ABSTRACT Temperature, a major abiotic environmental factor, regulates various physiological functions in land snails and therefore determines their biogeographical distribution. Thus, species with different distributions may present different thermal tolerance limits. Additionally, the intense reactivation of snail metabolic rate upon arousal from hibernation or estivation may provoke stress. Land snails, Helix lucorum , display a wide altitudinal distribution resulting in populations being exposed to different seasonal temperature variations. The aim of the present study was to investigate the expression of heat shock proteins (Hsps), mitogen activated protein kinases (MAPKs) and proteins that are related to apoptosis (Bcl-2, ubiquitin), that have ‘cytoprotective’ roles and are also considered to be reliable indicators of stress because of their crucial role in maintaining cellular homeostasis. These proteins were assessed in H. lucorum individuals from two different populations, one at Axios (sea level, 0 m) and the other at Kokkinopilos (Olympus, 1250 m), as well as after mutual population exchanges, in order to find out whether the different responses of these stress-related proteins depend solely on the environmental temperature. The results showed seasonally altered levels in all studied proteins in the hepatopancreas and foot of snails, both among different populations and between the same populations exposed to varying altitudes. However, individuals of the same population in their native habitat or acclimatized to a different habitat showed a relatively similar pattern of expression, supporting the induction of the specific proteins according to the life history of each species.

ABSTRACT Naked mole-rats reduce their metabolic requirements to tolerate severe hypoxia. However, the regulatory mechanisms that underpin this metabolic suppression have yet to be elucidated. 5′-AMP-activated protein kinase (AMPK) is the cellular ‘master’ energy effector and we hypothesized that alterations in the AMPK pathway contribute to metabolic reorganization in hypoxic naked mole-rat skeletal muscle. To test this hypothesis, we exposed naked mole-rats to 4 h of normoxia (21% O 2 ) or severe hypoxia (3% O 2 ), while indirectly measuring whole-animal metabolic rate and fuel preference. We then isolated skeletal muscle and assessed protein expression and post-translational modification of AMPK, and downstream changes in key glucose and fatty acid metabolic proteins mediated by AMPK, including acetyl-CoA carboxylase (ACC1), glycogen synthase (GS) and glucose transporters (GLUTs) 1 and 4. We found that in hypoxic naked mole-rats (1) metabolic rate decreased ∼80% and fuel use switched to carbohydrates, and that (2) levels of activated phosphorylated AMPK and GS, and GLUT4 expression were downregulated in skeletal muscle, while ACC1 was unchanged. To explore the regulatory mechanism underlying this hypometabolic state, we used RT-qPCR to examine 55 AMPK-associated microRNAs (miRNAs), which are short non-coding RNA post-transcriptional silencers. We identified changes in 10 miRNAs (three upregulated and seven downregulated) implicated in AMPK downregulation. Our results suggest that miRNAs and post-translational mechanisms coordinately reduce AMPK activity and downregulate metabolism in naked mole-rat skeletal muscle during severe hypoxia. This novel mechanism may support tissue-specific prioritization of energy for more essential organs in hypoxia.

ABSTRACT From squid at the bottom of the ocean to humans at the top of mountains, animals have adapted to diverse oxygen-limited environments. Surviving these challenging conditions requires global metabolic reorganization that is orchestrated, in part, by microRNAs that can rapidly and reversibly target all biological functions. Herein, we review the involvement of microRNAs in natural models of anoxia and hypoxia tolerance, with a focus on the involvement of oxygen-responsive microRNAs (OxymiRs) in coordinating the metabolic rate depression that allows animals to tolerate reduced oxygen levels. We begin by discussing animals that experience acute or chronic periods of oxygen deprivation at the ocean's oxygen minimum zone and go on to consider more elevated environments, up to mountain plateaus over 3500 m above sea level. We highlight the commonalities and differences between OxymiR responses of over 20 diverse animal species, including invertebrates and vertebrates. This is followed by a discussion of the OxymiR adaptations, and maladaptations, present in hypoxic high-altitude environments where animals, including humans, do not enter hypometabolic states in response to hypoxia. Comparing the OxymiR responses of evolutionarily disparate animals from diverse environments allows us to identify species-specific and convergent microRNA responses, such as miR-210 regulation. However, it also sheds light on the lack of a single unified response to oxygen limitation. Characterizing OxymiRs will help us to understand their protective roles and raises the question of whether they can be exploited to alleviate the pathogenesis of ischemic insults and boost recovery. This Review takes a comparative approach to addressing such possibilities.

