Effects of the anti-inflammatory drug budesonide on the gut microbiota and cytokine production of 13-lined ground squirrels during pre-hibernation fattening.

The gut microbiome is essential for maintaining organismal health. Gut microbiota may be disrupted through external factors like dietary change, which can lead to gut inflammation resulting in obesity. Hibernating mammals develop low-grade gut inflammation when they accumulate fat deposits in preparation for hibernation, making them useful models for studying the relationship between the microbiome, inflammation, and weight gain. Nonsteroidal anti-inflammatory drugs and steroids are commonly used in humans to target gut inflammation, but how these drugs affect the gut microbiome and its stability is unclear. We investigated the effect of the glucocorticoid drug budesonide on the gut microbiome and cytokine levels of an obligate hibernator, the 13-lined ground squirrel, during the fattening season. We used 16S rRNA gene sequencing to characterize bacterial communities in the lumen and mucosa of the cecum and colon and measured pro-inflammatory (TNF-α/ IL-6) and anti-inflammatory (IL-10) cytokine levels. Budesonide affected the microbiome only in the cecum lumen, where bacterial diversity was higher in the control group and communities significantly differed between treatments. Across gut sections, Marvinbrianthia and Enterococcus were significantly higher in the budesonide group while Sarcina was higher in the control group. TNF-α and IL-6 levels were higher in control squirrels compared to the budesonide group, but there was no difference in IL-10 levels. Overall, budesonide treatment affected the microbial community and diversity of 13-lined ground squirrels in the cecum lumen. Our study presents another step toward developing ground squirrels as a model for studying the interaction between the microbiota and host inflammation.

Remodeling of skeletal muscle myosin metabolic states in hibernating mammals.

Hibernation is a period of metabolic suppression utilized by many small and large mammal species to survive during winter periods. As the underlying cellular and molecular mechanisms remain incompletely understood, our study aimed to determine whether skeletal muscle myosin and its metabolic efficiency undergo alterations during hibernation to optimize energy utilization. We isolated muscle fibers from small hibernators, Ictidomys tridecemlineatus and Eliomys quercinus and larger hibernators, Ursus arctos and Ursus americanus. We then conducted loaded Mant-ATP chase experiments alongside X-ray diffraction to measure resting myosin dynamics and its ATP demand. In parallel, we performed multiple proteomics analyses. Our results showed a preservation of myosin structure in U. arctos and U. americanus during hibernation, whilst in I. tridecemlineatus and E. quercinus, changes in myosin metabolic states during torpor unexpectedly led to higher levels in energy expenditure of type II, fast-twitch muscle fibers at ambient lab temperatures (20 °C). Upon repeating loaded Mant-ATP chase experiments at 8 °C (near the body temperature of torpid animals), we found that myosin ATP consumption in type II muscle fibers was reduced by 77-107% during torpor compared to active periods. Additionally, we observed Myh2 hyper-phosphorylation during torpor in I. tridecemilineatus, which was predicted to stabilize the myosin molecule. This may act as a potential molecular mechanism mitigating myosin-associated increases in skeletal muscle energy expenditure during periods of torpor in response to cold exposure. Altogether, we demonstrate that resting myosin is altered in hibernating mammals, contributing to significant changes to the ATP consumption of skeletal muscle. Additionally, we observe that it is further altered in response to cold exposure and highlight myosin as a potentially contributor to skeletal muscle non-shivering thermogenesis.

Many animals use hibernation as a tactic to survive harsh winters. During this dormant, inactive state, animals reduce or limit body processes, such as heart rate and body temperature, to minimise their energy use. To conserve energy during hibernation, animals can use different approaches. For example, garden dormice undergo periodic states of extremely low core temperatures (down to 4­8^oC); whereas Eurasian brown bears see milder temperature drops (down to 23­25^oC). An important organ that changes during hibernation is skeletal muscle. Skeletal muscle typically uses large amounts of energy, making up around 50% of body mass. To survive, hibernating animals must change how their skeletal muscle uses energy. Traditionally, active myosin ­ a protein found in muscles that helps muscles to contract ­ was thought to be responsible for most of the energy use by skeletal muscle. But, more recently, resting myosin has also been found to use energy when muscles are relaxed. Lewis et al. studied myosin and skeletal muscle energy use changes during hibernation and whether they could impact the metabolism of hibernating animals. Lewis et al. assessed myosin changes in muscle samples from squirrels, dormice and bears during hibernation and during activity. Experiments showed changes in resting myosin in squirrels and dormice (whose temperature drops to 4­8^oC during hibernation) but not in bears. Further analysis revealed that cooling samples from non-hibernating muscle to 4­8^oC increased energy use in resting myosin, thereby generating heat. However, no increase in energy use was found after cooling hibernating muscle samples to 4­8^oC. This suggest that resting myosin generates heat at cool temperatures ­ a mechanism that is switched off in hibernating animals to allow them to cool their body temperature. These findings reveal key insights into how animals conserve energy during hibernation. In addition, the results show that myosin regulates energy use in skeletal muscles, which indicates myosin may be a potential drug target in metabolic diseases, such as obesity.

