Identification of shared gene expression programs activated in multiple modes of torpor across vertebrate clades.

Torpor encompasses diverse adaptations to extreme environmental stressors such as hibernation, aestivation, brumation, and daily torpor. Here we introduce StrokeofGenus, an analytic pipeline that identifies distinct transcriptomic states and shared gene expression patterns across studies, tissues, and species. We use StrokeofGenus to study multiple and diverse forms of torpor from publicly-available RNA-seq datasets that span eight species and two classes. We identify three transcriptionally distinct states during the cycle of heterothermia: euthermia, torpor, and interbout arousal. We also identify torpor-specific gene expression patterns that are shared both across tissues and between species with over three hundred million years of evolutionary divergence. We further demonstrate the general sharing of gene expression patterns in multiple forms of torpor, implying a common evolutionary origin for this process. Although here we apply StrokeofGenus to analysis of torpor, it can be used to interrogate any other complex physiological processes defined by transient transcriptomic states.

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.

Mitochondrial microRNA profiles are altered in thirteen-lined ground squirrels (Ictidomys tridecemlineatus) during hibernation.

Thirteen-lined ground squirrels (TLGSs) are obligate hibernators that cycle between torpor (low metabolic rate and body temperature) and interbout euthermia (IBE; typical euthermic body temperature and metabolism) from late autumn to spring. Many physiological changes occur throughout hibernation, including a reduction in liver mitochondrial metabolism during torpor, which is reversed during arousal to interbout euthermia. Nuclear-encoded microRNA (miRNA, small posttranscriptional regulator molecules) differ in abundance throughout TLGS hibernation and have been shown to regulate mitochondrial gene expression in mammalian cell culture (where they are referred to as mitomiRs). This study characterized differences in mitomiR profiles from TLGS liver mitochondria isolated during summer, torpor, and IBE, and predicted their mitochondrial targets. Using small RNA sequencing, differentially abundant mitomiRs were identified between hibernation states, and using quantitative PCR analysis, we quantified the expression of predicted mitochondrial mRNA targets. Most differences in mitomiR abundances were seasonal (i.e., between summer and winter) with only one mitomiR differentially abundant between IBE and torpor. Multiple factor analysis (MFA) revealed three clusters divided by hibernation states, where clustering was predominantly driven by mitomiR abundances. Nine of these differentially abundant mitomiRs had predicted mitochondrial RNA targets, including subunits of electron transfer system complexes I and IV, 12S rRNA, and two tRNAs. Overall, mitomiRs were predicted to suppress the expression of their mitochondrial targets and may have some involvement in regulating protein translation in mitochondria. This study found differences in mitomiR abundances between seasons and hibernation states of TLGS and suggests potential mechanisms for regulating the mitochondrial electron transfer system.NEW & NOTEWORTHY During the hibernation season, thirteen-lined ground squirrels periodically increase metabolism remarkably between torpor and interbout euthermia (IBE). This process involves rapid reactivation of mitochondrial respiration. We predicted that mitochondrial microRNA (mitomiRs) might be altered during this response. We found that the abundance of 38 liver mitomiRs differs based on hibernation state (summer, IBE, and torpor). Small RNA sequencing identified mitomiR profiles, including some mitomiRs that are predicted to bind to mitochondrial RNAs.

Effects of hibernation on two important contractile tissues in tibetan frogs, Nanorana parkeri: a perspective from transcriptomics and metabolomics approaches.

