an inability to effectively regulate proteolysis led to a modest rate of degradation that is not as affected by changes incurred by torpor state or changes in Tb. NSF and Department of Education (GAANN). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cold resistance of mammalian hibernators ∼ a matter of ferroptosis?

Most mammals adapt thermal physiology around 37°C and large deviations from their range, as observed in severe hypothermia and hyperthermia, resulting in organ dysfunction and individual death. A prominent exception is mammalian hibernation. Mammalian hibernators resist the long-term duration of severe low body temperature that is lethal to non-hibernators, including humans and mice. This cold resistance is supported, at least in part, by intrinsic cellular properties, since primary or immortalized cells from several hibernator species can survive longer than those from non-hibernators when cultured at cold temperatures. Recent studies have suggested that cold-induced cell death fulfills the hallmarks of ferroptosis, a type of necrotic cell death that accompanies extensive lipid peroxidation by iron-ion-mediated reactions. In this review, we summarize the current knowledge of cold resistance of mammalian hibernators at the cellular and molecular levels to organ and systemic levels and discuss key pathways that confer cold resistance in mammals.

Physiological and behavioural adaptations by big brown bats hibernating in dry rock crevices.

Winter energy stores are finite and factors influencing patterns of activity are important for overwintering energetics and survival. Hibernation patterns (e.g., torpor bout duration and arousal frequency) often depend on microclimate, with more stable hibernacula associated with greater energy savings than less stable hibernacula. We monitored hibernation patterns of individual big brown bats (Eptesicus fuscus; Palisot de Beauvois, 1796) overwintering in rock-crevices that are smaller, drier, and less thermally stable than most known cave hibernacula. While such conditions would be predicted to increase arousal frequency in many hibernators, we did not find support for this. We found that bats were insensitive to changes in hibernacula microclimate (temperature and humidity) while torpid. We also found that the probability of arousal from torpor remained under circadian influence, likely because throughout the winter during arousals, bats commonly exit their hibernacula. We calculated that individuals spend most of their energy on maintaining a torpid body temperature a few degrees above the range of ambient temperatures during steady-state torpor, rather than during arousals as is typical of other small mammalian hibernators. Flight appears to be an important winter activity that may expedite the benefits of euthermic periods and allow for short, physiologically effective arousals. Overall, we found that big brown bats in rock crevices exhibit different hibernation patterns than conspecifics hibernating in buildings and caves.

Slow decrease in temperature produces readthrough transcripts in mammalian hibernation

Accumulating evidence suggests that various cellular stresses interfere with the end processing of mRNA synthesis and lead to the production of abnormally long transcripts, known as readthrough transcripts (RTTs), which extend beyond the termination sites. Small mammalian hibernators repeatedly enter a state referred to as deep torpor (DT), where the metabolic rate, respiration rate, and core body temperature become extremely low, which produces various types of cellular stresses and therefore induces RTTs. However, the types of stresses and processes around the DT that cause RTTs are unclear. In the present study, we showed that RTTs are produced from different gene loci in the livers of Syrian hamsters under DT and summer-like conditions. Moreover, in vitro analysis using hamster primary hepatocytes revealed that DT-specific RTTs are induced by a slow decline in temperature, as seen in body temperature in the entrance phase of DT, but not by rapid cold treatment or hypoxia. In addition, it was observed that RTTs were not elongated under a significantly cold temperature (4 °C). These results indicate that DT-specific RTTs are produced during the entrance phase of torpor by a slow decrease in body temperature.

The interaction between gut microbiota and hibernation in mammals.

Hibernation, an evolved survival trait among animals, enables them to endure frigid temperatures and food scarcity during the winter months, and it is a widespread phenomenon observed in mammals. The gut microbiota, a crucial component of animal nutrition and health, exhibits particularly dynamic interactions in hibernating mammals. This manuscript comprehensively evaluates the impacts of fasting, hypothermia, and hypometabolism on the gut microbiota of hibernating mammals. It suggests that alterations in the gut microbiota may contribute significantly to the maintenance of energy metabolism and intestinal immune function during hibernation, mediated by their metabolites. By delving into these intricacies, we can gain a deeper understanding of how hibernating mammals adapt to their environments and the consequences of dietary modifications on the symbiotic relationship between the gut microbiota and the host. Additionally, this knowledge can inform our comprehension of the protective mechanisms underlying long-term fasting in non-hibernating species, including humans, providing valuable insights into nutritional strategies and health maintenance.