Changes in microRNA expression related to ischemia-reperfusion injury in the kidney of the thirteen-lined ground squirrel during torpor.

During the hibernation season, the thirteen-lined ground squirrel undergoes cyclical torpor and arousal periods. The decrease and restoration of metabolic rate and oxygen delivery during torpor and arousal, respectively, may cause reperfusion-ischemia injury in the kidneys. In order to maintain the structural integrity of the kidneys necessary for renal function resumption during arousal, the thirteen-lined ground squirrel has developed adaptive methods to prevent and repair kidney injury. In this present study, computational methods were used to clean and analyze sequenced kidney RNA samples. Significantly differentially expressed microRNAs and enriched gene sets were also determined. From the gene set analysis, the results showed an increase in ubiquitin-related processes and p53 signaling pathways which suggested the occurrence of kidney damage during torpor. There was also an observed increase in cell cycle processes and the anchoring junction cellular compartment which may lend to the prevention of kidney injury. From the differentially expressed microRNAs, miR-27a (log2FC = 1.639; p-value = 0.023), miR-129 (log2FC = 2.516; p-value = 0.023), miR-let-7b (log2FC = 2.360; p-value = 0.025), miR-let-7c (log2FC = 2.291; p-value = 0.037) and miR-let-7i (log2FC = 1.564; p-value = 0.039) were found to be significantly upregulated. These biochemical adaptations may allow the thirteen-lined ground squirrel to maintain kidney structure and function during hibernation.

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.

Peripheral circadian gene activity is altered during hibernation in the thirteen-lined ground squirrel.

Many small mammals living in seasonally cold environments rely on hibernation, utilizing strong metabolic rate suppression and a slow consumption of adipose reserves to survive the winter months. The circannual rhythm of hibernation is well known but less is known about the role of the circadian clock while animals are in torpor for weeks at a time. We hypothesized that due to strong global suppression of transcription and translation in the torpid state, that circadian clock activity would likewise be suppressed in peripheral tissues during hibernation. However, the present study indicates that peripheral circadian clock activity persists during torpor. Using 13-lined ground squirrels (Ictidomys tridecemlineatus) as the model, this study analyzed transcript and protein responses by clock components, comparing euthermic control animals with squirrels in deep torpor for >3 days (subcutaneous body temperature 5-8 °C). The data show tissue specific responses by mRNA transcript levels: (a) no significant changes in transcript abundance in liver of control versus torpid squirrels, (b) a strong increase in Nr1d1 levels in white adipose during torpor, and (c) five significant transcript changes in skeletal muscle during torpor (increased Bmal1, Clock, Cry1 and Nr1d1 but decreased Per1). Levels of core clock proteins (BMAL1, CRY2, PER2, and casein kinases CK1δ and CK1ε) were also assessed across five time points of the torpor/arousal cycle showing both tissue- and time-dependent changes in clock proteins that were most prominent in liver and white adipose and indicating that peripheral clocks are still active in tissues over the torpor/arousal cycle.

Gut microbiome is affected by gut region but robust to host physiological changes in captive active-season ground squirrels.