ABSTRACT Mole-rats are champions of self-preservation, with increased longevity compared with other rodents their size, strong antioxidant capabilities and specialized defenses against endogenous oxidative stress. However, how the brains of these subterranean mammals handle acute in vivo hypoxia is poorly understood. This study is the first to examine the molecular response to low oxygen in six different species of hypoxia-tolerant mole-rats from sub-Saharan Africa. Protein carbonylation, a known marker of DNA damage (hydroxy-2′-deoxyguanosine), and antioxidant capacity did not change following hypoxia but HIF-1 protein levels increased significantly in the brains of two species. Nearly 30 miRNAs known to play roles in hypoxia tolerance were differentially regulated in a species-specific manner. The miRNAs exhibiting the strongest response to low oxygen stress inhibit apoptosis and regulate neuroinflammation, likely providing neuroprotection. A principal component analysis (PCA) using a subset of the molecular targets assessed herein revealed differences between control and hypoxic groups for two solitary species ( Georychus capensis and Bathyergus suillus ), which are ecologically adapted to a normoxic environment, suggesting a heightened sensitivity to hypoxia relative to species that may experience hypoxia more regularly in nature. By contrast, all molecular data were included in the PCA to detect a difference between control and hypoxic populations of eusocial Heterocephalus glaber , indicating they may require many lower-fold changes in signaling pathways to adapt to low oxygen settings. Finally, none of the Cryptomys hottentotus subspecies showed a statistical difference between control and hypoxic groups, presumably due to hypoxia tolerance derived from environmental pressures associated with a subterranean and social lifestyle.

ABSTRACT The antioxidant defense protects turtles from oxidative stress caused by adverse environment conditions, such as acute thermal fluctuations. However, it remains unclear how these defenses work. The present study examined changes in key enzymes of the enzymatic antioxidant system and the glutathione (GSH) system at both the mRNA and enzyme activity levels during acute cold exposure and recovery in juvenile Chinese soft-shelled turtles, Pelodiscus sinensis . Transcript levels of the upstream regulator NF-E2-related factor 2 (Nrf2) were also measured. Turtles were acclimated at 28°C (3 weeks), then given acute cold exposure (8°C, 12 h) and finally placed in recovery (28°C, 24 h). The mRNA levels of cerebral and hepatic Nrf2 and of genes encoding downstream antioxidant enzymes did not change, whereas levels of nephric Nrf2 , manganese superoxide dismutase ( MnSOD ) and glutathione peroxidase 4 ( GPx4 ) mRNA decreased upon cold exposure. During recovery, Nrf2 mRNA remained stable in all three tissues, hepatic Cu / ZnSOD , MnSOD and catalase ( CAT ) mRNA levels increased, and nephric MnSOD and GPx4 mRNAs did not change from the values during cold exposure. In the GSH system, mRNA levels of most enzymes remained constant during cold exposure and recovery. Unmatched with changes in mRNA level, high and stable constitutive antioxidant enzyme activities were maintained throughout, whereas GPx activity significantly reduced in the kidney during cold exposure, and in liver and kidney during recovery. Our results suggest that the antioxidant defense regulation in response to acute cold exposure in P. sinensis may not be achieved at the transcriptional level, but may rely mainly on high constitutive antioxidant enzyme activities.

