Hibernation Phenotype Research Articles

Serum Immune-Related Proteins are Differentially Expressed during Hibernation in the American Black Bear

Hibernation is an adaptation to conserve energy in the face of extreme environmental conditions and low food availability that has risen in several animal phyla. This phenomenon is characterized by reduced metabolic rate (∼25% of the active basal metabolic rate in hibernating bears) and energy demand, while other physiological adjustments are far from clear. The profiling of the serum proteome of the American black bear (Ursus americanus) may reveal specific proteins that are differentially modulated by hibernation, and provide insight into the remarkable physiological adaptations that characterize ursid hibernation. In this study, we used differential gel electrophoresis (DIGE) analysis, liquid chromatography coupled to tandem mass spectrometry, and subsequent MASCOT analysis of the mass spectra to identify candidate proteins that are differentially expressed during hibernation in captive black bears. Seventy serum proteins were identified as changing by ±1.5 fold or more, out of which 34 proteins increased expression during hibernation. The majority of identified proteins are involved in immune system processes. These included α2-macroglobulin, complement components C1s and C4, immunoglobulin μ and J chains, clusterin, haptoglobin, C4b binding protein, kininogen 1, α2-HS-glycoprotein, and apoplipoproteins A-I and A-IV. Differential expression of a subset of these proteins identified by proteomic analysis was also confirmed by immunodetection. We propose that the observed serum protein changes contribute to the maintenance of the hibernation phenotype and health, including increased capacities for bone maintenance and wound healing during hibernation in bears.

Seasonal and Regional Differences in Gene Expression in the Brain of a Hibernating Mammal

Mammalian hibernation presents a unique opportunity to study naturally occurring neuroprotection. Hibernating ground squirrels undergo rapid and extreme physiological changes in body temperature, oxygen consumption, and heart rate without suffering neurological damage from ischemia and reperfusion injury. Different brain regions show markedly different activity during the torpor/arousal cycle: the cerebral cortex shows activity only during the periodic returns to normothermia, while the hypothalamus is active over the entire temperature range. Therefore, region-specific neuroprotective strategies must exist to permit this compartmentalized spectrum of activity. In this study, we use the Illumina HiSeq platform to compare the transcriptomes of these two brain regions at four collection points across the hibernation season: April Active, October Active, Torpor, and IBA. In the cerebral cortex, 1,085 genes were found to be differentially expressed across collection points, while 1,063 genes were differentially expressed in the hypothalamus. Comparison of these transcripts indicates that the cerebral cortex and hypothalamus implement very different strategies during hibernation, showing less than 20% of these differentially expressed genes in common. The cerebral cortex transcriptome shows evidence of remodeling and plasticity during hibernation, including transcripts for the presynaptic cytomatrix proteins bassoon and piccolo, and extracellular matrix components, including laminins and collagens. Conversely, the hypothalamic transcriptome displays upregulation of transcripts involved in damage response signaling and protein turnover during hibernation, including the DNA damage repair gene RAD50 and ubiquitin E3 ligases UBR1 and UBR5. Additionally, the hypothalamus transcriptome also provides evidence of potential mechanisms underlying the hibernation phenotype, including feeding and satiety signaling, seasonal timing mechanisms, and fuel utilization. This study provides insight into potential neuroprotective strategies and hibernation control mechanisms, and also specifically shows that the hibernator brain exhibits both seasonal and regional differences in mRNA expression.

Metabolic Hormone FGF21 Is Induced in Ground Squirrels during Hibernation but Its Overexpression Is Not Sufficient to Cause Torpor

