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.

Plasticity changes in iron homeostasis in hibernating Daurian ground squirrels (Spermophilus dauricus) may counteract chronically inactive skeletal muscle atrophy.

Disuse-induced muscular atrophy is frequently accompanied by iron overload. Hibernating animals are a natural animal model for resistance to disuse muscle atrophy. In this paper, we explored changes in skeletal muscle iron content of Daurian ground squirrels (Spermophilus dauricus) during different periods of hibernation as well as the regulatory mechanisms involved. The results revealed that compared with the summer active group (SA), iron content in the soleus muscle (SOL) decreased (- 65%) in the torpor group (TOR), but returned to normal levels in the inter-bout arousal (IBA); splenic iron content increased in the TOR group (vs. SA, + 67%), decreased in the IBA group (vs. TOR, - 37%). Expression of serum hepcidin decreased in the TOR group (vs. SA, - 22%) and returned to normal levels in the IBA groups; serum ferritin increased in the TOR group (vs. SA, + 31%), then recovered in the IBA groups. Soleus muscle transferrin receptor 1 (TfR1) expression increased in the TOR group (vs. SA, + 83%), decreased in the IBA group (vs. TOR, - 30%); ferroportin 1 increased in the IBA group (vs. SA, + 55%); ferritin increased in the IBA group (vs. SA, + 42%). No significant differences in extensor digitorum longus in iron content or iron metabolism-related protein expression were observed among the groups. Significantly, all increased or decreased indicators in this study returned to normal levels after the post-hibernation group, showing remarkable plasticity. In summary, avoiding iron overload may be a potential mechanism for hibernating Daurian ground squirrels to avoid disuse induced muscular atrophy. In addition, the different skeletal muscle types exhibited unique strategies for regulating iron homeostasis.

The effects of nickel chloride on papillary muscle contractility under normothermic and hypothermic conditions: Comparison of active and hibernating ground squirrels (Urocitellus undulatus) with Wistar rats.

Extracellular Ca2+ plays a pivotal role in the regulation of cardiac contractility under normal and extreme conditions. Here, by using nickel chloride (NiCl2), a non-specific blocker of extracellular Ca2+ influx, we studied the input of extracellular Ca2+ on the regulation of papillary muscle (PM) contractility under normal and hypothermic conditions in ground squirrels (GS), and rats. By measuring isometric force of contraction, we studied how NiCl2 affects force-frequency relationship and the rest effect in PM of these species at 30 °C and 10 °C. We found that at 30 °C 1.5 mM NiCl2 significantly reduced force of contraction across entire frequency range in active GS and rats, whereas in hibernating GS force of contraction was reduced at low and high frequency range. Additionally, NiCl2 evoked spontaneous contractility in rats but not GS PM. The rest effect was significantly reduced by NiCl2 for active GS and rats but not hibernating GS. At 10 °C, NiCl2 fully reduced contractility in active GS and, to a lesser extent, in rats, whereas in hibernating GS it was significant only at 0.3 Hz. The rest effect was significantly reduced by NiCl2 in both active and hibernating GS, whereas it was unmasked in rats that had high contractility under hypothermic conditions in control. Our results show a significant contribution of extracellular Ca2+ to myocardial contractility in GS not only in active but also in hibernating states, especially under hypothermic conditions, whereas limitation of extracellular Ca2+ influx in rats under hypothermia can play protective role for myocardial contractility.

Integrated Redox-Metabolic Orchestration Sustains Life in Hibernating Ground Squirrels.