ABSTRACT Some animals must endure prolonged periods of oxygen deprivation to survive. One such extreme model is the northern crayfish ( Orconectes virilis ), that regularly survives year-round hypoxic and anoxic stresses in its warm stagnant summer waters and in its cold, ice-locked winter waters. To elucidate the molecular underpinnings of anoxia resistance in this natural model, we surveyed the expression profiles of 76 highly conserved microRNAs in crayfish hepatopancreas and tail muscle from normoxic, acute 2 h anoxia, and chronic 20 h anoxia exposures. MicroRNAs are known to regulate a diverse array of cellular functions required for environmental stress adaptations, and here we explored their role in anoxia tolerance. The tissue-specific anoxia responses observed herein, with 22 anoxia-responsive microRNAs in the hepatopancreas and only four in muscle, suggest that microRNAs facilitate a reprioritization of resources to preserve crucial organ functions. Bioinformatic microRNA target enrichment analysis predicted that the anoxia-downregulated microRNAs in hepatopancreas targeted Hippo signalling, suggesting that cell proliferation and apoptotic signalling are highly regulated in this liver-like organ during anoxia. Compellingly, miR-125-5p , miR-33-5p and miR-190-5p , all known to target the master regulator of oxygen deprivation responses HIF1 (hypoxia inducible factor-1), were anoxia downregulated in the hepatopancreas. The anoxia-increased transcript levels of the oxygen-dependent subunit HIF1α highlight a potential critical role for miRNA-HIF targeting in facilitating a successful anoxia response. Studying the cytoprotective mechanisms in place to protect against the challenges associated with surviving in oxygen-poor environments is critical to elucidating the vast and substantial role of microRNAs in the regulation of metabolism and stress in aquatic invertebrates.

ABSTRACT Wood frogs inhabit a broad range across North America, extending from the southern tip of the Appalachian Mountains to the northern boreal forest. Remarkably, they can survive the winter in a frozen state, where as much as 70% of their body water is converted into ice. Whilst in the frozen state, their hearts cease to pump blood, causing their cells to experience ischemia, which can dramatically increase the production of reactive oxygen species within the cell. To overcome this, wood frogs have elevated levels of glutathione, a primary antioxidant. We examined the regulation of glutathione reductase, the enzyme involved in recycling glutathione, in both the frozen and unfrozen (control) state. Glutathione reductase activity from both the control and frozen state showed a dramatic reduction in substrate specificity ( K m ) for oxidized glutathione (50%) when measured in the presence of glucose (300 mmol l −1 ) and a increase (157%) when measured in the presence of levels of urea (75 mmol l −1 ) encountered in the frozen state. However, when we tested the synergistic effect of urea and glucose simultaneously, we observed a substantial reduction in the K m for oxidized glutathione (43%) to a value similar to that with glucose alone. In fact, we found no observable differences in the kinetic and structural properties of glutathione reductase between the two states. Therefore, a significant increase in the affinity for oxidized glutathione in the presence of endogenous levels of glucose suggests that increased glutathione recycling may occur as a result of passive regulation of glutathione reductase by rising levels of glucose during freezing.

ABSTRACT Hibernating mammals conserve energy in the winter by undergoing prolonged bouts of torpor, interspersed with brief arousals back to euthermia. These bouts are accompanied by a suite of reversible physiological and biochemical changes; however, much remains to be discovered about the molecular mechanisms involved. Given the seasonal nature of hibernation, it stands to reason that underlying plastic epigenetic mechanisms should exist. One such form of epigenomic regulation involves the reversible modification of cytosine bases in DNA by methylation. DNA methylation is well known to be a mechanism that confers upon DNA its cellular identity during differentiation in response to innate developmental cues. However, it has recently been hypothesized that DNA methylation also acts as a mechanism for adapting genome function to changing external environmental and experiential signals over different time scales, including during adulthood. Here, we tested the hypothesis that DNA methylation is altered during hibernation in adult wild animals. This study evaluated global changes in DNA methylation in response to hibernation in the liver and skeletal muscle of thirteen-lined ground squirrels along with changes in expression of DNA methyltransferases (DNMT1/3B) and methyl binding domain proteins (MBDs). A reduction in global DNA methylation occurred in muscle during torpor phases whereas significant changes in DNMTs and MBDs were seen in both tissues. We also report dynamic changes in DNA methylation in the promoter of the myocyte enhancer factor 2C ( mef2c ) gene, a candidate regulator of metabolism in skeletal muscle. Taken together, these data show that genomic DNA methylation is dynamic across torpor–arousal bouts during winter hibernation, consistent with a role for this regulatory mechanism in contributing to the hibernation phenotype.