Does the expensive brain hypothesis apply to amphibians and reptiles?

Vertebrate brains show extensive variation in relative size. The expensive brain hypothesis argues that one important source of this variation is linked to a species’ ability to generate the energy required to sustain the brain, especially during periods of unavoidable food scarcity. Here we ask whether this hypothesis, tested so far in endothermic vertebrates, also applies to ectotherms, where ambient temperature is an additional major aspect of energy balance. Phylogenetic comparative analyses of reptiles and amphibians support the hypothesis. First, relative brain size increases with higher body temperature in those species active during the day that can gain free energy by basking. Second, relative brain size is smaller among nocturnal species, which generally face less favorable energy budgets, especially when maintaining high body temperature. However, we do not find an effect of seasonal variation in ambient temperature or food on brain size, unlike in endotherms. We conclude that the factors affecting energy balance in ectotherms and endotherms are overlapping but not identical. We therefore discuss the idea that when body temperatures are seasonally very low, cognitive benefits may be thwarted and selection on larger brain size may be rare. Indeed, mammalian hibernators may show similarities to ectotherms.

Weakened Contractile Performance and Mitochondrial Respiratory Complex Activity in Skeletal Muscle Improve during Interbout Arousal in Hibernating Daurian Ground Squirrel, Spermophilus dauricus

Mammalian hibernation is composed of multiple episodes of torpor bout, separated by phases of interbout arousal. During torpor, the skeletal muscles of mammals are undoubtedly inactive, but it has been proven to mitigate disuse atrophy. While interbout arousal has been implicated in the prevention of muscle atrophy, the underlying mechanisms sustaining muscle contraction remain to be explored. In the present study, Daurian ground squirrels (Spermophilus dauricus) were divided into four groups: pre-hibernation (PRE), torpor (TOR), interbout arousal (IBA), and post-hibernation (POST). The contractile performance of slow-twitch soleus muscle (SOL) and fast-twitch extensor digitorum longus muscle (EDL) was detected both in situ and in vitro. Concurrently, mitochondrial respiratory chain complex activity in these muscles was quantified. Our findings revealed that in situ contractile properties of both muscles, including force, power output, time duration, and force development/relaxation rates of twitch contraction, and force and power output of tetanic contraction declined in the TOR group compared to the PRE group, but improved in the IBA and POST groups. Fatigue resistance of muscles, determined by the power output of repetitive tetanic contractions in situ, decreased in the TOR group but recovered in the IBA and POST groups. In vitro studies demonstrated that tetanic contraction power output in isolated muscles increased with muscle temperature in both TOR and IBA groups. However, at the same temperature, power output was consistently lower in the TOR group compared to the IBA group. Moreover, the activity of the mitochondrial respiratory chain complex, especially Complexes I and II, decreased in the TOR group but showed recovery in the IBA and POST groups. These findings suggest that both the contractile performance and fatigue resistance of mammalian skeletal muscle are compromised during torpor but can be improved during interbout arousal and post-hibernation. The rebound in body temperature and rise in mitochondrial respiratory chain complex activity in skeletal muscle are involved in enhancing contractile performance and fatigue resistance. This study suggests that interbout arousal functions as a vital temporal interval during which skeletal muscles can transition from the inactivity induced by torpor to a state of restored contractile functionality. Thus, interbout arousal serves as a behavioral safeguard against disuse-induced damage to skeletal muscles during hibernation.

Spontaneous recurrence of a summer-like diel rhythm in the body temperature of the Syrian hamster after hibernation.