BACKGROUND: Thirteen-lined ground squirrels (Ictidomys tridecemlineatus) are obligate hibernators and are only active 4-5 months annually. During this period, squirrels rapidly acquire fat for use during hibernation. We investigated how the gut microbiome changed over the active season in the mucosa and lumen of two gut sections: the cecum and ileum. We sequenced the 16S rRNA gene to assess diversity and composition of the squirrel gut microbiome and used differential abundance and network analyses to identify relationships among gut sections. RESULTS: Microbial composition significantly differed between the cecum and ileum, and within the ileum between the mucosa and lumen. Cecum mucosa and lumen samples did not differ in alpha diversity and composition, and clustered by individual squirrel. Ileum mucosa and lumen samples differed in community composition, which can likely be attributed to the transient nature of food-associated bacteria in the lumen. We did not detect a shift in microbiome diversity and overall composition over the duration of the active season, indicating that the squirrel microbiome may be relatively robust to changes in physiology. CONCLUSIONS: Overall, we found that the 13-lined ground squirrel microbiome is shaped by microenvironment during the active season. Our results provide baseline data for new avenues of research, such as investigating potential differences in microbial function among these physiologically unique gut environments.

Liver Transcriptome Dynamics During Hibernation Are Shaped by a Shifting Balance Between Transcription and RNA Stability.

Hibernators dramatically lower metabolism to save energy while fasting for months. Prolonged fasting challenges metabolic homeostasis, yet small-bodied hibernators emerge each spring ready to resume all aspects of active life, including immediate reproduction. The liver is the body's metabolic hub, processing and detoxifying macromolecules to provide essential fuels to brain, muscle and other organs throughout the body. Here we quantify changes in liver gene expression across several distinct physiological states of hibernation in 13-lined ground squirrels, using RNA-seq to measure the steady-state transcriptome and GRO-seq to measure transcription for the first time in a hibernator. Our data capture key timepoints in both the seasonal and torpor-arousal cycles of hibernation. Strong positive correlation between transcription and the transcriptome indicates that transcriptional control dominates the known seasonal reprogramming of metabolic gene expression in liver for hibernation. During the torpor-arousal cycle, however, discordance develops between transcription and the steady-state transcriptome by at least two mechanisms: 1) although not transcribed during torpor, some transcripts are unusually stable across the torpor bout; and 2) unexpectedly, on some genes, our data suggest continuing, slow elongation with a failure to terminate transcription across the torpor bout. While the steady-state RNAs corresponding to these read through transcripts did not increase during torpor, they did increase shortly after rewarming despite their simultaneously low transcription. Both of these mechanisms would assure the immediate availability of functional transcripts upon rewarming. Integration of transcriptional, post-transcriptional and RNA stability control mechanisms, all demonstrated in these data, likely initiate a serial gene expression program across the short euthermic period that restores the tissue and prepares the animal for the next bout of torpor.

Suppression of mitochondrial respiration by hydrogen sulfide in hibernating 13-lined ground squirrels.

Hibernating mammals may suppress their basal metabolic rate during torpor by up to 95% to reduce energy expenditure during winter, but the underlying mechanisms remain poorly understood. Here we show that hydrogen sulfide (H2S), a ubiquitous signaling molecule, is a powerful inhibitor of respiration of liver mitochondria isolated from torpid 13-lined ground squirrels, but has a weak effect on mitochondria isolated during summer and hibernation arousals, where metabolic rate is normal. Consistent with these in vitro effects, we find strong seasonal variations of in vivo levels of H2S in plasma and increases of H2S levels in the liver of squirrels during torpor compared to levels during arousal and summer. The in vivo changes of liver H2S levels correspond with low activity of the mitochondrial H2S oxidizing enzyme sulfide:quinone oxidoreductase (SQR) during torpor. Taken together, these results suggest that during torpor, H2S accumulates in the liver due to a low SQR activity and contributes to inhibition of mitochondrial respiration, while during arousals and summer these effects are reversed, H2S is degraded by active SQR and mitochondrial respiration rates increase. This study provides novel insights into mechanisms underlying mammalian hibernation, pointing to SQR as a key enzyme involved in the control of mitochondrial function.

Markers of tissue remodeling and inflammation in the white and brown adipose tissues of a model hibernator.