ABSTRACT Living animals are constantly faced with various environmental stresses that challenge normal life, including: oxygen limitation, very low or high temperature, as well as restriction of water and food. It has been well established that in response to these stresses, tolerant organisms regularly respond with a distinct suite of cellular modifications that involve transcriptional, translational and post-translational modification. In recent years, a new mechanism of rapid and reversible transcriptome regulation, via the action of non-coding RNA molecules, has emerged into post-transcriptional regulation and has since been shown to be part of the survival response. However, these RNA-based mechanisms by which tolerant organisms respond to stressed conditions are not well understood. Recent studies have begun to show that non-coding RNAs control gene expression and translation of mRNA to protein, and can also have regulatory influence over major cellular processes. For example, select microRNAs have been shown to have regulatory influence over the cell cycle, apoptosis, signal transduction, muscle atrophy and fatty acid metabolism during periods of environmental stress. As we are on the verge of dissecting the roles of non-coding RNA in environmental stress adaptation, this Commentary summarizes the hallmark alterations in microRNA expression that facilitate stress survival.

For many animals, survival of severe environmental stress (e.g. to extremes of heat or cold, drought, oxygen limitation, food deprivation) is aided by entry into a hypometabolic state. Strong depression of metabolic rate, often to only 1–20% of normal resting rate, is a core survival strategy of multiple forms of hypometabolism across the animal kingdom, including hibernation, anaerobiosis, aestivation and freeze tolerance. Global biochemical controls are needed to suppress and reprioritize energy use; one such well-studied control is reversible protein phosphorylation. Recently, we turned our attention to the idea that mechanisms previously associated mainly with epigenetic regulation can also contribute to reversible suppression of gene expression in hypometabolic states. Indeed, situations as diverse as mammalian hibernation and turtle anoxia tolerance show coordinated changes in histone post-translational modifications (acetylation, phosphorylation) and activities of histone deacetylases, consistent with their use as mechanisms for suppressing gene expression during hypometabolism. Other potential mechanisms of gene silencing in hypometabolic states include altered expression of miRNAs that can provide post-transcriptional suppression of mRNA translation and the formation of ribonuclear protein bodies in the nucleus and cytoplasm to allow storage of mRNA transcripts until animals rouse themselves again. Furthermore, mechanisms first identified in epigenetic regulation (e.g. protein acetylation) are now proving to apply to many central metabolic enzymes (e.g. lactate dehydrogenase), suggesting a new layer of regulatory control that can contribute to coordinating the depression of metabolic rate.

The jumbo squid, Dosidicus gigas , can survive extended forays into the oxygen minimum zone (OMZ) of the Eastern Pacific Ocean. Previous studies have demonstrated reduced oxygen consumption and a limited anaerobic contribution to ATP production, suggesting the capacity for substantial metabolic suppression during hypoxic exposure. Here, we provide a more complete description of energy metabolism and explore the expression of proteins indicative of transcriptional and translational arrest that may contribute to metabolic suppression. We demonstrate a suppression of total ATP demand under hypoxic conditions (1% oxygen, P O 2 =0.8 kPa) in both juveniles (52%) and adults (35%) of the jumbo squid. Oxygen consumption rates are reduced to 20% under hypoxia relative to air-saturated controls. Concentrations of arginine phosphate (Arg-P) and ATP declined initially, reaching a new steady state (~30% of controls) after the first hour of hypoxic exposure. Octopine began accumulating after the first hour of hypoxic exposure, once Arg-P breakdown resulted in sufficient free arginine for substrate. Octopine reached levels near 30 mmol g −1 after 3.4 h of hypoxic exposure. Succinate did increase through hypoxia but contributed minimally to total ATP production. Glycogenolysis in mantle muscle presumably serves to maintain muscle functionality and balance energetics during hypoxia. We provide evidence that post-translational modifications on histone proteins and translation factors serve as a primary means of energy conservation and that select components of the stress response are altered in hypoxic squids. Reduced ATP consumption under hypoxia serves to maintain ATP levels, prolong fuel store use and minimize the accumulation of acidic intermediates of anaerobic ATP-generating pathways during prolonged diel forays into the OMZ. Metabolic suppression likely limits active, daytime foraging at depth in the core of the OMZ, but confers an energetic advantage over competitors that must remain in warm, oxygenated surface waters. Moreover, the capacity for metabolic suppression provides habitat flexibility as OMZs expand as a result of climate change.