Significance: The ultimate manifestations of life, birth, survival under various environmental pressures and death are based on bioenergetics. Hibernation is a unique survival strategy for many small mammals that is characterised by severe metabolic depression and transition from euthermia to hypothermia (torpor) at body temperatures close to 0°C. These manifestations of life were made possible by the remarkable "social" behavior of biomolecules during billions of years of evolution: the evolution of life with oxygen. Oxygen was necessary for energy production and the evolutionary explosion of aerobic organisms. Recent Advances: Nevertheless, reactive oxygen species, formed through oxidative metabolism, are dangerous-they can kill a cell and, on the other hand, play a plethora of fundamentally valuable roles. Therefore, the evolution of life depended on energy metabolism and redox-metabolic adaptations. The more extreme the conditions for survival are, the more sophisticated the adaptive responses of organisms become. Hibernation is a beautiful illustration of this principle. Hibernating animals use evolutionarily conserved molecular mechanisms to survive adverse environmental conditions, including reducing body temperature to ambient levels (often to ∼0°C) and severe metabolic depression. This long-built secret of life lies at the intersection of oxygen, metabolism, and bioenergetics, and hibernating organisms have learned to exploit all the underlying capacities of molecular pathways to survive. Critical Issues: Despite such drastic changes in phenotype, tissues and organs of hibernators sustain no metabolic or histological damage during hibernation or upon awakening from hibernation. This was made possible by the fascinating integration of redox-metabolic regulatory networks whose molecular mechanisms remain undisclosed to this day. Future Directions: Discovering these molecular mechanisms is not warranted only to understand hibernation in itself but to help explain complex medical conditions (hypoxia/reoxygenation, organ transplantation, diabetes, and cancer) and to even help overcome limitations associated with space travel. This is a review of integrated redox-metabolic orchestration in hibernation. Antioxid. Redox Signal. 40, 345-368.

Integrated metabolomics and proteomics analysis to understand muscle atrophy resistance in hibernating Spermophilus dauricus.

Hibernating Spermophilus dauricus experiences minor muscle atrophy, which is an attractive anti-disuse muscle atrophy model. Integrated metabolomics and proteomics analysis was performed on the hibernating S. dauricus during the pre-hibernation (PRE) stage, torpor (TOR) stage, interbout arousal (IBA) stage, and post-hibernation (POST) stage. Time course stage transition-based (TOR vs. PRE, IBA vs. TOR, POST vs. IBA) differential expression analysis was performed based on the R limma package. A total of 14 co-differential metabolites were detected. Among these, l-cystathionine, l-proline, ketoleucine, serine, and 1-Hydroxy-3,6,7-Trimethoxy-2, 8-Diprenylxanthone demonstrated the highest levels in the TOR stage; Beta-Nicotinamide adenine dinucleotide, Dihydrozeatin, Pannaric acid, and Propionylcarnitine demonstrated the highest levels in the IBA stage; Adrenosterone, PS (18:0/14,15-EpETE), S-Carboxymethylcysteine, TxB2, and 3-Phenoxybenzylalcohol demonstrated the highest levels in the POST stage. Kyoto Encyclopedia of Genes and Genomes pathways annotation analysis indicated that biosynthesis of amino acids, ATP-binding cassette transporters, and cysteine and methionine metabolism were co-differential metabolism pathways during the different stages of hibernation. The stage-specific metabolism processes and integrated enzyme-centered metabolism networks in the different stages were also deciphered. Overall, our findings suggest that (1) the periodic change of proline, ketoleucine, and serine contributes to the hindlimb lean tissue preservation; and (2) key metabolites related to the biosynthesis of amino acids, ATP-binding cassette transporters, and cysteine and methionine metabolism may be associated with muscle atrophy resistance. In conclusion, our co-differential metabolites, co-differential metabolism pathways, stage-specific metabolism pathways, and integrated enzyme-centered metabolism networks are informative for biologists to generate hypotheses for functional analyses to perturb disuse-induced muscle atrophy.

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

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

Differential bone remodeling mechanism in hindlimb unloaded and hibernating Daurian ground squirrels: a comparison between artificial and natural disuse within the same species.