BACKGROUND: In response to seasonal cold and food shortage, the Xizang plateau frogs, Nanorana parkeri (Anura: Dicroglossidae), enter a reversible hypometabolic state where heart rate and oxygen consumption in skeletal muscle are strongly suppressed. However, the effect of winter hibernation on gene expression and metabolic profiling in these two tissues remains unknown. In the present study, we conducted transcriptomic and metabolomic analyses of heart and skeletal muscle from summer- and winter-collected N. parkeri to explore mechanisms involved in seasonal hibernation. RESULTS: We identified 2407 differentially expressed genes (DEGs) in heart and 2938 DEGs in skeletal muscle. Enrichment analysis showed that shared DEGs in both tissues were enriched mainly in translation and metabolic processes. Of these, the expression of genes functionally categorized as "response to stress", "defense mechanisms", or "muscle contraction" were particularly associated with hibernation. Metabolomic analysis identified 24 and 22 differentially expressed metabolites (DEMs) in myocardium and skeletal muscle, respectively. In particular, pathway analysis showed that DEMs in myocardium were involved in the pentose phosphate pathway, glycerolipid metabolism, pyruvate metabolism, citrate cycle (TCA cycle), and glycolysis/gluconeogenesis. By contrast, DEMs in skeletal muscle were mainly involved in amino acid metabolism. CONCLUSIONS: In summary, natural adaptations of myocardium and skeletal muscle in hibernating N. parkeri involved transcriptional alterations in translation, stress response, protective mechanisms, and muscle contraction processes as well as metabolic remodeling. This study provides new insights into the transcriptional and metabolic adjustments that aid winter survival of high-altitude frogs N. parkeri.

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.

c-fos induction in the choroid plexus, tanycytes and pars tuberalis is an early indicator of spontaneous arousal from torpor in a deep hibernator.

Hibernation is an extreme state of seasonal energy conservation, reducing metabolic rate to as little as 1% of the active state. During the hibernation season, many species of hibernating mammals cycle repeatedly between the active (aroused) and hibernating (torpid) states (T-A cycling), using brown adipose tissue (BAT) to drive cyclical rewarming. The regulatory mechanisms controlling this process remain undefined but are presumed to involve thermoregulatory centres in the hypothalamus. Here, we used the golden hamster (Mesocricetus auratus), and high-resolution monitoring of BAT, core body temperature and ventilation rate, to sample at precisely defined phases of the T-A cycle. Using c-fos as a marker of cellular activity, we show that although the dorsomedial hypothalamus is active during torpor entry, neither it nor the pre-optic area shows any significant changes during the earliest stages of spontaneous arousal. Contrastingly, in three non-neuronal sites previously linked to control of metabolic physiology over seasonal and daily time scales - the choroid plexus, pars tuberalis and third ventricle tanycytes - peak c-fos expression is seen at arousal initiation. We suggest that through their sensitivity to factors in the blood or cerebrospinal fluid, these sites may mediate metabolic feedback-based initiation of the spontaneous arousal process.

Comparative transcriptomic evidence of physiological changes and potential relationships in vertebrates under different dormancy states.

Dormancy represents a fascinating adaptive strategy for organisms to survive in unforgiving environments. After a period of dormancy, organisms often exhibit exceptional resilience. This period is typically divided into hibernation and aestivation based on seasonal patterns. However, the mechanisms by which organisms adapt to their environments during dormancy, as well as the potential relationships between different states of dormancy, deserve further exploration. Here, we selected Perccottus glenii and Protopterus annectens as the primary subjects to study hibernation and aestivation, respectively. Based on histological and transcriptomic analysis of multiple organs, we discovered that dormancy involved a coordinated functional response across organs. Enrichment analyses revealed noteworthy disparities between the two dormant species in their responses to extreme temperatures. Notably, similarities in gene expression patterns pertaining to energy metabolism, neural activity, and biosynthesis were noted during hibernation, suggesting a potential correlation between hibernation and aestivation. To further explore the relationship between these two phenomena, we analyzed other dormancy-capable species using data from publicly available databases. This comparative analysis revealed that most orthologous genes involved in metabolism, cell proliferation, and neural function exhibited consistent expression patterns during dormancy, indicating that the observed similarity between hibernation and aestivation may be attributable to convergent evolution. In conclusion, this study enhances our comprehension of the dormancy phenomenon and offers new insights into the molecular mechanisms underpinning vertebrate dormancy.

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.

