Does hypometabolism constrain innate immune defense?

Many animals routinely make energetic trade-offs to adjust to environmental demands and these trade-offs often have significant implications for survival. For example, environmental hypoxia is commonly experienced by many organisms and is an energetically challenging condition because reduced oxygen availability constrains aerobic energy production, which can be lethal. Many hypoxia-tolerant species downregulate metabolic demands when oxygen is limited; however, certain physiological functions are obligatory and must be maintained despite the need to conserve energy in hypoxia. Of particular interest is immunity (including both constitutive and induced immune functions) because mounting an immune response is among the most energetically expensive physiological processes but maintaining immune function is critical for survival in most environments. Intriguingly, physiological responses to hypoxia and pathogens share key molecular regulators such as hypoxia-inducible factor-1α, through which hypoxia can directly activate an immune response. This raises an interesting question: do hypoxia-tolerant species mount an immune response during periods of hypoxia-induced hypometabolism? Unfortunately, surprisingly few studies have examined interactions between immunity and hypometabolism in such species. Therefore, in this review, we consider mechanistic interactions between metabolism and immunity, as well as energetic trade-offs between these two systems, in hypoxia-tolerant animals but also in other models of hypometabolism, including neonates and hibernators. Specifically, we explore the hypothesis that such species have blunted immune responses in hypometabolic conditions and/or use alternative immune pathways when in a hypometabolic state. Evidence to date suggests that hypoxia-tolerant animals do maintain immunity in low oxygen conditions, but that the sensitivity of immune responses may be blunted.

Status of the Janus Kinase-Signal Transducer and Activator of Transcription (JAK-STAT) pathway in liver and skin of the freeze tolerant wood frog.

The wood frog (Rana sylvatica) has adapted full-body freezing and thawing as a means of sub-zero winter survival and early-breeding in ephemeral pools. One such protective process implicated recently in freeze-thaw tolerance is that of anti-apoptotic signaling, which has been proposed to play a cytoprotective role by modulating stress-induced death signals. This study employed the use of immunoblotting to examine response of a potent cell cycle and apoptosis regulator, known as the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway, to freezing and thawing in the liver and skin of the wood frog. This pathway demonstrably exhibits factor- and tissue-specific changes between non-frozen, 24 h-frozen, and 8 h-thawed conditions. There were few changes in JAK-STAT proteins in frozen frogs, but protective changes were observed upon thaw: Elevated levels of pJAK3 and nuclear localization of pSTAT3 and pSTAT5 suggested an increase in anti-apoptotic signaling after thaw. By contrast, both STAT1 and STAT3 signaling appeared to increase in frozen skin, suggesting frogs use homeostatic regulation of apoptotic- and anti-apoptotic signals, in an antagonistic and compensatory manner. As such, these findings support that JAK-STAT pathway signaling modulation is a plausible adaptation that contributes to fast and reversible manipulation of anti-apoptotic signals, thus assisting in freeze survival of the wood frog.

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

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

Isoflurane and low-level carbon monoxide exposures increase expression of pro-survival miRNA in neonatal mouse heart.

Anesthetics such as isoflurane are known to cause apoptosis in the developing mammalian brain. However, isoflurane may have protective effects on the heart via relieving ischemia and downregulating genes related to apoptosis. Ischemic preconditioning, e.g. through the use of low levels of carbon monoxide (CO), has promise in preventing ischemia-reperfusion injury and cell death. However, it is still unclear how it either triggers the stress response in neonatal hearts. For this reason, thirty-three microRNAs (miRNAs) known to be differentially expressed following anesthesia and/or ischemic or hypoxic heart damage were investigated in the hearts from neonatal mice exposed to isoflurane or low level of CO, using an air-exposed control group. Only miR-93-5p increased with isoflurane exposure, which may be associated with the suppression of cell death, autophagy, and inflammation. By contrast, twelve miRNAs were differentially expressed in the heart following CO treatment. Many miRNAs previously shown to be responsible for suppressing cell death, autophagy, and myocardial hypertrophy were upregulated (e.g., 125b-3p, 19-3p, and 21a-5p). Finally, some miRNAs (miR-103-3p, miR-1a-3p, miR-199a-1-5p) which have been implicated in regulating energy balance and cardiac contraction were also differentially expressed. Overall, this study demonstrated that CO-mediated miRNA regulation may promote ischemic preconditioning and cardioprotection based on the putative protective roles of the differentially expressed miRNAs explored herein and the consistency of these results with those that have shown positive effects of CO on heart viability following anesthesia and ischemia-reperfusion stress.