Loss of bone mass can occur in mammals after prolonged disuse but the situation for hibernators that are in a state of torpor for many months of the year is not yet fully understood. The present study assesses the bone remodeling mechanisms present in Daurian ground squirrels (Spermophilus dauricus) during hibernation as compared with a model of hindlimb disuse. Differences in microstructure, mechanical properties, bone remodeling-related proteins (Runx2, OCN, ALP, RANKL, CTK and MMP-9) and key proteins of Wnt/ß-catenin signaling pathway (GSK-3ß and phospho-ß-catenin) were evaluated in ground squirrels under 3 conditions: summer active (SA) vs. hibernation (HIB) vs. hindlimb unloaded (HLU). The results indicated that the body weight in HLU ground squirrels was lower than the SA group, and the middle tibia diameter in the HLU group was lower than that in SA and HIB groups. The thickness of cortical and trabecular bone in femurs from HLU ground squirrels was lower than in SA and HIB groups. Most parameters of the tibia in the HLU group were lower than those in SA and HIB groups, which indicated cortical bone loss in ground squirrels. Moreover, our data showed that the changes in microscopic parameters in the femur were more obvious than those in the tibia in HLU and HIB ground squirrels. The levels of Runx2 and ALP were lower in HLU ground squirrels than SA and HIB groups. The protein levels of OCN were unchanged in the three groups, but the protein levels of ALP were lower in the HLU group than in SA and HIB groups. RANKL, CTK and MMP-9 protein levels were significantly decreased in tibia of HLU ground squirrels as compared with SA and HIB groups. In addition, the protein expression levels of RANKL, CTK and MMP-9 showed no statistical difference between SA and HIB ground squirrels. Thus, the mechanisms involved in the balance between bone formation and resorption in hibernating and hindlimb unloading ground squirrels may be different. The present study showed that in femur, the Wnt signaling pathway was inhibited, the protein level of GSK-3ß was increased, and the protein expression of phospho-ß-catenin was decreased in the HIB group as compared with the SA group, which indicates that the Wnt signaling pathway has a great influence on the femur of the HIB group. In conclusion, the natural anti-osteoporosis properties of Daurian ground squirrels are seasonal. The squirrels do not experience bone loss when they are inactive for a long time during hibernation, but the mechanisms of anti-osteoporosis did not work in HLU summer active squirrels.

Investigating the effects of chronic low-dose radiation exposure in the liver of a hypothermic zebrafish model.

Mankind's quest for a manned mission to Mars is placing increased emphasis on the development of innovative radio-protective countermeasures for long-term space travel. Hibernation confers radio-protective effects in hibernating animals, and this has led to the investigation of synthetic torpor to mitigate the deleterious effects of chronic low-dose-rate radiation exposure. Here we describe an induced torpor model we developed using the zebrafish. We explored the effects of radiation exposure on this model with a focus on the liver. Transcriptomic and behavioural analyses were performed. Radiation exposure resulted in transcriptomic perturbations in lipid metabolism and absorption, wound healing, immune response, and fibrogenic pathways. Induced torpor reduced metabolism and increased pro-survival, anti-apoptotic, and DNA repair pathways. Coupled with radiation exposure, induced torpor led to a stress response but also revealed maintenance of DNA repair mechanisms, pro-survival and anti-apoptotic signals. To further characterise our model of induced torpor, the zebrafish model was compared with hepatic transcriptomic data from hibernating grizzly bears (Ursus arctos horribilis) and active controls revealing conserved responses in gene expression associated with anti-apoptotic processes, DNA damage repair, cell survival, proliferation, and antioxidant response. Similarly, the radiation group was compared with space-flown mice revealing shared changes in lipid metabolism.

Transcriptomic and proteomic time-course analyses based on Metascape reveal mechanisms against muscle atrophy in hibernating Spermophilus dauricus.