Hibernation is a natural adaptation that allows certain mammals to survive physiological extremes that are lethal to humans. Near freezing body temperatures, heart rates of 3–10 beats per minute, absence of food consumption, and depressed metabolism are characteristic of hibernation torpor bouts that are periodically interrupted by brief interbout arousals (IBAs). The molecular basis of torpor induction is unknown, however starved mice overexpressing the metabolic hormone fibroblast growth factor 21 (FGF21) promote fat utilization, reduce body temperature, and readily enter torpor–all hallmarks of mammalian hibernation. In this study we cloned FGF21 from the naturally hibernating thirteen-lined ground squirrel (Ictidomys tridecemlineatus) and found that levels of FGF21 mRNA in liver and FGF21 protein in serum are elevated during hibernation torpor bouts and significantly elevated during IBAs compared to summer active animals. The effects of artificially elevating circulating FGF21 concentrations 50 to 100-fold via adenoviral-mediated overexpression were examined at three different times of the year. This is the first time that a transgenic approach has been used in a natural hibernator to examine mechanistic aspects of hibernation. Surgically implanted transmitters measured various metrics of the hibernation phenotype over a 7-day period including changes in motor activity, heart rate and core body temperature. In April fed-state animals, FGF21 overexpression decreased blood insulin and free fatty acid concentrations, effects similar to those seen in obese mice. However, elevated FGF21 concentrations did not cause torpor in these fed-state animals nor did they cause torpor or affect metabolic parameters in fasted-state animals in March/April, August or October. We conclude that FGF21 is strongly regulated during torpor and IBA but that its overexpression is not sufficient to cause torpor in naturally hibernating ground squirrels.

Deep sequencing the transcriptome reveals seasonal adaptive mechanisms in a hibernating mammal.

Mammalian hibernation is a complex phenotype involving metabolic rate reduction, bradycardia, profound hypothermia, and a reliance on stored fat that allows the animal to survive for months without food in a state of suspended animation. To determine the genes responsible for this phenotype in the thirteen-lined ground squirrel (Ictidomys tridecemlineatus) we used the Roche 454 platform to sequence mRNA isolated at six points throughout the year from three key tissues: heart, skeletal muscle, and white adipose tissue (WAT). Deep sequencing generated approximately 3.7 million cDNA reads from 18 samples (6 time points ×3 tissues) with a mean read length of 335 bases. Of these, 3,125,337 reads were assembled into 140,703 contigs. Approximately 90% of all sequences were matched to proteins in the human UniProt database. The total number of distinct human proteins matched by ground squirrel transcripts was 13,637 for heart, 12,496 for skeletal muscle, and 14,351 for WAT. Extensive mitochondrial RNA sequences enabled a novel approach of using the transcriptome to construct the complete mitochondrial genome for I. tridecemlineatus. Seasonal and activity-specific changes in mRNA levels that met our stringent false discovery rate cutoff (1.0×10−11) were used to identify patterns of gene expression involving various aspects of the hibernation phenotype. Among these patterns are differentially expressed genes encoding heart proteins AT1A1, NAC1 and RYR2 controlling ion transport required for contraction and relaxation at low body temperatures. Abundant RNAs in skeletal muscle coding ubiquitin pathway proteins ASB2, UBC and DDB1 peak in October, suggesting an increase in muscle proteolysis. Finally, genes in WAT that encode proteins involved in lipogenesis (ACOD, FABP4) are highly expressed in August, but gradually decline in expression during the seasonal transition to lipolysis.

Shotgun Proteomics Analysis of Hibernating Arctic Ground Squirrels

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

Identification of qRT-PCR reference genes for analysis of opioid gene expression in a hibernator

Previous work has suggested that central and peripheral opioid signaling are involved in regulating torpor behavior and tissue protection associated with the hibernation phenotype. We used quantitative real-time PCR (qRT-PCR) to measure mRNA levels of opioid peptide precursors and receptors in the brain and heart of summer ground squirrels (Ictidomys tridecemlineatus) and winter hibernating squirrels in the torpid or interbout arousal states. The use of appropriate reference genes for normalization of qRT-PCR gene expression data can have profound effects on the analysis and interpretation of results. This may be particularly important when experimental subjects, such as hibernating animals, undergo significant morphological and/or functional changes during the study. Therefore, an additional goal of this study was to identify stable reference genes for use in qRT-PCR studies of the 13-lined ground squirrel. Expression levels of 10 potential reference genes were measured in the small intestine, liver, brain, and heart, and the optimal combinations of the most stable reference genes were identified by the GeNorm Excel applet. Based on this analysis, we provide recommendations for reference genes to use in each tissue that would be suitable for comparative studies among different activity states. When appropriate normalization of mRNA levels was used, there were no changes in opioid-related genes in heart among the three activity states; in brain, DOR expression was highest during torpor, lowest in interbout arousal and intermediate in summer. The results support the idea that changes in DOR expression may regulate the level of neuronal activity in brain during the annual hibernation cycle and may contribute to hibernation-associated tissue protection.

