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.

Evaluation of a New NT-proBNP Immunoassay on an Automated Core Laboratory System.

BACKGROUND: Heart failure remains a major cause of morbidity and mortality despite improvements in treatment. This study aimed to evaluate the Alere N-terminal pro B-type natriuretic peptide (NT-proBNP) immunoassay on the Abbott Alinity i platform. METHODS: The analytical performance including precision, linearity, limit of quantitation (LOQ), carryover, dilution-recovery, and stability was evaluated. A method comparison between the Abbott Alere NT-proBNP assay and Roche Elecsys proBNP II assay was performed using 70 residual plasma samples. RESULTS: Total imprecision was 4.1%, 3.5%, and 2.3% for low (120.9 ng/L), medium (333.9 ng/L), and high (4767.4 ng/L) QC levels, respectively. The manufacturer's claimed LOQ of 8.3 ng/L was verified. Method comparison between the Alere NT-proBNP assay and the Elecsys proBNP II assay showed good agreement between assays with an R value of 0.998, a slope of 1.05 (95% CI, 1.03-1.06), and an intercept of 45.81 (95% CI, -46.6.84 to 138.22). The Bland-Altman plot showed an absolute bias of 250 ng/L or 6.02%. Subrange analysis (NT-proBNP <2000 ng/L) showed good agreement with an R value of 0.998, a slope of 1.04 (95% CI, 1.02-1.06), and an intercept of -4.83 (95% CI, -26.95 to 17.28), with a mean bias of 26 ng/L or 3.2%. The stability of NT-proBNP was also verified in lithium heparin plasma samples stored at 4°C over a 7-day period. Hemolysis and lipemia interference thresholds were verified, but icterus impacted NT-proBNP recovery by >20% at low analyte concentrations. CONCLUSIONS: The Alere NT-proBNP assay demonstrated acceptable analytical performance and very good clinical concordance with the Elecsys proBNP II assay.

Advances in preeclampsia testing.

Preeclampsia is a multisystem hypertensive disorder and one of the leading causes of maternal and fetal morbidity and mortality. The clinical hallmarks such as hypertension and proteinuria, and additional laboratory tests currently available including liver enzyme testing, are neither specific nor sufficiently sensitive. Therefore, biomarkers for timely and accurate identification of patients at risk of developing preeclampsia are extremely valuable to improve patient outcomes and safety. In this chapter, we will first discuss the clinical characteristics of preeclampsia and current evidence of the role of angiogenic factors, such as placental growth factor (PlGF) and soluble FMS like tyrosine kinase 1 (sFlt-1) in the pathogenesis of preeclampsia. Second, we will review the clinical practice guidelines for preeclampsia diagnostic criteria and their recommendations on laboratory testing. Third, we will review the currently available PlGF and sFlt-1 assays in terms of their methodologies, analytical performance, and clinical diagnostic values. Finally, we will discuss the future research needs from both an analytical and clinical perspective.

Components of the Nucleotide Salvage Pathway Increase Frog Virus 3 (FV3) Replication.

Viruses are obligate intracellular parasites that alter host metabolic machinery to obtain energy and macromolecules that are pivotal for replication. Ranavirus, including the type species of the genus frog virus 3 (FV3), represent an ecologically important group of viruses that infect fish, amphibians, and reptiles. It was established that fatty acid synthesis, glucose, and glutamine metabolism exert roles during iridovirus infections; however, no information exists regarding the role of purine metabolism. In this study, we assessed the impact of exogenously applied purines adenine, adenosine, adenosine 5'-monophosphate (AMP), inosine 5'-monophosphate (IMP), inosine, S-adenosyl-L-homocysteine (SAH), and S-adenosyl-L-methionine (SAM) on FV3 replication. We found that all compounds except for SAH increased FV3 replication in a dose-dependent manner. Of the purines investigated, adenine and adenosine produced the most robust response, increasing FV3 replication by 58% and 51%, respectively. While all compounds except SAH increased FV3 replication, only adenine increased plaque area. This suggests that the stimulatory effect of adenine on FV3 replication is mediated by a mechanism that is at least in part independent from the other compounds investigated. Our results are the first to report a response to exogenously applied purines and may provide insight into the importance of purine metabolism during iridoviral infection.

Factors to consider with estimated low-density lipoprotein cholesterol using the new Sampson/NIH equation.