Hibernating Spermophilus dauricus is resistant to muscle atrophy. Comprehensive transcriptome and proteome time-course analyses based on Metascape can further reveal the underlying processes (pre-hibernation stage, PRE; torpor stage, TOR; interbout arousal stage, IBA; and post-hibernation stage, POST). Transcriptome analysis showed that the cellular responses to growth factor stimulus and discrete oxygen levels continuously changed during hibernation. Proteomic analysis showed that neutrophil degranulation, sulfur compound metabolic process, and generation of precursor metabolites and energy continuously changed during hibernation. Molecular complex detection (MCODE) analysis in both transcriptome and proteome indicated that smooth muscle contraction was involved in the POST versus IBA stage, and peroxisome proliferator-activated receptor delta (Ppard), Myc proto-oncogene (Myc), Sp1 transcription factor (Sp1), and nuclear factor Kappa B subunit 1 (NFκB1) are the common TFs during the hibernation process. Integrated transcriptome and proteome analyses found 18 molecules in the TOR versus PRE stage, 1 molecule in the IBA versus TOR stage, and 16 molecules in the POST versus IBA stage. Among these molecules, carnitine palmitoyltransferase 1A (Cpt1a), SET and MYND domain containing 2 (Smyd2), four and a half LIM domains 1(Fhl1), reactive oxygen species modulator 1 (Romo1), and translocase of the inner mitochondrial membrane 50 (Timm50) were testified by Western blot. In conclusion, novel muscle atrophy resistance mechanisms can be deciphered by time-course transcriptome and proteome analyses based on Metascape.

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.

Body Temperature Patterns and Energy Balance Hormones in Free-Living Thirteen-Lined Ground Squirrels (Ictidomys tridecemlineatus) from Different Latitudes.

AbstractThis article examines hormone concentrations and body temperature (Tb) patterns of free-living thirteen-lined ground squirrels (TLGSs) across the majority of their latitudinal range in the United States (from Texas to Minnesota). Free-living TLGSs (n=40) were implanted with Tbdata loggers in 2019 before they entered hibernation. Three adult female TLGSs, one each from Oklahoma (low latitude), Iowa (middle latitude), and Minnesota (high latitude), were recaptured in 2020 after the hibernation season. Although this provides an n of 1 for each location and therefore no statistically supported conclusions can be drawn, the hibernation season was longest in the animal from the highest latitude with coldest winter soil temperatures (Minnesota) and shortest in the animal retrapped at the lowest latitude (Oklahoma). Torpor bouts were generally longer when soil temperatures were lower. The Iowa and Minnesota squirrels had a prolonged period of short torpor bouts with Tb near 20°C at the beginning of the hibernation season. Concentrations of the orexigenic hormone ghrelin and the sex hormones estradiol and testosterone were also compared in populations from different latitudes. In general, Minnesota males had higher testosterone than males from other populations, possibly due to a later breeding season relative to other squirrel populations. Animals trapped in early summer had significantly lower concentrations of ghrelin than those captured in midsummer, potentially driving the fat-storing period before the hibernation season. Together, these results suggest latitudinal variation in physiological regulation of circannual rhythms.

The Protective Effects on Ischemia-Reperfusion Injury Mechanisms of the Thoracic Aorta in Daurian Ground Squirrels (Spermophilus dauricus) over the Torpor-Arousal Cycle of Hibernation.

Hibernators are a natural model of vascular ischemia-reperfusion injury; however, the protective mechanisms involved in dealing with such an injury over the torpor-arousal cycle are unclear. The present study aimed to clarify the changes in the thoracic aorta and serum in summer-active (SA), late-torpor (LT) and interbout-arousal (IBA) Daurian ground squirrels (Spermophilus dauricus). The results show that total antioxidant capacity (TAC) was unchanged, but malondialdehyde (MDA), hydrogen peroxide (H2O2), interleukin-1ß (IL-1ß) and tumor necrosis factor α (TNFα) were significantly increased for the LT group, whereas the levels of superoxide dismutase (SOD) and interleukin-10 (IL-10) were significantly reduced in the LT group as compared with the SA group. Moreover, the levels of MDA and IL-1ß were significantly reduced, whereas SOD and IL-10 were significantly increased in the IBA group as compared with the SA group. In addition, the lumen area of the thoracic aorta and the expression of the smooth muscle cells (SMCs) contractile marker protein 22α (SM22α) were significantly reduced, whereas the protein expression of the synthetic marker proteins osteopontin (OPN), vimentin (VIM) and proliferating cell nuclear antigen (PCNA) were significantly increased in the LT group as compared with the SA group. Furthermore, the smooth muscle layer of the thoracic aorta was significantly thickened, and PCNA protein expression was significantly reduced in the IBA group as compared with the SA group. The contractile marker proteins SM22α and synthetic marker protein VIM underwent significant localization changes in both LT and IBA groups, with localization of the contractile marker protein α-smooth muscle actin (αSMA) changing only in the IBA group as compared with the SA group. In tunica intima, the serum levels of heparin sulfate (HS) and syndecan-1 (Sy-1) in the LT group were significantly reduced, but the serum level of HS in the IBA group increased significantly as compared with the SA group. Protein expression and localization of endothelial nitric oxide synthase (eNOS) was unchanged in the three groups. In summary, the decrease in reactive oxygen species (ROS) and pro-inflammatory factors and increase in SOD and anti-inflammatory factors during the IBA period induced controlled phenotypic switching of thoracic aortic SMCs and restoration of endothelial permeability to resist ischemic and hypoxic injury during torpor of Daurian ground squirrels.