Mammalian hibernation is a survival strategy characterized by metabolic suppression and drastically lowering body temperature (Tb), used during harsh seasons with food shortages and cold. The Syrian hamster commences hibernation in response to a short photoperiod and cold but spontaneously concludes hibernation after several months without environmental cues. Little is known about the changes in diel rhythms during hibernation. Using long-term and high-resolution Tb data, we analysed the diel Tb rhythm time-course changes in Syrian hamsters raised under summer-like conditions (long photoperiod (LP) and warm; LP-warm) and transferred to winter-like conditions (short photoperiod (SP) and cold; SP-cold). The diel Tb rhythm was undetectable during the hibernation period (HIBP), reappearing after the HIBP. The phase of this returning rhythm reverted to the LP entrainment phase characteristics despite the ambient SP and then re-entrained to the ambient SP as if the hamsters were transferred from the LP-warm to SP-cold conditions. The diel Tb rhythm reverted from the SP- to LP-type in a hibernation-dependent manner. Under constant dark and cold conditions, the circadian Tb rhythm recovered without photic stimuli following the HIBP. These findings suggest that hibernation involves a program that anticipates the ambient photoperiod when animals emerge from hibernation.

Integrated transcriptomics and metabolomics reveal protective effects on heart of hibernating Daurian ground squirrels.

Hibernating mammals are natural models of resistance to ischemia, hypoxia-reperfusion injury, and hypothermia. Daurian ground squirrels (spermophilus dauricus) can adapt to endure multiple torpor-arousal cycles without sustaining cardiac damage. However, the molecular regulatory mechanisms that underlie this adaptive response are not yet fully understood. This study investigates morphological, functional, genetic, and metabolic changes that occur in the heart of ground squirrels in three groups: summer active (SA),late torpor (LT),and interbout arousal (IBA).Morphological and functional changes in the heart were measured using hematoxylin-eosin (HE)staining, Masson staining, echocardiography, and enzyme-linked immunosorbent assay (ELISA).Results showed significant changes in cardiac function in the LT group as compared with SA or IBA groups, but no irreversible damage occurred. To understand the molecular mechanisms underlying these phenotypic changes, transcriptomic and metabolomic analyses were conducted to assess differential changes in gene expression and metabolite levels in the three groups of ground squirrels, with a focus on GOand KEGG pathway analysis. Transcriptomic analysis showed that differentially expressed genes were involved in the remodeling of cytoskeletal proteins, reduction in protein synthesis, and downregulation of the ubiquitin-proteasome pathway during hibernation (including LT and IBA groups), as compared with the SA group. Metabolomic analysis revealed increased free amino acids, activation of the glutathione antioxidant system, altered cardiac fatty acid metabolic preferences, and enhanced pentose phosphate pathway activity during hibernation as compared with the SA group. Combining the transcriptomic and metabolomic data, active mitochondrial oxidative phosphorylation and creatine-phosphocreatine energy shuttle systems were observed, as well as inhibition of ferroptosis signaling pathways during hibernation as compared with the SA group. In conclusion, these results provide new insights into cardio-protection in hibernators from the perspective of gene and metabolite changes and deepen our understanding of adaptive cardio-protection mechanisms in mammalian hibernators.

Seasonal variation in glucose and insulin is modulated by food and temperature conditions in a hibernating primate.

Feast-fast cycles allow animals to live in seasonal environments by promoting fat storage when food is plentiful and lipolysis when food is scarce. Fat-storing hibernators have mastered this cycle over a circannual schedule, by undergoing extreme fattening to stockpile fuel for the ensuing hibernation season. Insulin is intrinsic to carbohydrate and lipid metabolism and is central to regulating feast-fast cycles in mammalian hibernators. Here, we examine glucose and insulin dynamics across the feast-fast cycle in fat-tailed dwarf lemurs, the only obligate hibernator among primates. Unlike cold-adapted hibernators, dwarf lemurs inhabit tropical forests in Madagascar and hibernate under various temperature conditions. Using the captive colony at the Duke Lemur Center, we determined fasting glucose and insulin, and glucose tolerance, in dwarf lemurs across seasons. During the lean season, we maintained dwarf lemurs under stable warm, stable cold, or fluctuating ambient temperatures that variably included food provisioning or deprivation. Overall, we find that dwarf lemurs can show signatures of reversible, lean-season insulin resistance. During the fattening season prior to hibernation, dwarf lemurs had low glucose, insulin, and HOMA-IR despite consuming high-sugar diets. In the active season after hibernation, glucose, insulin, HOMA-IR, and glucose tolerance all increased, highlighting the metabolic processes at play during periods of weight gain versus weight loss. During the lean season, glucose remained low, but insulin and HOMA-IR increased, particularly in animals kept under warm conditions with daily food. Moreover, these lemurs had the greatest glucose intolerance in our study and had average HOMA-IR values consistent with insulin resistance (5.49), while those without food under cold (1.95) or fluctuating (1.17) temperatures did not. Remarkably low insulin in dwarf lemurs under fluctuating temperatures raises new questions about lipid metabolism when animals can passively warm and cool rather than undergo sporadic arousals. Our results underscore that seasonal changes in insulin and glucose tolerance are likely hallmarks of hibernating mammals. Because dwarf lemurs can hibernate under a range of conditions in captivity, they are an emerging model for primate metabolic flexibility with implications for human health.