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

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

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

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

The Ratio of Linoleic and Linolenic Acid in the Pre-hibernation Diet Influences NFκB Signaling in Garden Dormice During Torpor.

The fatty acid composition of a pre-hibernation diet can influence the depth and duration of metabolic suppression achieved by hibernators. More specifically, a diet high in n-6 polyunsaturated fatty acids (PUFAs) relative to n-3 PUFAs is essential to maximize torpor expression. However, few studies have investigated how diets with different n-6/n-3 PUFA ratios change stress-inducible cell signaling. Garden dormice (Eliomys quercinus) were fed one of three diets designed with different ratios of n-6 PUFA linoleic acid (LA) and n-3 PUFA linolenic acid (ALA). Then, NFκB signaling was assessed in the white adipose, brown adipose, and liver tissues of euthermic and hibernating dormice via multiplex and RT-qPCR analyses of relative protein and transcript levels, respectively. Dormice fed a high LA diet regulated NFκB signaling in a protective manner in all tissues. NFκB signaling was generally decreased in the high LA group, with significant decreases in the protein levels of NFκB mediators IKKα/ß, IκBα, and downstream pro-apoptotic protein FADD. Liver and white adipose from torpid dormice fed a high LA diet increased sod2 expression relative to the other diets or relative to euthermic controls, indicating protection against ROS generated from potentially increased ß-oxidation of n-6 PUFAs. The low LA diet increased biomarkers for apoptosis relative to other diets and relative to euthermia, suggesting low LA diets may be detrimental to hibernator health. Overall, this study suggests that changes in the ratio of n-6/ n-3 PUFAs in the diet influences apoptotic and antioxidant responses in white adipose, brown adipose, and liver of hibernating garden dormice.

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

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

The brains of six African mole-rat species show divergent responses to hypoxia.

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.

Regulation of Peroxisome Proliferator-Activated Receptor Pathway During Torpor in the Garden Dormouse, Eliomys quercinus.

Differential levels of n-6 and n-3 essential polyunsaturated fatty acids (PUFAs) are incorporated into the hibernator's diet in the fall season preceding prolonged, multi-days bouts of torpor, known as hibernation. Peroxisome proliferator-activated receptor (PPAR) transcriptional activators bind lipids and regulate genes involved in fatty acid transport, beta-oxidation, ketogenesis, and insulin sensitivity; essential processes for survival during torpor. Thus, the DNA-binding activity of PPARα, PPARδ, PPARγ, as well as the levels of PPARγ coactivator 1α (PGC-1α) and L-fatty acid binding protein (L-FABP) were investigated in the hibernating garden dormouse (Eliomys quercinus). We found that dormice were hibernating in a similar way regardless of the n-6/n-3 PUFA diets fed to the animals during the fattening phase prior to hibernation. Further, metabolic rates and body mass loss during hibernation did not differ between dietary groups, despite marked differences in fatty acid profiles observed in white adipose tissue prior and at mid-hibernation. Overall, maintenance of PPAR DNA-binding activity was observed during torpor, and across three n-6/n-3 ratios, suggesting alternate mechanisms for the prioritization of lipid catabolism during torpor. Additionally, while no change was seen in L-FABP, significantly altered levels of PGC-1α were observed within the white adipose tissue and likely contributes to enhanced lipid metabolism when the diet favors n-6 PUFAs, i.e., high n-6/n-3 ratio, in both the torpid and euthermic state. Altogether, the maintenance of lipid metabolism during torpor makes it likely that consistent activity or levels of the investigated proteins are in aid of this metabolic profile.

The Torpid State: Recent Advances in Metabolic Adaptations and Protective Mechanisms†.