Tissue-specific response of the RB-E2F1 complex during mammalian hibernation.

Metabolic rate depression during prolonged bouts of torpor is characteristic of mammalian hibernation, reducing energy expenditures over the winter. Cell cycle arrest is observed in quiescent cells during dormancy, partly due to the retinoblastoma (Rb) protein at G1 /S, given cell division and proliferation are metabolic-costly processes. Rb binds to E2F transcription factors and recruits corepressors (e.g., SUV39H1) to E2F target genes, blocking their transcription and cell cycle passage. Phosphorylation by cyclin-CDK complexes at S780 or S795 abolishes Rb-mediated repression, allowing transition into S phase. The present study compares Rb-E2F1 responses between euthermic and torpid states in five organs (brain, heart, kidney, liver, skeletal muscle) of 13-lined ground squirrels (Ictidomys tridecemlineatus). Immunoblotting assessed the expression of Rb, pRb (S780, S795), E2F1, and SUV39H1. Our findings demonstrate multi-tissue upregulation of Rb and SUV39H1 during torpor, with tissue-specific changes to E2F1 and pRb (S780), suggesting Rb-E2F1 contributes to cell cycle control in hibernation.

Changing lanes: seasonal differences in cellular metabolism of adipocytes in grizzly bears (Ursus arctos horribilis).

Obesity is among the most prevalent of health conditions in humans leading to a multitude of metabolic pathologies such as type 2 diabetes and hyperglycemia. However, there are many wild animals that have large seasonal cycles of fat accumulation and loss that do not result in the health consequences observed in obese humans. One example is the grizzly bear (Ursus arctos horribilis) that can have body fat content > 40% that is then used as the energy source for hibernation. Previous in vitro studies found that hibernation season adipocytes exhibit insulin resistance and increased lipolysis. Yet, other aspects of cellular metabolism were not addressed, leaving this in vitro model incomplete. Thus, the current studies were performed to determine if the cellular energetic phenotype-measured via metabolic flux-of hibernating bears was retained in cultured adipocytes and to what extent that was due to serum or intrinsic cellular factors. Extracellular acidification rate and oxygen consumption rate were used to calculate proton efflux rate and total ATP defined as both ATP from glycolysis and from mitochondrial respiration. Hibernation adipocytes treated with hibernation serum produced less ATP and exhibited lower maximal respiration and glycolysis rates than active season adipocytes. These effects were reversed with serum from the opposite season. Insulin had little influence on total ATP production and lipolysis in both hibernation and active serum-treated adipocytes. Together, these results suggest that the metabolic suppression occurring in hibernation adipocytes are downstream of insulin signaling and likely due to a combined reduction in mitochondria number and/or function and glycolytic processes. Future elucidation of the serum components and the cellular mechanisms that enable alterations in mitochondrial function could provide a novel avenue for the development of treatments for human metabolic diseases.

Variations in oxidative stress and antioxidant defense level during different phases of hibernation in common Asian toad, Duttaphrynus melanostictus.