A role for nuclear receptors in mammalian hibernation

Hibernation is one of the most dramatic examples of phenotypic plasticity in mammals. During periods of food shortage and/or reduced ambient temperatures hibernating mammals become heterothermic, allowing their body temperature to decrease while entering an energy-conserving torpid state. In order to survive the multi-month hibernation season many species engage in hyperphagy, dramatically increasing adipose stores prior to the onset of hibernation. Nuclear receptors are a superfamily of transcription factors many of which bind lipophilic molecules as ligands. They regulate a variety of processes including energy homeostasis, carbohydrate and lipid metabolism, inflammation and circadian rhythm. Given that lipids are integral in the hibernation phenotype they may play important regulatory roles through their interactions with nuclear receptors. Here we review current knowledge and suggest possible roles in mammalian hibernation for peroxisome proliferator-activated receptors (PPARs), farnesoid X receptors (FXRs), liver X receptors (LXRs), retinoid-related orphan receptors (RORs) and Rev-ERBs.

Hibernation and plasma from hibernating animals modulate the cardioprotective delta opioid system

Our goal is to harness the protective effects of mammalian hibernation to prevent and to treat cardiovascular disease (CVD). CVD is among the leading causes of morbidity and mortality in the United States. The specific objective of this study was to determine the effect of hibernation on the cardiac delta opioid system, and to determine the mechanisms by which plasma from hibernating woodchucks (HWP) is cardioprotective. Hearts were collected from summer woodchucks and squirrels, and from animals at defined times in the hibernation cycle. Tissues were analyzed by real‐time PCR for gene expression and by Western blot analysis for protein levels. Immunofluorescence microscopy was used to determine the effects of HWP on cellular localization of the delta opioid receptor (DOR). Hibernation induced dynamic changes in cardiac expression of opioid genes, and altered protein levels of the delta opioid precursor proenkephalin and of DOR. HWP increased localization of DOR to the plasma membrane of primary human vascular cells, suggesting that mobilization of DOR to cell surfaces may contribute to cardioprotection. These data support a role for delta opioids and their receptor in the hibernation phenotype, and provide insights that will further the development of therapies based on the natural protective processes of hibernation. Funded by the Department of Emergency Medicine, The University of Iowa.

Mammalian hibernation: differential gene expression and novel application of epigenetic controls

This review highlights current information about the regulatory mechanisms that govern gene expression during mammalian hibernation, in particular the potential role of epigenetic controls in coordinating the global suppression of transcription. Hibernation is characterized by long periods of deep torpor (when core body temperature drops to near ambient) that are interspersed with brief arousal periods back to euthermia. Entry into torpor requires coordinated controls which strongly suppress and reprioritize all metabolic functions, including global controls on both transcription and translation. At the same time, however, selected hibernation-specific genes are up-regulated under the control of specific transcription factors to support the torpid state; this includes genes that encode proteins involved in lipid fuel catabolism and in long term cytoprotection (e.g. antioxidants, chaperones). We evaluate the currently available information on global transcriptional suppression in hibernation and propose that epigenetic mechanisms such as DNA methylation, histone modification, SUMOylation and the actions of sirtuins play crucial roles in transcriptional suppression during torpor. Global controls providing translational suppression also occur during hibernation including reversible phosphorylation control of ribosomal initiation and elongation factors as well as polysome dissociation. We also present initial data that mRNA transcripts are regulated via inhibitory interactions with microRNA species during torpor and provide the first evidence of differential expression of miRNAs in hibernators. When taken together, these mechanisms provide hibernators with multiple layers of regulatory controls that achieve both global repression of gene expression and selected enhancement of genes/proteins that achieve the hibernation phenotype.

Analysis of the hibernation cycle using LC-MS-based metabolomics in ground squirrel liver

A hallmark of hibernation in mammals is metabolic flexibility, which is typified by reversible bouts of metabolic depression (torpor) and the seasonal shift from predominantly carbohydrate to lipid metabolism from summer to winter. To provide new insight into the control and consequences of hibernation, we used LC/MS-based metabolomics to measure differences in small molecules in ground squirrel liver in five activity states: summer, entering torpor, late torpor, arousing from torpor, and interbout arousal. There were significant alterations both seasonally and within torpor-arousal cycles in enzyme cofactor metabolism, amino acid catabolism, and purine and pyrimidine metabolism, with observed metabolites reduced during torpor and increased upon arousal. Multiple lipids also changed, including 1-oleoyllysophosphatidylcholine, cholesterol sulfate, and sphingosine, which tended to be lowest during torpor, and hexadecanedioic acid, which accumulated during a torpor bout. The results reveal the dramatic alterations that occur in several classes of metabolites, highlighting the value of metabolomic analyses in deciphering the hibernation phenotype.