Torpor and hibernation are powerful strategies enabling animals to survive periods of low resource availability. The state of torpor results from an active and drastic reduction of an individual's metabolic rate (MR) associated with a relatively pronounced decrease in body temperature. To date, several forms of torpor have been described in all three mammalian subclasses, i.e., monotremes, marsupials, and placentals, as well as in a few avian orders. This review highlights some of the characteristics, from the whole organism down to cellular and molecular aspects, associated with the torpor phenotype. The first part of this review focuses on the specific metabolic adaptations of torpor, as it is used by many species from temperate zones. This notably includes the endocrine changes involved in fat- and food-storing hibernating species, explaining biomedical implications of MR depression. We further compare adaptive mechanisms occurring in opportunistic vs. seasonal heterotherms, such as tropical and sub-tropical species. Such comparisons bring new insights into the metabolic origins of hibernation among tropical species, including resistance mechanisms to oxidative stress. The second section of this review emphasizes the mechanisms enabling heterotherms to protect their key organs against potential threats, such as reactive oxygen species, associated with the torpid state. We notably address the mechanisms of cellular rehabilitation and protection during torpor and hibernation, with an emphasis on the brain, a central organ requiring protection during torpor and recovery. Also, a special focus is given to the role of an ubiquitous and readily-diffusing molecule, hydrogen sulfide (H2S), in protecting against ischemia-reperfusion damage in various organs over the torpor-arousal cycle and during the torpid state. We conclude that (i) the flexibility of torpor use as an adaptive strategy enables different heterothermic species to substantially suppress their energy needs during periods of severely reduced food availability, (ii) the torpor phenotype implies marked metabolic adaptations from the whole organism down to cellular and molecular levels, and (iii) the torpid state is associated with highly efficient rehabilitation and protective mechanisms ensuring the continuity of proper bodily functions. Comparison of mechanisms in monotremes and marsupials is warranted for understanding the origin and evolution of mammalian torpor.

Angiogenic signaling in the lungs of a metabolically suppressed hibernating mammal (Ictidomys tridecemlineatus).

To conserve energy in times of limited resource availability, particularly during cold winters, hibernators suppress even the most basic of physiologic processes. Breathing rates decrease from 40 breaths/minute to less than 1 breath/min as they decrease body temperature from 37 °C to ambient. Nevertheless, after months of hibernation, these incredible mammals emerge from torpor unscathed. This study was conducted to better understand the protective and possibly anti-inflammatory adaptations that hibernator lungs may use to prevent damage associated with entering and emerging from natural torpor. We postulated that the differential protein expression of soluble protein receptors (decoy receptors that sequester soluble ligands to inhibit signal transduction) would help identify inhibited inflammatory signaling pathways in metabolically suppressed lungs. Instead, the only two soluble receptors that responded to torpor were sVEGFR1 and sVEGFR2, two receptors whose full-length forms are bound by VEGF-A to regulate endothelial cell function and angiogenesis. Decreased sVEGFR1/2 correlated with increased total VEGFR2 protein levels. Maintained or increased levels of key γ-secretase subunits suggested that decreased sVEGFR1/2 protein levels were not due to decreased levels of intramembrane cleavage complex subunits. VEGF-A protein levels did not change, suggesting that hibernators may regulate VEGFR1/2 signaling at the level of the receptor instead of increasing relative ligand abundance. A panel of angiogenic factors used to identify biomarkers of angiogenesis showed a decrease in FGF-1 and an increase in BMP-9. Torpid lungs may use VEGF and BMP-9 signaling to balance angiogenesis and vascular stability, possibly through the activation of SMAD signaling for adaptive tissue remodeling.

Temperature and serine phosphorylation regulate glycerol-3-phosphate dehydrogenase in skeletal muscle of hibernating Richardson's ground squirrels.

Glycerol-3-phosphate dehydrogenase (G3PDH) bridges carbohydrate and lipid metabolism by interconverting glycerol-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP). This reversible reaction converts G3P derived from triglyceride hydrolysis to DHAP that can then enter glycolysis or gluconeogenesis and, in the reverse reaction, makes G3P for use in triglyceride biosynthesis. Small hibernating mammals rely almost exclusively on triglyceride reserves as their fuel for energy production during torpor and the recovery of glycerol after lipolysis is an important source of carbohydrate over the nonfeeding winter months. G3PDH (∼37 kDa) was purified from skeletal muscle of euthermic and hibernating Richardson's ground squirrels (Urocitellus richardsonii) using three column chromatography steps. Analysis of enzyme kinetic properties revealed that G3PDH from hibernator muscle had higher affinities for G3P and NAD at low (5 °C) assay temperature compared with high (21 or 37 °C) and a greater stability in the presence of denaturing agents (urea, guanidine hydrochloride) or high temperature (50 °C). Immunoblotting showed that hibernator muscle G3PDH had a higher phosphoserine content than the enzyme from euthermic controls and incubation studies showed that enzyme affinity for G3P changed significantly by stimulating endogenous protein kinases or phosphatases. Overall, this study suggests that the properties of ground squirrel muscle G3PDH are modulated by temperature and post-translational phosphorylation to alter enzyme function under euthermic versus hibernating states.