To assess redox status during hibernation with metabolic depression, oxidative stress parameters and antioxidant defense were assessed during different phases of hibernation including active period, hibernation, arousal, and post-arousal period, in the liver and brain tissues of Duttaphrynus melanostictus. We hypothesized low levels of oxidative stress and antioxidant defense during the hibernation period in comparison to the summer active period, due to hypometabolism and their subsequent increase during the arousal period following an increase in body temperature and metabolism. Contrary to our hypothesis, increased oxidative stress with significantly higher lipid peroxidation, protein carbonylation, oxidized glutathione (GSSG): glutathione (GSH) ratio, and elevated antioxidants defense consisting of higher catalase activity and high ascorbic acid content to control oxidative stress were found during hibernation. However, GSH and uric acid levels were found low with super oxide dismutase (SOD) activities at a steady level during hibernation. Supporting our hypothesis, increased oxidative stress with high lipid peroxidation and GSSG:GSH ratio were found during arousal from hibernation owing to increased oxygen consumption and rewarming. Augmented catalase and SOD activities and nonenzymatic antioxidants (GSH, ascorbic acid, and uric acid) level were found to counteract oxidative stress during arousal periods as it was expected. A steady level of protein carbonylation, indicating no oxidative damage during arousal from hibernation due to elevated antioxidant defense, shows the significance of hibernation to overcome food and water scarcity and cold climatic condition. Decrease in antioxidants levels accompanying coming down of lipid peroxidation, protein carbonylation, and GSSG:GSH ratio to their lower levels during the post-arousal period showing normalcy in redox status as it was during active period indicates controllability of oxidative stress in hibernating toads.

Immunoreactivities of AR, ERα, ERß and aromatase in the nuptial pad of Chinese brown frog (Rana dybowskii) during pre-hibernation and the breeding period.

There is a prominent local raised pad called nuptial pad on the forelimb of Chinese brown frog (Rana dybowskii), which is hypothetically concluded as an enhancement of the grip and a spreader of pheromone during the amplexus. In this study, we investigated the immunolocalization and protein expression levels of AR, ERα, ERß and aromatase in the nuptial pad of R. dybowskii during pre-hibernation and the breeding period. Histologically, the annual development of the nuptial pad in R. dybowskii is manifested as the larger area of specialized mucous gland and the longer length of papillary epidermal projection during the breeding period. AR, ERα, ERß and aromatase are present in the stratum granulosum, stratum spinosum, stratum basale and the secretory portion of specialized mucous glands during both periods. Western blotting results confirmed that AR, ERα and ERß protein levels are higher during pre-hibernation than those during the breeding season. These results suggest that nuptial pad is the direct target organ of androgen and estrogen. Androgen may participate in the regulation of annual development and glandular function of nuptial pad, and estrogen may play an endocrine, autocrine or paracrine role during pre-hibernation and the breeding period.

Seasonal changes in adenosine kinase in tanycytes of the Arctic ground squirrel (Urocitellus parryii).

Hibernation is a seasonal strategy to conserve energy, characterized by modified thermoregulation, an increase in sleep pressure and drastic metabolic changes. Glial cells such as astrocytes and tanycytes are the brain metabolic sensors, but it remains unknown whether they contribute to seasonal expression of hibernation. The onset of hibernation is controlled by an undefined endogenous circannual rhythm in which adenosine plays a role through the activation of the A1 adenosine receptor (A1AR). Seasonal changes in brain levels of adenosine may contribute to an increase in A1AR sensitivity leading to the onset of hibernation. The primary regulator of extracellular adenosine concentration is adenosine kinase, which is located in astrocytes. Using immunohistochemistry to localize and quantify adenosine kinase in Arctic ground squirrels' brain collected during different seasons, we report lower expression of adenosine kinase in the third ventricle tanycytes in winter compared to summer; a similar change was not seen in astrocytes. Moreover, for the first time, we describe adenosine kinase expression in tanycyte cell bodies in the hypothalamus and in the area postrema, both brain regions involved in energy homeostasis. Next we describe seasonal changes in tanycyte morphology in the hypothalamus. Although still speculative, our findings contribute to a model whereby adenosine kinase in tanycytes regulates seasonal changes in extracellular concentration of adenosine underling the seasonal expression of hibernation.

