Hypoxia impairs blood glucose homeostasis in naked mole-rat adult subordinates but not queens.

Naked mole-rats (NMRs) are among the most hypoxia-tolerant mammals and metabolize only carbohydrates in hypoxia. Glucose is the primary building block of dietary carbohydrates, but how blood glucose is regulated during hypoxia has not been explored in NMRs. We hypothesized that NMRs mobilize glucose stores to support anaerobic energy metabolism in hypoxia. To test this, we treated newborn, juvenile and adult (subordinate and queen) NMRs in normoxia (21% O2) or hypoxia (7, 5 or 3% O2), while measuring metabolic rate, body temperature and blood [glucose]. We also challenged animals with glucose, insulin or insulin-like growth factor-1 (IGF-1) injections and measured the rate of glucose clearance in normoxia and hypoxia. We found that: (1) blood [glucose] increases in moderate hypoxia in queens and pups, but only in severe hypoxia in adult subordinates and juveniles; (2) glucose tolerance is similar between developmental stages in normoxia, but glucose clearance times are 2- to 3-fold longer in juveniles and subordinates than in queens or pups in hypoxia; and (3) reoxygenation accelerates glucose clearance in hypoxic subordinate adults. Mechanistically, (4) insulin and IGF-1 reduce blood [glucose] in subordinates in both normoxia but only IGF-1 impacts blood [glucose] in hypoxic queens. Our results indicate that insulin signaling is impaired by hypoxia in NMRs, but that queens utilize IGF-1 to overcome this limitation and effectively regulate blood glucose in hypoxia. This suggests that sexual maturation impacts blood glucose handling in hypoxic NMR queens, which may allow queens to spend longer periods of time in hypoxic nest chambers.

Enhanced mitochondrial buffering prevents Ca2+ overload in naked mole-rat brain.

Deleterious Ca2+ accumulation is central to hypoxic cell death in the brain of most mammals. Conversely, hypoxia-mediated increases in cytosolic Ca2+ are retarded in hypoxia-tolerant naked mole-rat brain. We hypothesized that naked mole-rat brain mitochondria have an enhanced capacity to buffer exogenous Ca2+ and examined Ca2+ handling in naked mole-rat cortical tissue. We report that naked mole-rat brain mitochondria buffer >2-fold more exogenous Ca2+ than mouse brain mitochondria, and that the half-maximal inhibitory concentration (IC50 ) at which Ca2+ inhibits aerobic oxidative phosphorylation is >2-fold higher in naked mole-rat brain. The primary driving force of Ca2+ uptake is the mitochondrial membrane potential (Δψm ), and the IC50 at which Ca2+ decreases Δψm is ∼4-fold higher in naked mole-rat than mouse brain. The ability of naked mole-rat brain mitochondria to safely retain large volumes of Ca2+ may be due to ultrastructural differences that support the uptake and physical storage of Ca2+ in mitochondria. Specifically, and relative to mouse brain, naked mole-rat brain mitochondria are larger and have higher crista density and increased physical interactions between adjacent mitochondrial membranes, all of which are associated with improved energetic homeostasis and Ca2+ management. We propose that excessive Ca2+ influx into naked mole-rat brain is buffered by physical storage in large mitochondria, which would reduce deleterious Ca2+ overload and may thus contribute to the hypoxia and ischaemia-tolerance of naked mole-rat brain. KEY POINTS: Unregulated Ca2+ influx is a hallmark of hypoxic brain death; however, hypoxia-mediated Ca2+ influx into naked mole-rat brain is markedly reduced relative to mice. This is important because naked mole-rat brain is robustly tolerant against in vitro hypoxia, and because Ca2+ is a key driver of hypoxic cell death in brain. We show that in hypoxic naked mole-rat brain, oxidative capacity and mitochondrial membrane integrity are better preserved following exogenous Ca2+ stress. This is due to mitochondrial buffering of exogenous Ca2+ and is driven by a mitochondrial membrane potential-dependant mechanism. The unique ultrastructure of naked mole-rat brain mitochondria, as a large physical storage space, may support increased Ca2+ buffering and thus hypoxia-tolerance.