Brevinin-1SY is the only described antimicrobial peptide (AMP) of Rana sylvatica . As AMPs are important innate immune molecules that inhibit microbes, this study examined brevinin-1SY regulation during development and in adult frogs in response to environmental stress. The brevinin-1SY nucleotide sequence was identified and used for protein modeling. Brevinin-1SY was predicted to be an amphipathic, hydrophobic, alpha helical peptide that inserts into a lipid bilayer. Brevinin-1SY transcripts were detected in tadpoles and were significantly increased during the later stages of development. Effects of environmental stress (24 h anoxia, 40% dehydration or 24 h frozen) on the mRNA levels of brevinin-1SY in the dorsal and ventral skin were examined. The brevinin-1SY mRNA levels were increased in dorsal and ventral skin of dehydrated frogs, and in ventral skin of anoxic frogs, compared with controls (non-stressed). Brevinin-1SY protein levels in peptide extracts of dorsal skin showed a similar, but not significant, trend to that of brevinin-1SY mRNA levels. Antimicrobial activity of skin extracts from control and stressed animals were assessed for Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Botrytis cinerea, Rhizopus stolonifer and Pythium sulcatum using disk diffusion assays. Peptide extracts of dorsal skin from anoxic, frozen and dehydrated animals showed significantly higher inhibition of E. coli and P. sulcatum than from control animals. In ventral skin peptide extracts, significant growth inhibition was observed in frozen animals for E. coli and P. sulcatum , and in anoxic animals for B. cinerea , compared with controls. Environmental regulation of brevinin-1SY may have important implications for defense against pathogens.

SUMMARY Hibernation is a crucial strategy of winter survival used by many mammals. During hibernation, thirteen-lined ground squirrels, Ictidomys tridecemlineatus , cycle through a series of torpor bouts, each lasting more than a week, during which the animals are largely immobile. Previous hibernation studies have demonstrated that such natural models of skeletal muscle disuse cause limited or no change in either skeletal muscle size or contractile performance. However, work loop analysis of skeletal muscle, which provides a realistic assessment of in vivo power output, has not previously been undertaken in mammals that undergo prolonged torpor during hibernation. In the present study, our aim was to assess the effects of 3 months of hibernation on contractile performance (using the work loop technique) and several biochemical properties that may affect performance. There was no significant difference in soleus muscle power output–cycle frequency curves between winter (torpid) and summer (active) animals. Total antioxidant capacity of gastrocnemius muscle was 156% higher in torpid than in summer animals, suggesting one potential mechanism for maintenance of acute muscle performance. Soleus muscle fatigue resistance was significantly lower in torpid than in summer animals. Gastrocnemius muscle glycogen content was unchanged. However, state 3 and state 4 mitochondrial respiration rates were significantly suppressed, by 59% and 44%, respectively, in mixed hindlimb skeletal muscle from torpid animals compared with summer controls. These findings in hindlimb skeletal muscles suggest that, although maximal contractile power output is maintained in torpor, there is both suppression of ATP production capacity and reduced fatigue resistance.

SUMMARY Ectotherms overwintering in temperate ecosystems must survive low temperatures while conserving energy to fuel post-winter reproduction. Freeze-tolerant wood frogs, Rana sylvatica , have an active response to the initiation of ice formation that includes mobilising glucose from glycogen and circulating it around the body to act as a cryoprotectant. We used flow-through respirometry to measure CO 2 production ( ) in real time during cooling, freezing and thawing. CO 2 production increases sharply at three points during freeze–thaw: at +1°C during cooling prior to ice formation (total of 104±17 μl CO 2 frog −1 event −1 ), at the initiation of freezing (565±85 μl CO 2 frog −1 freezing event −1 ) and after the frog has thawed (564±75 μ l CO 2 frog −1 freezing event −1 ). We interpret these increases in metabolic rate to represent the energetic costs of preparation for freezing, the response to freezing and the re-establishment of homeostasis and repair of damage after thawing, respectively. We assumed that frogs metabolise lipid when unfrozen and that carbohydrate fuels metabolism during cooling, freezing and thawing, and when frozen. We then used microclimate temperature data to predict overwinter energetics of wood frogs. Based on the freezing and melting points we measured, frogs in the field were predicted to experience as many as 23 freeze–thaw cycles in the winter of our microclimate recordings. Overwinter carbohydrate consumption appears to be driven by the frequency of freeze–thaw events, and changes in overwinter climate that affect the frequency of freeze–thaw will influence carbohydrate consumption, but changes that affect mean temperatures and the frequency of winter warm spells will modify lipid consumption.