The squirrel with the lagging eIF2: Global suppression of protein synthesis during torpor.

Hibernating mammals use strong metabolic rate depression and a reduction in body temperature to near-ambient to survive the cold winter months. During torpor, protein synthesis is suppressed but can resume during interbout arousals. The current study aimed to identify molecular targets responsible for the global suppression of protein synthesis during torpor as well as possible mechanisms that could allow for selective protein translation to continue over this time. Relative changes in protein expression and/or phosphorylation levels of key translation factors (ribosomal protein S6, eIF4E, eIF2α, eEF2) and their upstream regulators (mTOR, TSC2, p70 S6K, 4EBP) were analyzed in liver and kidney of 13-lined ground squirrels (Ictidomys tridecemlineatus) sampled from six points over the torpor-arousal cycle. The results indicate that both organs reduce protein synthesis during torpor by decreasing mTOR and TSC2 phosphorylation between 30 and 70% of control levels. Translation resumes during interbout arousal when p-p70 S6K, p-rpS6, and p-4EBP levels returned to control values or above. Only liver translation factors were activated or disinhibited during periods of torpor itself, with >3-fold increases in total eIF2α and eEF2 protein levels, and a decrease in p-eEF2 (T56) to as low as 16% of the euthermic control value. These data shed light on a possible molecular mechanism involving eIF2α that could enable the translation of key transcripts during times of cell stress.

Pro-inflammatory AGE-RAGE signaling is activated during arousal from hibernation in ground squirrel adipose.

BACKGROUND: Inflammation is generally suppressed during hibernation, but select tissues (e.g. lung) have been shown to activate both antioxidant and pro-inflammatory pathways, particularly during arousal from torpor when breathing rates increase and oxidative metabolism fueling the rewarming process produces more reactive oxygen species. Brown and white adipose tissues are now understood to be major hubs for the regulation of immune and inflammatory responses, yet how these potentially damaging processes are regulated by fat tissues during hibernation has hardly been studied. The advanced glycation end-product receptor (RAGE) can induce pro-inflammatory responses when bound by AGEs (which are glycated and oxidized proteins, lipids, or nucleic acids) or damage associated molecular pattern molecules (DAMPs, which are released from dying cells). METHODS: Since gene expression and protein synthesis are largely suppressed during torpor, increases in AGE-RAGE pathway proteins relative to a euthermic control could suggest some role for these pro-inflammatory mediators during hibernation. This study determined how the pro-inflammatory AGE-RAGE signaling pathway is regulated at six major time points of the torpor-arousal cycle in brown and white adipose from a model hibernator, Ictidomys tridecemlineatus. Immunoblotting, RT-qPCR, and a competitive ELISA were used to assess the relative gene expression and protein levels of key regulators of the AGE-RAGE pathway during a hibernation bout. RESULTS: The results of this study revealed that RAGE is upregulated as animals arouse from torpor in both types of fat, but AGE and DAMP levels either remain unchanged or decrease. Downstream of the AGE-RAGE cascade, nfat5 was more highly expressed during arousal in brown adipose. DISCUSSION: An increase in RAGE protein levels and elevated mRNA levels of the downstream transcription factor nfat5 during arousal suggest the pro-inflammatory response is upregulated in adipose tissue of the hibernating ground squirrel. It is unlikely that this cascade is activated by AGEs or DAMPs. This research sheds light on how a fat-but-fit organism with highly regulated metabolism may control the pro-inflammatory AGE-RAGE pathway, a signaling cascade that is often dysregulated in other obese organisms.

Potential role for microRNA in regulating hypoxia-induced metabolic suppression in jumbo squids.