Adapting to stress: The effects of hibernation and hibernacula temperature on the hepatic transcriptome of Rhinolophus pusillus.

Hibernation, a survival strategy in mammals for extreme climates, induces physiological phenomena such as ischemia-reperfusion and metabolic shifts that hold great potential for advancements in modern medicine. Despite this, the molecular mechanisms underpinning hibernation remain largely unclear. This study used RNA-seq and Iso-seq techniques to investigate the changes in liver transcriptome expression of Rhinolophus pusillus during hibernation and active periods, as well as under different microhabitat temperatures. We identified 11 457 differentially expressed genes during hibernation and active periods, of which 395 showed significant differential expression. Genes associated with fatty acid catabolism were significantly upregulated during hibernation, whereas genes related to carbohydrate metabolism and glycogen synthesis were downregulated. Conversely, immune-related genes displayed differential expression patterns: genes tied to innate immunity were significantly upregulated, while those linked to adaptive immunity and inflammatory response were downregulated. The analysis of transcriptomic data obtained from different microhabitat temperatures revealed that R. pusillus exhibited an upregulation of genes associated with lipid metabolism in lower microhabitat temperature. This upregulation facilitated an enhanced utilization rate of triglyceride, ultimately resulting in increased energy provision for the organism. Additionally, R. pusillus upregulated gluconeogenesis-related genes regardless of the microhabitat temperature, demonstrating the importance of maintaining blood glucose levels during hibernation. Our transcriptomic data reveal that these changes in liver gene expression optimize energy allocation during hibernation, suggesting that liver tissue adaptively responds to the inherent stress of its function during hibernation. This study sheds light on the role of differential gene expression in promoting more efficient energy allocation during hibernation. It contributes to our understanding of how liver tissue adapts to the stressors associated with this state.

Comparative transcriptomics of the garden dormouse hypothalamus during hibernation.

Torpor or heterothermy is an energy-saving mechanism used by endotherms to overcome harsh environmental conditions. During winter, the garden dormouse (Eliomys quercinus) hibernates with multiday torpor bouts and body temperatures of a few degrees Celsius, interrupted by brief euthermic phases. This study investigates gene expression within the hypothalamus, the key brain area controlling energy balance, adding information on differential gene expression potentially relevant to orchestrate torpor. A de novo assembled transcriptome of the hypothalamus was generated from garden dormice hibernating under constant darkness without food and water at 5 °C. Samples were collected during early torpor, late torpor, and interbout arousal. During early torpor, 765 genes were differentially expressed as compared with interbout arousal. Twenty-seven pathways were over-represented, including pathways related to hemostasis, extracellular matrix organization, and signaling of small molecules. Only 82 genes were found to be differentially expressed between early and late torpor, and no pathways were over-represented. During late torpor, 924 genes were differentially expressed relative to interbout arousal. Despite the high number of differentially expressed genes, only 10 pathways were over-represented. Of these, eight were also observed to be over-represented when comparing early torpor and interbout arousal. Our results are largely consistent with previous findings in other heterotherms. The addition of a transcriptome of a novel species may help to identify species-specific and overarching torpor mechanisms through future species comparisons.

Circadian gene transcription plays a role in cellular metabolism in hibernating brown bears, Ursus arctos.

Hibernation is a highly seasonal physiological adaptation that allows brown bears (Ursus arctos) to survive extended periods of low food availability. Similarly, daily or circadian rhythms conserve energy by coordinating body processes to optimally match the environmental light/dark cycle. Brown bears express circadian rhythms in vivo and their cells do in vitro throughout the year, suggesting that these rhythms may play important roles during periods of negative energy balance. Here, we use time-series analysis of RNA sequencing data and timed measurements of ATP production in adipose-derived fibroblasts from active and hibernation seasons under two temperature conditions to confirm that rhythmicity was present. Culture temperature matching that of hibernation body temperature (34 °C) resulted in a delay of daily peak ATP production in comparison with active season body temperatures (37 °C). The timing of peaks of mitochondrial gene transcription was altered as were the amplitudes of transcripts coding for enzymes of the electron transport chain. Additionally, we observed changes in mean expression and timing of key metabolic genes such as SIRT1 and AMPK which are linked to the circadian system and energy balance. The amplitudes of several circadian gene transcripts were also reduced. These results reveal a link between energy conservation and a functioning circadian system in 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 GO and 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.