Central nervous system regulation of mammalian hibernation: implications for metabolic suppression and ischemia tolerance

Torpor during hibernation defines the nadir of mammalian metabolism where whole animal rates of metabolism are decreased to as low as 2% of basal metabolic rate. This capacity to decrease profoundly the metabolic demand of organs and tissues has the potential to translate into novel therapies for the treatment of ischemia associated with stroke, cardiac arrest or trauma where delivery of oxygen and nutrients fails to meet demand. If metabolic demand could be arrested in a regulated way, cell and tissue injury could be attenuated. Metabolic suppression achieved during hibernation is regulated, in part, by the central nervous system through indirect and possibly direct means. In this study, we review recent evidence for mechanisms of central nervous system control of torpor in hibernating rodents including evidence of a permissive, hibernation protein complex, a role for A1 adenosine receptors, mu opiate receptors, glutamate and thyrotropin-releasing hormone. Central sites for regulation of torpor include the hippocampus, hypothalamus and nuclei of the autonomic nervous system. In addition, we discuss evidence that hibernation phenotypes can be translated to non-hibernating species by H(2)S and 3-iodothyronamine with the caveat that the hypothermia, bradycardia, and metabolic suppression induced by these compounds may or may not be identical to mechanisms employed in true hibernation.

Metabolomic analysis of plasma from hibernating and active ground squirrels

Mammals that hibernate survive seasonal food shortages by using extended periods of torpor to conserve energy. The hibernation phenotype is associated with resilience to several physiological challenges, such as hypoxia, ischemia/reperfusion injury, and obesity. Thirteen-lined ground squirrels (Spermophilus tridecemlineatus) during interbout arousals (IBA) within the hibernation season have enhanced resistance to a hypovolemic challenge relative to summer-active (SA) squirrels or rats at the same body temperature. To better understand the molecular mechanisms of this resistance and provide insight into the hibernation phenotype in general, plasma from IBA and SA squirrels was analyzed by LC-MS. Using accurate mass and chromatographic retention time information, thousands of features were characterized. Several identified features differed significantly between IBA and SA squirrels. These include certain peroxidized lipids that were enriched in IBA relative to SA, whereas SA squirrels had higher enrichment of certain amino acids, vitamins and other nutrients. This approach permits a molecular characterization of the hibernation phenotype in ground squirrels and will provide candidate molecules responsible for hibernation-induced protection. Supported by DARPA W81XWH-05-02-0016.

Hibernation confers resistance to intestinal ischemia-reperfusion injury

The damaging effects of intestinal ischemia-reperfusion (I/R) on the gut and remote organs can be attenuated by subjecting the intestine to a prior, less severe I/R insult, a process known as preconditioning. Because intestines of hibernating ground squirrels experience repeated cycles of hypoperfusion and reperfusion, we examined whether hibernation serves as a model for natural preconditioning against I/R-induced injury. We induced intestinal I/R in either the entire gut or in isolated intestinal loops using rats, summer ground squirrels, and hibernating squirrels during natural interbout arousals (IBA; body temperature 37-39 degrees C). In both models, I/R induced less mucosal damage in IBA squirrels than in summer squirrels or rats. Superior mesenteric artery I/R increased MPO activity in the gut mucosa and lung of rats and summer squirrels and the liver of rats but had no effect in IBA squirrels. I/R in isolated loops increased luminal albumin levels, suggesting increased gut permeability in rats and summer squirrels but not IBA squirrels. The results suggest that the hibernation phenotype is associated with natural protection against intestinal I/R injury.

Therapeutic approaches to organ preservation injury

The long-term preservation of organ explants and protection from cold ischaemia and subsequent rewarming reperfusion injury is essential to successful allografting and instrumental in enlarging the international donor pool. Current hypothermic preservation solutions may have reached the limit of what is possible in terms of explant longevity and viability, and research attention has been turned to novel organoprotective pharmacotherapeutics that could provide the next generation of explant/transplant nurture. Organoprotection is equally paramount during surgical procedures requiring a period of ischaemia as in coronary artery bypass grafting, angioplasty or stenting. Similarly, the phenomenon of non-freezing cold injury (NFCI), associated with microcirculatory and neuronal damage in exposed and often immersed extremities, also involves ischaemia–reperfusion injury. Whilst additions to cold storage solutions of antioxidants and modulators of cellular metabolism may extend their preservative efficacy, pharmacological triggers of ischaemic preconditioning and the hibernation phenotype may offer breakthroughs in the prevention of ischaemia–reperfusion injury.