The thirteen-lined ground squirrel is a model fat-storing hibernator that nearly doubles its weight in the fall to fuel metabolism with triglycerides throughout the winter months. Hibernator brown and white adipose tissue (BAT, WAT) are important to study in terms of their inflammatory profile and tissue remodeling mechanisms since controlled and natural regulation of these processes could inform new pharmacological interventions that limit oxidative stress and inflammation in the adipose tissues of humans suffering from obesity, promote non-shivering thermogenesis-mediated weight loss, or prevent tissue damage in transplantable organs emerging from cold-storage. Thus, markers of inflammation like cytokines and soluble receptors and tissue remodeling proteins such as matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) were investigated in normothermic, torpid, and arousing ground squirrels. Multiplex protein assays and western blotting revealed fewer changes in WAT compared to BAT. Pro-inflammatory IL-1α levels increased during torpor and soluble epidermal growth factor receptor protein levels increased during arousal in BAT. Given their known roles in other model systems, these proteins could regulate processes like adipogenesis, lipid catabolism, or cell motility. Decreased TIMP2 levels combined with maintained MMP2 or MMP3 protein levels suggested that BAT may avoid tissue remodeling until arousal. No changes in WAT inflammatory cytokines or soluble receptors as well as decreased MMP2 levels during torpor and arousal suggested inflammation and modification to the extracellular matrix is likely suppressed in WAT. This study emphasizes the fat-but-fit nature of the hibernating ground squirrel and the ability of its fat stores to suppress inflammation.

MicroRNA expression patterns in the brown fat of hibernating 13-lined ground squirrels.

The sequence diversity of microRNAs (miRNAs) allows these potent regulators of mRNA fate to bind multiple transcripts, giving them the power to inhibit diverse cellular processes. Therefore, miRNAs may regulate metabolic rate suppression (also termed torpor), an adaptation used by capable species to reduce energy expenditure, minimize tissue damage, and prolong life. Small RNA-sequencing of brown fat from control (37 °C) and torpid (5-8 °C) ground squirrels revealed a central role for miRNAs in torpor. Unsupervised clustering analysis of all 319 conserved miRNAs showed separation of control and torpor samples, which was supported by PCA analysis. Of the 76 miRNAs that were differentially expressed, 45 were upregulated during torpor. KEGG and GO analyses suggested these miRNAs inhibit genes within the ribosome, oxidative phosphorylation, and glycolysis/gluconeogenesis pathways. Some of the most downregulated miRNAs (miR-1-3p, miR-206 and miR-133a/b) had significant Pearson correlation coefficients, suggesting these myomiRs may be co-expressed in control animals. Only 3 of the 16 enriched KEGG pathways were less targeted by miRNAs during torpor, including cytokine-cytokine receptor interactions and the coagulation and complement cascades, suggesting epigenetic or post-translation modifications may inhibit these potentially damaging processes. Alternatively, their activation could promote damage sensing, wound repair, and improve tissue homeostasis. Overall, miRNA-seq analysis of brown fat revealed a strong role for miRNAs in the downregulation of central metabolic processes necessary for MRS, and highlighted miRNAs that could be inhibited by antagomiRs to promote brown fat activity in potential obesity treatments, or that could be used to replicate torpor in non-hibernating mammals.

Elevated ambient temperature accelerates aspects of torpor phenology in an obligate hibernator.

The thirteen-lined ground squirrel (Ictidomys tridecemlineatus) is assumed to be an obligate hibernator - commencing and terminating hibernation on a circannual rhythm, regardless of environmental conditions - but, until now, this assumption had never been fully tested. We housed three groups of captive-born ground squirrels from Aug. 2017 to Aug. 2018 under constant photoperiod (12 h L:12 h D) at 5, 16 or 25 °C, and monitored hibernation using body temperature loggers. At 5 and 16 °C all animals hibernated from autumn to spring with no differences in date of first/last torpor or duration of interbout euthermic periods (IBE), but torpor bout duration was 25% shorter at 16 °C. One of 4 animals housed at 25 °C did not hibernate. For the other three 25 °C animals, the first torpor date did not differ from the other groups, but the last torpor bout (5 Feb.) occurred almost 8 weeks earlier. These animals aroused from torpor more frequently and IBE lasted significantly longer, so the total time spent torpid was less than 50% of the other groups. Unlike the 5 or 16 °C animals, 25 °C animals re-entered torpor in late spring 2018. Taken together these data suggest that this species is an obligate hibernator, but that high ambient temperatures can accelerate the endogenous circannual hibernation rhythm.

Inflammasome signaling could be used to sense and respond to endogenous damage in brown but not white adipose tissue of a hibernating ground squirrel.