Blood transcriptomics mirror regulatory mechanisms during hibernation-a comparative analysis of the Djungarian hamster with other mammalian species.

Hibernation enables many species of the mammalian kingdom to overcome periods of harsh environmental conditions. During this physically inactive state metabolic rate and body temperature are drastically downregulated, thereby reducing energy requirements (torpor) also over shorter time periods. Since blood cells reflect the organism´s current condition, it was suggested that transcriptomic alterations in blood cells mirror the torpor-associated physiological state. Transcriptomics on blood cells of torpid and non-torpid Djungarian hamsters and QIAGEN Ingenuity Pathway Analysis (IPA) revealed key target molecules (TMIPA), which were subjected to a comparative literature analysis on transcriptomic alterations during torpor/hibernation in other mammals. Gene expression similarities were identified in 148 TMIPA during torpor nadir among various organs and phylogenetically different mammalian species. Based on TMIPA, IPA network analyses corresponded with described inhibitions of basic cellular mechanisms and immune system-associated processes in torpid mammals. Moreover, protection against damage to the heart, kidney, and liver was deduced from this gene expression pattern in blood cells. This study shows that blood cell transcriptomics can reflect the general physiological state during torpor nadir. Furthermore, the understanding of molecular processes for torpor initiation and organ preservation may have beneficial implications for humans in extremely challenging environments, such as in medical intensive care units and in space.

Feeding during hibernation shifts gene expression toward active season levels in brown bears (Ursus arctos).

Hibernation in bears involves a suite of metabolical and physiological changes, including the onset of insulin resistance, that are driven in part by sweeping changes in gene expression in multiple tissues. Feeding bears glucose during hibernation partially restores active season physiological phenotypes, including partial resensitization to insulin, but the molecular mechanisms underlying this transition remain poorly understood. Here, we analyze tissue-level gene expression in adipose, liver, and muscle to identify genes that respond to midhibernation glucose feeding and thus potentially drive postfeeding metabolical and physiological shifts. We show that midhibernation feeding stimulates differential expression in all analyzed tissues of hibernating bears and that a subset of these genes responds specifically by shifting expression toward levels typical of the active season. Inferences of upstream regulatory molecules potentially driving these postfeeding responses implicate peroxisome proliferator-activated receptor gamma (PPARG) and other known regulators of insulin sensitivity, providing new insight into high-level regulatory mechanisms involved in shifting metabolic phenotypes between hibernation and active states.

A multi-tissue gene expression dataset for hibernating brown bears.

OBJECTIVES: Complex physiological adaptations often involve the coordination of molecular responses across multiple tissues. Establishing transcriptomic resources for non-traditional model organisms with phenotypes of interest can provide a foundation for understanding the genomic basis of these phenotypes, and the degree to which these resemble, or contrast, those of traditional model organisms. Here, we present a one-of-a-kind gene expression dataset generated from multiple tissues of two hibernating brown bears (Ursus arctos). DATA DESCRIPTION: This dataset is comprised of 26 samples collected from 13 tissues of two hibernating brown bears. These samples were collected opportunistically and are typically not possible to attain, resulting in a highly unique and valuable gene expression dataset. In combination with previously published datasets, this new transcriptomic resource will facilitate detailed investigation of hibernation physiology in bears, and the potential to translate aspects of this biology to treat human disease.

Heat shock factor 1 induces a short burst of transcription of the clock gene Per2 during interbout arousal in mammalian hibernation.