Adenosine and γ-aminobutyric acid partially regulate metabolic and ventilatory responses of Damaraland mole-rats to acute hypoxia.

Fossorial Damaraland mole-rats (Fukomys damarensis) mount a robust hypoxic metabolic response (HMR) but a blunted hypoxic ventilatory response (HVR) to acute hypoxia. Although these reflex physiological responses have been described previously, the underlying signalling pathways are entirely unknown. Of particular interest are contributions from γ-aminobutyric acid (GABA), which is the primary inhibitory neurotransmitter in the nervous system of most adult mammals, and adenosine, the accumulation of which increases during hypoxia as a breakdown product of ATP. Therefore, we hypothesized that GABAergic and/or adenosinergic signalling contributes to the blunted HVR and robust HMR in Damaraland mole-rats. To test this hypothesis, we injected adult animals with saline alone (controls), or 100 mg kg-1 aminophylline or 1 mg kg-1 bicuculline, to block adenosine or GABAA receptors, respectively. We then used respirometry, plethysmography and thermal RFID probes to non-invasively measure metabolic, ventilator and thermoregulatory responses, respectively, to acute hypoxia (1 h in 5 or 7% O2) in awake and freely behaving animals. We found that bicuculline had relatively minor effects on metabolism and thermoregulation but sensitized ventilation such that the HVR became manifest at 7% instead of 5% O2 and was greater in magnitude. Aminophylline increased metabolic rate, ventilation and body temperature in normoxia, and augmented the HMR and HVR. Taken together, these findings indicate that adenosinergic and GABAergic signalling play important roles in mediating the robust HMR and blunted HVR in Damaraland mole-rats.

Physiological responses to hypoxia are constrained by environmental temperature in heterothermic tenrecs.

Malagasy tenrecs are placental hibernating mammals that seal the entrances to their burrows and hibernate either singly or in groups for 8-9 months, which is likely to create a hypoxic and hypercapnic burrow environment. Therefore, we hypothesized that tenrecs are tolerant to environmental hypoxia and hypercapnia. Many hypoxia- and hypercapnia-tolerant fossorial mammals respond to hypoxia by decreasing metabolic rate and thermogenesis, and have blunted ventilatory responses to both environmental hypoxia and hypercapnia. However, tenrecs exhibit extreme metabolic and thermoregulatory plasticity, which exceeds that of most heterothermic mammals and approaches that of ectothermic reptiles. Thus, we predicted that tenrecs would have abnormal physiological responses to hypoxia and hypercapnia relative to other fossorial mammals. To test this, we exposed common tenrecs (Tenrec ecaudatus) to moderate and severe hypoxia (9 and 4% O2) or hypercapnia (5 and 10% CO2) in either 28 or 16°C while non-invasively measuring metabolic rate, thermogenesis and ventilation. We found that tenrecs exhibit robust metabolic decreases in both hypoxia and hypercapnia. Furthermore, tenrecs have blunted ventilatory responses to both hypoxia and hypercapnia, and these responses are highly temperature sensitive such that they are reduced or absent in 16°C. Thermoregulation was highly variable in 16°C but constrained in 28°C across all treatment conditions and was not impacted by hypoxia or hypercapnia, unlike in other heterothermic mammals. Taken together, our results indicate that physiological responses to hypoxia and hypercapnia in tenrecs are highly dependent on environmental temperature and differ from those of other mammalian heterotherms.

The glutamatergic drive to breathe is reduced in severe but not moderate hypoxia in Damaraland mole-rats.