Small mammalian hibernators use metabolic suppression to enhance survival during the winter. Torpor is punctuated by periods of euthermia used to clear metabolic by-products and damaged cell components. The current study was performed to determine if the innate immune system, specifically NLRP and AIM2 inflammasome signaling, may detect and respond to cell stress during hibernation. Nlrp3, Casp1, and Il1b genes were significantly upregulated in brown adipose tissue (BAT) during arousal with respect to the euthermic control, suggesting increased NLRP3 inflammasome priming. NLRP3, IL-18, and gasdermin D protein levels increased during torpor, indicating a lag between inflammasome priming and formation. AIM2 and gasdermin D levels increased in BAT during arousal, as did caspase-1 activity. Thus, non-shivering thermogenesis may generate pro-inflammatory triggers of inflammasome signaling. This study is the first to support a role for inflammasome signaling in sensing cellular perturbations at various points of the torpor-arousal cycle, in metabolically-active BAT, but not white adipose tissue (WAT).

Cold-inducible RNA-binding protein Cirp, but not Rbm3, may regulate transcript processing and protection in tissues of the hibernating ground squirrel.

RNA-binding proteins (RBPs) have important roles in transcription, pre-mRNA processing/transport, mRNA degradation, translation, and non-coding RNA processing, among others. RBPs that are expressed in response to cold stress, such as Cirp and Rbm3, could regulate RNA stability and translation in hibernating mammals that reduce their body temperatures from 37 °C to as low as 0-5 °C during torpor bouts. RBPs including Cirp, Rbm3, and stress-inducible HuR translocate from the nucleus to stabilize mRNAs in the cytoplasm, and thereby could regulate which mRNA transcripts are protected from degradation and are translated, versus stored, for future protein synthesis or degraded by nucleases during cell stress associated with metabolic rate depression. This is the first study to explore the transcriptional/translational regulation, and subcellular localization of cold-inducible RBPs in a model hibernator, the 13-lined ground squirrel (Ictidomys tridecemlineatus). Cirp protein levels were upregulated in liver, skeletal muscle, and brown adipose tissue throughout the torpor-arousal cycle whereas Rbm3 protein levels stayed constant or decreased, suggesting an important role for Cirp, but likely not Rbm3, in the hibernator stress response. Increased cytoplasmic localization of Cirp in liver and muscle and HuR in liver during torpor, but no changes in the relative levels of Rbm3 in the cytoplasm, emphasizes a role for Cirp and possibly HuR in regulating mRNA processing during torpor. This study informs our understanding of the natural adaptations that extreme animals use in the face of stress, and highlight natural stress response mediators that could be used to bolster cryoprotection of human organs donated for transplant.

The association between increasing levels of O-GlcNAc and galectins in the liver tissue of hibernating thirteen-lined ground squirrels (Ictidomys tridecemlineatus).

Post-translational glycosylation of proteins with O-linked ß-N-acetylglucosamine (O-GlcNAcylation) and changes of galectin expression profiles are essential in many cellular stress responses. We examine this regulation in the liver tissue of hibernating thirteen-lined ground squirrels (Ictidomys tridecemlineatus) representing a biological model of hypometabolism and physiological stress resistance. The tissue levels of O-GlcNAcylated proteins as well as galectin-1 and galectin-3 proteins detected by immunodot blot assay were significantly lower by 4.6-5.4-, 2.2-2.3- and 2.5-2.9-fold, respectively, in the non-hibernating summer squirrels compared with those in winter, whether hibernating or aroused. However, there were no differences in the expression of genes encoding enzymes involved in O-GlcNAc cycle (O-GlcNAc transferase and O-GlcNAcase) and such galectins as LGALS1, LGALS2, LGALS3, LGALS4 and LGALS9. Only the expression of LGALS8 gene in the liver tissue was significantly decreased by 37.6 ± 0.1% in hibernating ground squirrels relative to summer animals. Considering that the expression of a proven genetic biomarker ELOVL6 encoding ELOVL fatty acid elongase 6 was readily upregulated in non-hibernating animals by 11.3-32.9-fold, marginal differential changes in the expression of galectin genes cannot be classified as biomarkers of hibernation. Thus, this study provides evidence that hibernation in Ictidomys tridecemlineatus is associated with increasing O-GlcNAcylation of liver proteins and suggests that the contribution of galectins deserves further studies at the protein level.

Dynamic RNA Regulation in the Brain Underlies Physiological Plasticity in a Hibernating Mammal.