At night, Humboldt squid (Dosidicus gigas) rise to the ocean's surface to feed, but come morning, they descend into the ocean's oxygen minimum zone where they can avoid predators but must deal with severe hypoxia, high pressure, and very cold water. To survive this extreme environment, squid use various adaptations to enter a hypometabolic state characterized by metabolic rate suppression by 35-52%, relative to normoxic conditions. The molecular mechanisms facilitating this metabolic flexibility have yet to be elucidated in hypometabolic squid. Herein, we report the first investigation of the role of microRNAs, a rapid and reversible post-transcriptional master regulator of virtually all biological functions, in cephalopods. We examined expression levels of 39 highly-conserved invertebrate microRNAs in D. gigas brain, mantle muscle, and branchial heart, comparing hypoxic and normoxic conditions. Hypoxia-inducible microRNAs are potentially involved in facilitating neuroprotection, anti-apoptosis, and regenerative mechanisms in brain; inhibiting apoptosis and cell proliferation while conserving energy in heart; and limiting damage by reactive oxygen species and apoptosis in muscle. Rather than orchestrate global metabolic rate depression, the majority of hypoxia-inducible microRNAs identified are involved in promoting cytoprotective mechanisms, suggesting a regulatory role for microRNA in hypoxic marine invertebrates that sets the stage for mechanistic analyses.

Tissue-specific response of carbohydrate-responsive element binding protein (ChREBP) to mammalian hibernation in 13-lined ground squirrels.

Mammalian hibernation is characterized by a general suppression of energy expensive processes and a switch to lipid oxidation as the primary fuel source. Glucose-responsive carbohydrate responsive element binding protein (ChREBP) has yet to be studied in hibernating organisms, which prepare for the cold winter months by feeding until they exhibit an obesity-like state that is accompanied by naturally-induced and completely reversible insulin resistance. Studying ChREBP expression and activity in the hibernating 13-lined ground squirrel is important to better understand the molecular mechanisms that regulate energy metabolism under cellular stress. Immunoblotting was used to determine the relative expression level and subcellular localization of ChREBP, as well as serine phosphorylation at 95 kDa, comparing euthermic and late torpid ground squirrel liver, kidney, heart and muscle. DNA-binding ELISAs and RT-PCR were used to explore ChREBP transcriptional activity during cold stress. ChREBP activity seemed generally suppressed in liver and kidney. During torpor, ChREBP total protein levels decreased to 44% of EC in liver, phosphoserine levels increased 2.1-fold of EC in kidney, and downstream Fasn/Pkl transcript levels decreased to <60% of EC in liver. By contrast, ChREBP activity generally increased during torpor in cardiac and skeletal muscle, where ChREBP total protein levels increased over 1.5-fold and 5-fold of EC in muscle and heart, respectively; where DNA-binding increased by ∼2-fold of EC in muscle; and where Fasn transcript levels increased over 3-fold and 7-fold in both muscle and heart, respectively. In summary, ChREBP has a tissue-specific role in regulating energy metabolism during hibernation.

Turn down genes for WAT? Activation of anti-apoptosis pathways protects white adipose tissue in metabolically depressed thirteen-lined ground squirrels.

During hibernation, the metabolic rate of thirteen-lined ground squirrels (Ictidomys tridecemlineatus) can drop to <5 % of normal resting rate at 37 °C, core body temperature can decrease to as low as 1-5 °C, and heart rate can fall from 350-400 to 5-10 bpm. Energy saved by hibernating allows squirrels to survive the winter when food is scarce, and living off lipid reserves in white adipose tissue (WAT) is crucial. While hibernating, some energy must be used to cope with conditions that would normally be damaging for mammals (e.g., low core body temperatures, ischemia) and could induce cell death via apoptosis. Cell survival is largely dependent on the relative amounts and activities of pro- and anti-apoptotic Bcl-2 family proteins. The present study analyzed how anti-apoptotic proteins respond to protect WAT cells during hibernation. Relative levels of several anti-apoptotic proteins were quantified in WAT via immunoblotting over six time points of the torpor-arousal cycle. These included anti-apoptotic Bcl-2 family members Bcl-2, Bcl-xL, and Mcl-l, as well as caspase inhibitors x-IAP and c-IAP. Changes in the relative protein levels and/or phosphorylation levels were also observed for various regulators of apoptosis (p-JAKs, p-STATs, SOCS, and PIAS). Mcl-1 and x-IAP protein levels increased whereas Bcl-xL, Bcl-2, and c-IAP protein/phosphorylation levels decreased signifying important roles for certain Bcl-2 family members in cell survival over the torpor-arousal cycle. Importantly, the relative phosphorylation of selected STAT proteins increased, suggesting a mechanism for Bcl-2 family activation. These results suggest that an increase in WAT cytoprotective mechanisms supports survival efforts during hibernation.