During winter hibernation, a diverse range of small mammals can enter prolonged torpor. They spend the nonhibernation season as a homeotherm but the hibernation season as a heterotherm. In the hibernation season, chipmunks (Tamias asiaticus) cycle regularly between 5 and 6 days-long deep torpor with a body temperature (Tb) of 5 to 7 °C and interbout arousal of ∼20 h, during which, their Tb returns to the normothermic level. Here, we investigated Per2 expression in the liver to elucidate the regulation of the peripheral circadian clock in a mammalian hibernator. In the nonhibernation season, as in mice, heat shock factor 1, activated by elevated Tb during the wake period, activated Per2 transcription in the liver, which contributed to synchronizing the peripheral circadian clock to the Tb rhythm. In the hibernation season, we determined that the Per2 mRNA was at low levels during deep torpor, but Per2 transcription was transiently activated by heat shock factor 1, which was activated by elevated Tb during interbout arousal. Nevertheless, we found that the mRNA from the core clock gene Bmal1 exhibited arrhythmic expression during interbout arousal. Since circadian rhythmicity is dependent on negative feedback loops involving the clock genes, these results suggest that the peripheral circadian clock in the liver is nonfunctional in the hibernation season.

Adrenal gene expression dynamics support hibernation in 13-lined ground squirrels.

Hibernation is a natural model of extreme physiology in a mammal. Throughout winter, small hibernators repeatedly undergo rapid, dramatic swings in body temperature, perfusion, and oxygen delivery. To gain insight into the molecular mechanisms that support homeostasis despite the numerous challenges posed by this dynamic physiology, we collected 13-lined ground squirrel adrenal glands from at least five individuals representing six key timepoints across the year using body temperature telemetry. Differentially expressed genes were identified using RNA-seq, revealing both strong seasonal and torpor-arousal cycle effects on gene expression. Two novel findings emerge from this study. First, transcripts encoding multiple genes involved in steroidogenesis decreased seasonally. Taken together with morphometric analyses, the data are consistent with preservation of mineralocorticoids but suppression of glucocorticoid and androgen output throughout winter hibernation. Second, a temporally orchestrated, serial gene expression program unfolds across the brief arousal periods. This program initiates during early rewarming with the transient activation of a set of immediate early response (IER) genes, comprised of both transcription factors and the RNA degradation proteins that assure their rapid turnover. This pulse in turn activates a cellular stress response program to restore proteostasis comprised of protein turnover, synthesis, and folding machinery. These and other data support a general model for gene expression across the torpor-arousal cycle that is facilitated in synchrony with whole body temperature shifts; induction of the immediate early response upon rewarming activates a proteostasis program followed by a restored tissue-specific gene expression profile enabling renewal, repair, and survival of the torpid state.NEW & NOTEWORTHY This pioneer study of adrenal gland gene expression dynamics in hibernating ground squirrels leverages the power of RNA-seq on multiple precisely timed samples to demonstrate: 1) steroidogenesis is seasonally reorganized to preserve aldosterone at the expense of glucocorticoids and androgens throughout winter hibernation; 2) a serial gene expression program unfolds during each short arousal whereby immediate early response genes induce the gene expression machinery that restores proteostasis and the cell-specific expression profile before torpor reentry.

Temporal dynamics of the bat wing transcriptome: Insight into gene-expression changes that enable protection against pathogen.