Hibernation is a physiological and behavioral phenotype that minimizes energy expenditure. Hibernators cycle between profound depression and rapid hyperactivation of multiple physiological processes, challenging our concept of mammalian homeostasis. How the hibernator orchestrates and survives these extremes while maintaining cell to organismal viability is unknown. Here, we enhance the genome integrity and annotation of a model hibernator, the 13-lined ground squirrel. Our new assembly brings this genome to near chromosome-level contiguity and adds thousands of previously unannotated genes. These new genomic resources were used to identify 6,505 hibernation-related, differentially-expressed and processed transcripts using RNA-seq data from three brain regions in animals whose physiological status was precisely defined using body temperature telemetry. A software tool, squirrelBox, was developed to foster further data analyses and visualization. SquirrelBox includes a comprehensive toolset for rapid visualization of gene level and cluster group dynamics, sequence scanning of k-mer and domains, and interactive exploration of gene lists. Using these new tools and data, we deconvolute seasonal from temperature-dependent effects on the brain transcriptome during hibernation for the first time, highlighting the importance of carefully timed samples for studies of differential gene expression in hibernation. The identified genes include a regulatory network of RNA binding proteins that are dynamic in hibernation along with the composition of the RNA pool. In addition to passive effects of temperature, we provide evidence for regulated transcription and RNA turnover during hibernation. Significant alternative splicing, largely temperature dependent, also occurs during hibernation. These findings form a crucial first step and provide a roadmap for future work toward defining novel mechanisms of tissue protection and metabolic depression that may 1 day be applied toward improving human health.

The heart of a hibernator: EGFR and MAPK signaling in cardiac muscle during the hibernation of thirteen-lined ground squirrels, Ictidomys tridecemlineatus.

BACKGROUND: Thirteen-lined ground squirrels (Ictidomys tridecemlineatus) experience dramatic changes in physiological and molecular parameters during winter hibernation. Notably, these animals experience reduced blood circulation during torpor, which can put numerous stresses on their hearts. The present study evaluates the role played by the epidermal growth factor receptor (EGFR) in signal transduction during hibernation at low body temperature to evaluate signaling mechanisms. By investigating the regulation of intracellular mitogen activated protein kinase (MAPK) pathway responses, anti-apoptosis signals, downstream transcription factors, and heat shock proteins in cardiac muscle we aim to determine the correlation between upstream tyrosine phosphorylation events and downstream outcomes. METHODS: Protein abundance of phosphorylated EGFR, MAPKs and downstream effector proteins were quantified using immunoblotting and Luminex® multiplex assays. RESULTS: Monitoring five time points over the torpor/arousal cycle, EGFR phosphorylation on T654, Y1068, Y1086 was found to increase significantly compared with euthermic control values particularly during the arousal process from torpor, whereas phosphorylation at Y1045 was reduced during torpor. Phosphorylation of intracellular MAPK targets (p-ERK 1/2, p-JNK, p-p38) also increased strongly during the early arousal stage with p-p38 levels also rising during prolonged torpor. However, of downstream MAPK effector kinases that were measured, only p-Elk-1 levels changed showing a decrease during interbout arousal (IA). Apoptosis markers revealed a strong reduction of the pro-apoptotic p-BAD protein during entrance into torpor that remained suppressed through torpor and IA. However, active caspase-9 protein rose strongly during IA. Levels of p-AKT were suppressed during the transition phases into and out of torpor. Of four heat shock proteins assessed, only HSP27 protein levels changed significantly (a 40% decrease) during torpor. CONCLUSION: We show evidence of EGFR phosphorylation correlating to activation of MAPK signaling and downstream p-ELK1 suppression during hibernation. We also demonstrate a reduction in p-BAD mediated pro-apoptotic signaling during hibernation with active caspase-9 protein levels increasing only during IA. I. tridecemlineatus has natural mechanisms of tissue protection during hibernation that is largely due to cellular regulation through phosphorylation-mediated signaling cascade. We identify a possible link between EGFR and MAPK signaling via p-ERK, p-p38, and p-JNK in the cardiac muscle of these hibernating mammals that correlates with an apparent reduction in caspase-9 apoptotic signaling. This reveals a piece of the mechanism behind how these mammals are resilient to cardiac stresses during hibernation that would otherwise be damaging.