Natural resistance to liver cold ischemia-reperfusion injury associated with the hibernation phenotype

The success of liver grafts is currently limited by the length of time organs are cold preserved before transplant. Novel insights to improve viability of cold-stored organs may emerge from studies with animals that naturally experience low body temperatures (T(b)) for extended periods. In this study, we tested whether livers from hibernating ground squirrels tolerate cold ischemia-warm reperfusion (cold I/R) for longer times and with better quality than livers from rats or summer squirrels. Hibernators were used when torpid (T(b) < 10 degrees C) or aroused (T(b) = 37 degrees C). Livers were stored at 4 degrees C in University of Wisconsin solution for 0-72 h and then reperfused with 37 degrees C buffer in vitro. Lactate dehydrogenase (LDH) release after 60 min was increased 37-fold in rat livers after 72 h cold I/R but only 10-fold in summer livers and approximately three- to sixfold in torpid and aroused hibernator livers, despite twofold higher total LDH content in livers from hibernators compared with rats or summer squirrels. Reperfusion for up to 240 min had the least effect on LDH release in livers from hibernators and the greatest effect in rats. Compared with rats or summer squirrels, livers from hibernators after 72 h cold I/R showed better maintenance of mitochondrial respiration, bile production, and sinusoidal lining cell viability, as well as lower vascular resistance and Kupffer cell phagocytosis. These results demonstrate that the hibernation phenotype in ground squirrels confers superior resistance to liver cold I/R injury compared with rats and summer squirrels. Because hibernation-induced protection is not dependent on animals being in the torpid state, the mechanisms responsible for this effect may provide new strategies for liver preservation in humans.

Mitogen-activated protein kinases and selected downstream targets display organ-specific responses in the hibernating ground squirrel

The responses of mitogen-activated protein kinase (MAPK) family members, including the extracellular signal-regulated kinases (ERKs), the c-jun NH2-terminal kinases (JNKs) and p38 MAPK, during mammalian hibernation were analyzed in five organs of Richardson's ground squirrels, Spermophilus richardsonii. Each kinase subfamily responded differently in torpor and each showed organ-specific patterns of response. ERK1/2 activities increased significantly in muscle and brain during hibernation but decreased in kidney and liver. JNK activity rose in four organs (except brain) during hibernation whereas active, phosphorylated p38 MAPK increased only in muscle and heart. Activities of ERK-activated kinases also responded to hibernation: MAPKAPK-1 rose in muscle and brain, MAPKAPK-2 decreased in liver and kidney but rose in the other three organs, and p70 S6K kinase activity decreased kidney and heart. Transcription factors, c-Jun and CREB, also showed organ-specific responses during torpor. The data suggest key roles for MAPKs in the regulation of the known organ-specific changes in gene expression and protein phosphorylation that define the hibernation phenotype.

Cold Ischemic Organ Preservation: Lessons from Natural Systems

Mammalian hibernators offer natural models for investigating solutions to the metabolic injuries that accrue during cold ischemic storage of human organs removed for transplant. Knowledge of the biochemical mechanisms that regulate and stabilize metabolism to ensure long-term viability in the hypometabolic, hypothermic state of hibernation could lead to applied treatments that could increase the time that excised organs can be maintained in cold storage and/or improve recovery of function after implantation. New research has documented the widespread role of reversible protein phosphorylation control of metabolism in achieving the coordinated suppression of metabolic rate that greatly extends viability during torpor. Analysis of hibernation-induced gene expression is proving to be of crucial importance for identifying the genes and proteins that are up-regulated to address organ-specific concerns during torpor. In particular, the power of complementary deoxyribonucleic acid (cDNA) array screening is identifying families of proteins that are up-regulated during hibernation (eg, serpins, heat shock proteins, antioxidants, membrane transporters) and highlighting previously unrecognized areas of cellular metabolism as contributing to the hibernation phenotype. These offer new targets for innovative applied treatments that could enhance cyto-protection and cold ischemia survival of organ explants.