INTRODUCTION: The most commonly utilized method for determining low-density lipoprotein cholesterol (LDLc) is by Friedewald estimation (FeLDLc). A new approach to better estimate LDLc has been proposed by Sampson et al. 2020, known as the Sampson/National Institutes of Health (NIH) estimation of LDLc (NeLDLc), to overcome the limitations of FeLDLc. Non-high-density lipoprotein-cholesterol (Non-HDLc), has equivalent cut-offs to LDLc, established by the 2021 Canadian Cardiovascular Society (CCS) guideline. We hypothesized that NeLDLc remains an inadequate substitute at high triglyceride levels when compared to Non-HDLc. METHODS: A retrospective analysis of 120,959 lipid profiles (47085 patients) spanning five years across a large academic medical center was utilized for comparison of NeLDLc and FeLDLc relative to Non-HDLc as a function of triglyceride content. Regression and concordance between calculated methods were determined at various triglyceride levels to determine optimal utilization of NeLDLc. RESULTS: NeLDLc is generally more correlated and has greater concordance than FeLDLc with Non-HDLc. NeLDLc with increasing triglycerides can produce negatively erroneous results, even with triglycerides < 4.52 mmol/L (400 mg/dL). The largest variation of NeLDLc results is notable at < 0.5 mmol/L (19 mg/dL). Currently, the 2021 CCS guideline recommends reliance on Non-HDLc when triglycerides are > 1.5 mmol/L (133 mg/dL). With the use of NeLDLc, this triglyceride cut-off can be increased to 1.7 mmol/L(150 mg/dL), making it consistent with the hypertriglyceridemia flagging limit. CONCLUSION: NeLDLc offers increased concordance and correlation to Non-HDLc when compared to FeLDLc. However, caution is warranted when triglycerides are > 4.5 mmol/L and when NeLDLc results are < 0.5 mmol/L. Adopting NeLDLc enables flagging at 1.7 mmol/L (vs. 1.5 mmol/L) of triglycerides to suggest reliance on Non-HDLc while simultaneoulsly indicating hypertriglyceridemia.

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.

Glucocorticoids Improve Enteral Feeding Tolerance in Pediatric Short Bowel Syndrome With Chronic Intestinal Inflammation.

OBJECTIVES: A group of short bowel syndrome (SBS) patients developed chronic intestinal inflammation while struggling weaning off parenteral nutrition (PN). They did not respond to standard management of SBS and food allergy. We treated them with glucocorticoids and described the outcome. METHODS: Our study is a retrospective descriptive study. We reviewed records from the intestinal rehabilitation program from 2006 to 2017. We identified 15 patients whose lab values, pathology results, and clinic notes were reviewed. RESULTS: We had more patients (n = 10) with diagnosis of gastroschisis, and more female patients (n = 9). Seven patients weaned off PN with median treatment duration of 5 months, 5 of which remained on budesonide for significant period of time (median: 7.5 months). One of these 7 patients relapsed, as the patient resumed glucocorticoids because of recurrence of chronic intestinal inflammation. Six of 15 children had significant eosinophils in their initial biopsy, 5 of these children weaned off PN whereas 1 child's gastrointestinal (GI) bleeding stopped. Four patients were not able to decrease PN calorie. Two of these patients' GI bleeding stopped, the other 2 had normalized histology. CONCLUSIONS: For SBS children with histologically confirmed chronic intestinal inflammation, glucocorticoids may help promote enteral feeding tolerance. Glucocorticoids regimen should be chosen individually. Patients are more likely to respond if initial histology has significant eosinophilic infiltration. Patients may need to remain on glucocorticoids for over 6 months.

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).

Repeat serial transverse enteroplasty leads to reduction in parenteral nutrition in children with short bowel syndrome.

BACKGROUND/PURPOSE: Following a serial transverse enteroplasty (STEP) procedure some children develop redilation of the small intestine leading to impaired enteral tolerance and inability to wean parenteral nutrition (PN). The benefit of a second STEP procedure (2STEP) has been controversial. METHODS: We performed a retrospective review of our experience (2008-2018) performing 2STEP, with comparative analysis of nutritional outcomes pre- and postsurgery. RESULTS: During this period 2STEP was performed in 23 patients (13 F:10 M) at a median (25%-75%) age of 2.2 (1.2-3.6) years. Median intestinal length was 68 (40-105) cm before and 85 (40-128) cm after 2STEP. Leading up to 2STEP, PN provided almost 75% of estimated calorie needs. By 24 weeks following 2STEP drops in mean PN percent approached statistical significance (p = 0.07) and at most recent follow up the mean PN percentage was statistically better than at the time of operation or 4 weeks prior to 2STEP, and was nearly significant compared with 12 weeks (p = 0.07) and 24 weeks (p = 0.06) prior. Thirteen children were completely off parenteral support. CONCLUSION: When small intestine redilation occurs following a STEP procedure and where PN cannot otherwise be weaned we believe these data support performing a 2STEP. We cannot predict preoperatively which children will ultimately benefit. LEVEL OF EVIDENCE: 3 (retrospective comparative study).

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.