Peptidomic Analysis Reveals Seasonal Neuropeptide and Peptide Hormone Changes in the Hypothalamus and Pituitary of a Hibernating Mammal.

During the winter, hibernating mammals undergo extreme changes in physiology, which allow them to survive several months without access to food. These animals enter a state of torpor, which is characterized by decreased metabolism, near-freezing body temperatures, and a dramatically reduced heart rate. The neurochemical basis of this regulation is largely unknown. Based on prior evidence suggesting that the peptide-rich hypothalamus plays critical roles in hibernation, we hypothesized that changes in specific cell-cell signaling peptides (neuropeptides and peptide hormones) underlie physiological changes during torpor/arousal cycles. To test this hypothesis, we used a mass spectrometry-based peptidomics approach to examine seasonal changes of endogenous peptides that occur in the hypothalamus and pituitary of a model hibernating mammal, the thirteen-lined ground squirrel (Ictidomys tridecemlineatus). In the pituitary, we observed changes in several distinct peptide hormones as animals prepared for torpor in October, exited torpor in March, and progressed from spring (March) to fall (August). In the hypothalamus, we observed an overall increase in neuropeptides in October (pre-torpor), a decrease as the animal entered torpor, and an increase in a subset of neuropeptides during normothermic interbout arousals. Notable changes were observed for feeding regulatory peptides, opioid peptides, and several peptides without well-established functions. Overall, our study provides critical insight into changes in endogenous peptides in the hypothalamus and pituitary during mammalian hibernation that were not available from transcriptomic measurements. Understanding the molecular basis of the hibernation phenotype may pave the way for future efforts to employ hibernation-like strategies for organ preservation, combating obesity, and treatment of stroke.

Mass spectrometry of the white adipose metabolome in a hibernating mammal reveals seasonal changes in alternate fuels and carnitine derivatives.

Mammalian hibernators undergo substantial changes in metabolic function throughout the seasonal hibernation cycle. We report here the polar metabolomic profile of white adipose tissue isolated from active and hibernating thirteen-lined ground squirrels (Ictidomys tridecemlineatus). Polar compounds in white adipose tissue were extracted from five groups representing different timepoints throughout the seasonal activity-torpor cycle and analyzed using hydrophilic interaction liquid chromatography-mass spectrometry in both the positive and negative ion modes. A total of 224 compounds out of 660 features detected after curation were annotated. Unsupervised clustering using principal component analysis revealed discrete clusters representing the different seasonal timepoints throughout hibernation. One-way analysis of variance and feature intensity heatmaps revealed metabolites that varied in abundance between active and torpid timepoints. Pathway analysis compared against the KEGG database demonstrated enrichment of amino acid metabolism, purine metabolism, glycerophospholipid metabolism, and coenzyme A biosynthetic pathways among our identified compounds. Numerous carnitine derivatives and a ketone that serves as an alternate fuel source, beta-hydroxybutyrate (BHB), were among molecules found to be elevated during torpor. Elevated levels of the BHB-carnitine conjugate during torpor suggests the synthesis of beta-hydroxybutyrate in white adipose mitochondria, which may contribute directly to elevated levels of circulating BHB during hibernation.