Skin acts as a mechanical barrier between the body and its surrounding environment and plays an important role in resistance to pathogens. However, we still know little regarding skin responses to physiological changes, particularly with regard to responses against potential pathogens. We herein executed RNA-seq on the wing of the Rhinolophus ferrumequinum to assess gene-expression variations at four physiological stages: pre-hibernation, hibernation (early-hibernation and late-hibernation), and post-hibernation, as well as the gene-expression patterns of infected and uninfected bats with the Pseudogymnoascus destructans (Pd). Our results showed that a greater number of differentially expressed genes between the more disparate physiological stages. Functional enrichment analysis showed that the down-regulated response pathways in hibernating bats included phosphorus metabolism and immune response, indicating metabolic suppression and decreased whole immune function. We also found up-regulated genes in post-hibernating bats that included C-type lectin receptor signalling, Toll-like receptor signalling pathway, and cell adhesion, suggesting that the immune response and skin integrity of the wing were improved after bats emerged from their hibernation and that this facilitated clearing Pd from the integument. Additionally, we found that the genes involved in cytokine or chemokine activity were up-regulated in late-hibernation compared to early-hibernation and that FOSB regulation of immune cell activation was differentially expressed in bats infected with Pd during late-hibernation, implying that the host's innate immune function was enhanced during late-hibernation so as to resist pathogenic infection. Our findings highlight the concept that maintenance of intrinsic immunity provides protection against pathogenic infections in highly resistant bats.

Epigenetic changes between the active and torpid states in the greater horseshoe bat (Rhinolophus ferrumequinum).

Dynamic epigenetic changes during hibernation occur in some hibernating rodents, but these changes are poorly understood in hibernating bats. Populations of the greater horseshoe bat (Rhinolophus ferrumequinum) in north China hibernate and provide an opportunity to study how epigenetic markers and modifiers differ in the active and torpid states of a chiropteran. We used fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) and qRT-PCR techniques to determine changes in the global DNA methylation levels and mRNA expression levels of methylation-related proteins. These included DNA methyltransferase (DNMTs), methyl-CpG-binding proteins (MBPs, including MBDs, UHRFs, and zinc-finger protein family) in active and torpid R. ferrumequinum. In the torpid state, both the relative global methylation and the relative mRNA expression levels of some DNMTs and MBPs, including dnmt3b and zbtb4, increased significantly compared to the expression levels of these in the active state. These changes may involve methylation or assist in regulation of a particular subset of genes according to hibernation status. This indicates that epigenetic mechanisms may exist and facilitate the hibernation process of R. ferrumequinum.

Differential bone metabolism and protein expression in mice fed a high-fat diet versus Daurian ground squirrels following natural pre-hibernation fattening.

This study compared the effects on bone metabolism and morphology of pathological obesity induced by excessive fat intake in a non-hibernator (mice) versus healthy obesity due to pre-hibernation fattening in a hibernator (ground squirrels). Kunming mice were fed a high-fat diet to provide a model of pathological obesity (OB group). Daurian ground squirrels fattened naturally in their pre-hibernation season (PRE group) were used as a healthy obesity model. Micro-computed tomography (micro-CT) and three-point bending tests were used to determine the microstructure and mechanical properties of bone. Western blots were used to analyze protein expression levels related to bone metabolism (Runt-related transcription factor 2 (RunX2), osteocalcin (OCN), alkaline phosphatase (ALP), osteoprotegerin (OPG), receptor activator of nuclear factor-|κB ligand (RANKL), cathepsin K, matrix metallopeptidase 9 (MMP9), patched protein homolog 1 (Ptch1), phosphorylated ß|-|catenin (P|-|ß|-|catenin), and glycogen synthase kinase-3ß (GSK-3ß)). Compared with controls, there was no obvious bone loss in the OB mice, and the stiffness of the femur was increased significantly. Compared with summer active squirrels, bone formation was enhanced but the mechanical properties did not change in the PRE group squirrels. In OB mice, western blots showed significantly increased expression levels of all proteins except RunX2, OPG, and Ptch1. PRE ground squirrels showed significantly increased expression of most proteins except OCN and Ptch1, which decreased significantly, and P|-|ß|-|catenin and OPG, which did not change. In conclusion, for non-hibernating mice, moderate obesity had a certain protective effect on bones, demonstrating two-way regulation, increasing both bone loss and bone formation. For pre-hibernating ground squirrels, the healthy obesity acquired before hibernation had a positive effect on the microstructure of bones, and also enhanced the expression levels of proteins related to bone formation, bone resorption, and Wnt signaling.