SUMMARY For many small mammals, survival over the winter months is a serious challenge because of low environmental temperatures and limited food availability. The solution for many species, such as thirteen-lined ground squirrels ( Ictidomys tridecemlineatus ), is hibernation, an altered physiological state characterized by seasonal heterothermy and entry into long periods of torpor that are interspersed with short arousals back to euthermia. During torpor, metabolic rate is strongly reduced to achieve major energy savings, and a coordinated depression of non-essential ATP-expensive functions such as protein synthesis takes place. This study examines the mammalian target of rapamycin (mTOR) signaling pathway, a crucial component of the insulin receptor network, over six stages of the torpor–arousal cycle of hibernation. Immunoblots showed that the phosphorylation state of mTOR Ser2448 was strongly reduced in skeletal muscle (by 55%) during late torpor but increased by 200% during early arousal compared with euthermia. However, the phosphorylation state of this residue remained relatively constant in cardiac muscle during torpor but was enhanced during entrance into torpor and early arousal from torpor stages (by 2.9- and 3.2-fold, respectively). Phosphorylation states of upstream regulators of mTOR, p-Akt Thr473 and p-TSC2 Thr1462 , were also suppressed in skeletal muscle by 55 and 51%, respectively, during late torpor, as were selected downstream substrates – p-4E-BP1 Thr46 and p-S6 Ser235 contents dropped by 74 and 41%, respectively. Overall, the results indicate suppressed mTOR signaling in skeletal muscle, but not cardiac muscle, during torpor. By contrast, activation of mTOR and other components of the mTORC1 complex (p-PRAS40 Thr246 and GβL) occurred during early arousal in both skeletal and cardiac muscle.

Summary Aestivation is a survival strategy used by many vertebrates and invertebrates to endure arid environmental conditions. Key features of aestivation include strong metabolic rate suppression, strategies to retain body water, conservation of energy and body fuel reserves, altered nitrogen metabolism, and mechanisms to preserve and stabilize organs, cells and macromolecules over many weeks or months of dormancy. Cell signaling is crucial to achieving both a hypometabolic state and reorganizing multiple metabolic pathways to optimize long-term viability during aestivation. This commentary examines the current knowledge about cell signaling pathways that participate in regulating aestivation, including signaling cascades mediated by the AMP-activated kinase, Akt, ERK, and FoxO1.

SUMMARY The terrestrial anuran Rana sylvatica tolerates extended periods of whole-body freezing during the winter. Freezing survival is facilitated by extensive glycogen hydrolysis and distribution of high concentrations of the cryoprotectant glucose into blood and all tissues. As glycogenesis is both an energy-expensive process and counter-productive to maintaining sustained high cryoprotectant levels, we proposed that glycogen synthase kinase-3 (GSK-3) would be activated when wood frogs froze and would phosphorylate its downstream substrates to inactivate glycogen synthesis. Western blot analysis determined that the amount of phosphorylated (inactive) GSK-3 decreased in all five tissues tested in 24 h frozen frogs compared with unfrozen controls. Total GSK-3 protein levels did not change, with the exception of heart GSK-3, indicating that post-translational modification was the primary regulatory mechanism for this kinase. Kinetic properties of skeletal muscle GSK-3 from control and frozen frogs displayed differential responses to a temperature change (22 versus 4°C) and high glucose. For example, when assayed at 4°C, the K m for the GSK-3 substrate peptide was ∼44% lower for frozen frogs than the corresponding value in control frogs, indicating greater GSK-3 affinity for its substrates in the frozen state. This indicates that at temperatures similar to the environment encountered by frogs, GSK-3 in frozen frogs will phosphorylate its downstream targets more readily than in unfrozen controls. GSK-3 from skeletal muscle of control frogs was also allosterically regulated. AMP and phosphoenolpyruvate activated GSK-3 whereas inhibitors included glucose, glucose 6-phosphate, pyruvate, ATP, glutamate, glutamine, glycerol, NH 4 Cl, NaCl and KCl. The combination of phosphorylation and allosteric control argues for a regulatory role of GSK-3 in inactivating glycogenesis to preserve high glucose cryoprotectant levels throughout each freezing bout.