Phase specific suppression of neutrophil function in hibernating Syrian hamster.

Hibernation consists of alternating periods of reduced metabolism (torpor) with brief periods of metabolism similar to summer euthermia (arousal). The function of the innate immune system is reduced during hibernation, of which the underlying mechanisms are incompletely understood. Here, we studied neutrophil functionality during hibernation in Syrian hamsters. The inflammatory response to LPS-induced endotoxemia is inhibited in hibernation, partly mediated by reduced IL-6 production in early arousal. Furthermore, neutrophil pathogen binding, phagocytosis and oxidative burst is profoundly reduced in early arousal. Functionality of both summer and early arousal neutrophils was repressed in plasma from early arousal and mixed plasma from early arousal and summer euthermic, but restored by summer euthermic plasma, signifying that a plasma factor in early arousal inhibits TLR-recognition. Identification of the inhibiting factor may offer a target to modulate neutrophil function with relevance to (auto-)inflammatory diseases.

Co-activation of Akt, Nrf2, and NF-κB signals under UPRER in torpid Myotis ricketti bats for survival.

Bats hibernate to survive stressful conditions. Examination of whole cell and mitochondrial proteomes of the liver of Myotis ricketti revealed that torpid bats had endoplasmic reticulum unfolded protein response (UPRER), global reduction in glycolysis, enhancement of lipolysis, and selective amino acid metabolism. Compared to active bats, torpid bats had higher amounts of phosphorylated serine/threonine kinase (p-Akt) and UPRER markers such as PKR-like endoplasmic reticulum kinase (PERK) and activating transcription factor 4 (ATF4). Torpid bats also had lower amounts of the complex of Kelch-like ECH-associated protein 1 (Keap1), nuclear factor erythroid 2-related factor 2 (Nrf2), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) (p65)/I-κBα. Cellular redistribution of 78 kDa glucose-regulated protein (GRP78) and reduced binding between PERK and GRP78 were also seen in torpid bats. Evidence of such was not observed in fasted, cold-treated, or normal mice. These data indicated that bats activate Akt, Nrf2, and NF-κB via the PERK-ATF4 regulatory axis against endoplasmic reticulum stresses during hibernation.

Losing seasonal patterns in a hibernating omnivore? Diet quality proxies and faecal cortisol metabolites in brown bears in areas with and without artificial feeding.

Bears are omnivores particularly well-adapted to variations in the nutritional composition, quality and availability of food resources. Artificial feeding practices have been shown to strongly influence diet composition and seasonality, as well as to cause alterations in wintering and movement in brown bears (Ursus arctos). In this study, we investigated seasonal differences (hypophagia vs hyperphagia) in food quality of two brown bear subpopulations in the Polish Carpathians using faecal nitrogen (FN) and carbon (FC) estimates. The subpopulations inhabit areas that differ in artificial feeding practices: no artificial feeding occurs in the western subpopulation (Tatra Mountains), while artificial food targeted to ungulates is provided and used year-round in the eastern subpopulation (Bieszczady Mountains). We also compared these results with faecal cortisol metabolites (FCM) to explore how FN and FC correlate with the hypothalamic-pituitary-adrenal axis activity and if the seasonal patterns are apparent. We found that in Tatra Mts bears fed on significantly higher quality diet, as shown by FN and FC values, and had significantly higher FC levels in hyperphagia, when they accumulate fat reserves for wintering. The pattern in FCM levels for Tatra subpopulation followed the changes in energy intake during the seasons of hypo- and hyperphagia, while in Bieszczady Mts, the area with intensive feeding, no seasonal patterns could be observed. Artificial feeding practices may disrupt nutrient phenology and seasonality, relative to subpopulations with natural diets. We showed that the availability of human-provided foods may alter not only the overall dietary quality, but also hormonal patterns linked to seasonal nutritional requirements. Combining FN, FC and FCM proved to be a useful tool for reconstructing diet quality and related physiological patterns.