Thermoneutral temperature reduces liver volume but increases fat content in a mammalian hibernator.

Hibernators survive challenging winters by entering torpor, which lowers body temperature (Tb) to ∼5 °C for 12-14 days, followed by spontaneous arousals where Tb increases to ∼37 °C for 10-12 h before entering another torpor bout. This Tb cycle is accompanied by significant fluctuations in metabolic rate. Little is known about the role of the liver in lipid metabolism during hibernation. In this study we measured the effect of ambient temperature on liver volume and lipid content in 13-lined ground squirrels (Ictidomys tridecemlineatus). We housed animals at thermoneutral (25 °C) or cold (5 °C) ambient temperatures, with the same photoperiod (12 h light:12 h dark) for an entire year. We determined volume and water-fat ratio of the liver using magnetic resonance imaging (MRI). Ambient temperature significantly affected both liver volume and fat content. From October to August squirrels housed at 25 °C had 25% smaller livers compared to the squirrels housed at 5 °C, but their average lipid content (13.3%) was 37% higher. Because the squirrels housed at 25 °C appeared to continue feeding throughout the winter but did not enter extended torpor, more carbohydrates may have been diverted to lipid stores. By contrast, animals housed at 5 °C did not appear to feed, and carbohydrates would likely be preferentially stored in the liver as glycogen to supply glucose for brain metabolism. These results suggest that the fat burden caused by hibernators preparing for winter can lead to symptoms of metabolic syndrome, but that these symptoms are reversible in the spring.

Rodent species as possible reservoirs of Borrelia burgdorferi in a prairie ecosystem.

Lyme borreliosis is the most commonly reported vector-borne disease in the United States and Europe. It is caused by a group of spirochete bacteria belonging to the Borrelia burgdorferi sensu lato complex. These pathogens are transmitted among vertebrate reservoir hosts through the bite of hard-bodied ticks. While the enzootic cycle of Borrelia transmission is well understood in its primary reservoir, the white-footed mouse, Peromyscus leucopus, far less is known about other reservoir hosts, particularly in grassland ecosystems. This study assessed the prevalence of B. burgdorferi s. l. among four non-Peromyscus rodents in a prairie ecosystem in the Midwestern United States over a four-year period. We found high prevalences of the bacteria in all four species studied. Our results help to support the roles of Microtus species as reservoirs of B. burgdorferi and add to the literature that suggests Zapus hudsonius may also be a reservoir. Additionally, we identified a previously unknown possible reservoir, Ictidomys tridecemlineatus. Our study also identifies the need to study the dynamics of Lyme borreliosis in habitats and areas outside of the typical range of P. leucopus.

Genes of the undead: hibernation and death display different gene profiles.

A degree of regulation continues into death according to post-mortem transcriptome studies, which have identified 'zombie genes' that come alive hours and days after organismal death. We hypothesized that hibernation, representing the closest natural mammalian phenomenon to death, would display similar gene expression profiles. Exploring zombie genes using qPCR and available transcriptomic resources from multiple torpid tissues in 13-lined ground squirrels showed little in common with gene profiles observed following death. Hibernators repress transcription, surviving only on the transcripts required during profound slowdowns of metabolic rate and of most physiological functions, therefore not requiring zombie gene expression that could be the cell's last resort during stress. This is the first study to explore zombie gene responses to a near-death situation in a living system.

Identification of a lipid-rich depot in the orbital cavity of the thirteen-lined ground squirrel.

We discovered a previously undescribed orbital lipid depot in the thirteen-lined ground squirrel during the first ever magnetic resonance image (MRI) of this common experimental model of mammalian hibernation. In animals housed at constant ambient temperatures (5°C or 25°C, 12 h:12 h light:dark photoperiod), the volume of this depot increased in the autumn and decreased in the spring, suggesting an endogenous circannual pattern. Water-fat MRI revealed that throughout the year this depot is composed of ∼40% lipid, similar to brown adipose tissue (BAT). During arousal from torpor, thermal images showed higher surface temperatures near this depot before the rest of the head warmed, suggesting a thermoregulatory function. This depot, however, does not contain uncoupling protein 1, a BAT biomarker, or uncoupling protein 3. Histology shows blood vessels in close proximity to each other, suggesting it may serve as a vascular rete, perhaps to preferentially warm the eye and brain during arousals.