SUMMARY Myostatin, a negative regulator of muscle mass, is elevated during disuse and starvation. Mammalian hibernation presents a unique scenario, where animals are hypocaloric and in torpor, but the extent of muscle protein loss is minimized. We hypothesized that myostatin expression, which is usually increased early in disuse and under hypocaloric conditions, could be suppressed in this unique model. Skeletal muscle was collected from thirteen-lined ground squirrels, Spermophilus tridecemlineatus , at six time points during hibernation: control euthermic (CON); entrance into hibernation (ENT), body temperature ( T b ) falling; early hibernation (EHib), stable T b in torpor for 24 h; late hibernation (LHib), stable T b in torpor for 3 days; early arousal (EAr), T b rising; and arousal (AR), T b restored to 34–37°C for about 18 h. There was no significant increase of myostatin during ENT, EHib or LHib. Unexpectedly, there were approximately sixfold increases in myostatin protein levels as squirrels arose from torpor. The elevation during EAr remained high in AR, which represented an interbout time period. Mechanisms that could release the suppression or promote increased levels of myostatin were assessed. SMAD2 and phosphorylated SMAD2 were increased during EHib, but only the phosphorylated SMAD2 during AR mirrored increases in myostatin. Follistatin, a negative regulator of myostatin, did not follow the same time course as myostatin or its signaling pathway, indicating more control of myostatin at the signaling level. However, SMAD7, an inhibitory SMAD, did not appear to play a significant role during deep hibernation. Hibernation is an excellent natural model to study factors involved in the endogenous intracellular mechanisms controlling myostatin.

SUMMARY In its native environment the African clawed frog, Xenopus laevis ,can experience seasonally arid conditions that impose dehydration stress. Activation of intracellular signal transduction cascades can mediate and coordinate biochemical responses to ameliorate dehydration stress. This study examines the extracellular signal-regulated kinase (ERK) signaling cascade,analyzing responses of both upstream and downstream components in six tissues of X. laevis experiencing medium and high levels of dehydration,16.6±1.59 and 28.0±1.6% of total body water lost, respectively. Immunoblotting was used to assess the three tiers in this mitogen-activated protein kinase (MAPK) cascade: the initiating MAPK kinase kinases (c-Raf,MEKK), the MAPK kinase (MEK1/2), and finally the MAPK (ERK1/2). The amount of active phosphorylated c-Raf Ser338 rose by 2- to 2.5-fold under high dehydration in muscle, lung and skin whereas MEKK protein levels rose in these organs and also increased 4-fold in liver. As a result, phosphorylated active MEK1/2 Ser217/221 increased significantly by 2- to 6-fold during dehydration which, in turn, led to 2- to 6-fold increases in phospho-ERK Thr202/Tyr204 content in all tissues except skin. Given this clear demonstration of ERK cascade activation, two downstream targets of ERK2 were then evaluated. The amount of phosphorylated active transcription factor, STAT3 Ser727 and p90 ribosomal S6 kinase(RSK Ser380 ) increased particularly in muscle, lung and kidney. Furthermore, RSK activation was correlated with a 5- to 8-fold increase in phosphorylation of its target, S6 ribosomal protein. Overall, the results show a strong conserved activation of the ERK cascade in X. laevis tissues in response to dehydration.

SUMMARY Entry into a hypometabolic state is an important survival strategy for many organisms when challenged by environmental stress, including low oxygen, cold temperatures and lack of food or water. The molecular mechanisms that regulate transitions to and from hypometabolic states, and stabilize long-term viability during dormancy, are proving to be highly conserved across phylogenic lines. A number of these mechanisms were identified and explored using anoxia-tolerant turtles as the model system, particularly from the research contributions made by Dr Peter L. Lutz in his explorations of the mechanisms of neuronal suppression in anoxic brain. Here we review some recent advances in understanding the biochemical mechanisms of metabolic arrest with a focus on ideas such as the strategies used to reorganize metabolic priorities for ATP expenditure, molecular controls that suppress cell functions (e.g. ion pumping, transcription, translation, cell cycle arrest),changes in gene expression that support hypometabolism, and enhancement of defense mechanisms (e.g. antioxidants, chaperone proteins, protease inhibitors) that stabilize macromolecules and promote long-term viability in the hypometabolic state.