Damaraland mole-rats (Fukomys damarensis) are a hypoxia-tolerant fossorial species that exhibit a robust hypoxic metabolic response (HMR) and blunted hypoxic ventilatory response (HVR). Whereas the HVR of most adult mammals is mediated by increased excitatory glutamatergic signalling, naked mole-rats, which are closely related to Damaraland mole-rats, do not utilize this pathway. Given their phylogenetic relationship and similar lifestyles, we hypothesized that the signalling mechanisms underlying physiological responses to acute hypoxia in Damaraland mole-rats are like those of naked mole-rats. To test this, we used pharmacological antagonists of glutamatergic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) and N-methyl-d-aspartate receptors (NMDARs), combined with plethysmography, respirometry and thermal RFID chips, to non-invasively evaluate the role of excitatory AMPAR and NMDAR signalling in mediating ventilatory, metabolic and thermoregulatory responses, respectively, to 1 h of 5 or 7% O2. We found that AMPAR or NMDAR antagonism have minimal impacts on the HMR or hypoxia-mediated changes in thermoregulation. Conversely, the 'blunted' HVR of Damaraland mole-rats is reduced by either AMPAR or NMDAR antagonism such that the onset of the HVR occurs in less severe hypoxia. In more severe hypoxia, antagonists have no impact, suggesting that these receptors are already inhibited. Together, these findings indicate that the glutamatergic drive to breathe decreases in Damaraland mole-rats exposed to severe hypoxia. These findings differ from other adult mammals, in which the glutamatergic drive to breathe increases with hypoxia.

The relationship between hypoxia exposure and circulating cortisol levels in social and solitary African mole-rats: An initial report.

Hypoxemia from exposure to intermittent and/or acute environmental hypoxia (lower oxygen concentration) is a severe stressor for many animal species. The response to hypoxia of the hypothalamic-pituitary-adrenal axis (HPA-axis), which culminates in the release of glucocorticoids, has been well-studied in hypoxia-intolerant surface-dwelling mammals. Several group-living (social) subterranean species, including most African mole-rats, are hypoxia-tolerant, likely due to regular exposure to intermittent hypoxia in their underground burrows. Conversely, solitary mole-rat species, lack many adaptive mechanisms, making them less hypoxia-tolerant than the social genera. To date, the release of glucocorticoids in response to hypoxia has not been measured in hypoxia-tolerant mammalian species. Consequently, this study exposed three social African mole-rat species and two solitary mole-rat species to normoxia, or acute hypoxia and then measured their respective plasma glucocorticoid (cortisol) concentrations. Social mole-rats had lower plasma cortisol concentrations under normoxia than the solitary genera. Furthermore, individuals of all three of the social mole-rat species exhibited significantly increased plasma cortisol concentrations after hypoxia, similar to those of hypoxia-intolerant surface-dwelling species. By contrast, individuals of the two solitary species had a reduced plasma cortisol response to acute hypoxia, possibly due to increased plasma cortisol under normoxia. If placed in perspective with other closely related surface-dwelling species, the regular exposure of the social African mole-rats to hypoxia may have reduced the basal levels of the components for the adaptive mechanisms associated with hypoxia exposure, including circulating cortisol levels. Similarly, the influence of body mass on plasma cortisol levels cannot be ignored. This study demonstrates that both hypoxia-tolerant rodents and hypoxia-intolerant terrestrial laboratory-bred rodents may possess similar HPA-axis responses from exposure to hypoxia. Further research is required to confirm the results from this pilot study and to further confirm how the cortisol concentrations may influence responses to hypoxia in African mole-rats.

Apocynin reduces dihydroethidium fluorescence in naked mole-rat cortex independently of NADPH oxidase.

Pharmacological agents that modulate cellular targets offer a powerful approach to interrogate the role of a given component in cellular signalling cascades. However, such drugs are often nonspecific and/or have unexpected off-target effects. One cellular target of interest is the NADPH oxidase (NOX) enzyme family, which consume oxygen and produce reactive oxygen species. Among the most widely used inhibitors of NOX is apocynin, but apocynin also has off-target effects that may interfere with detection assays of hydrogen peroxide (H2O2) or directly scavenge H2O2 in some cell lines. Nonetheless, apocynin remains widely used for in vivo studies of brain function. Therefore, we used apocynin and another widely-used NOX inhibitor - diphenyleneiodonium (DPI) - to study the role of NOX in ROS homeostasis of hypoxia-tolerant naked mole-rat cortical brain slices during a normoxia➔hypoxia➔reoxygenation protocol. Using fluorescence microscopy, we found that apocynin decreased dihydroethidium fluorescence from naked mole-rat cortex in all treatment conditions by 65-75% of pre-drug normoxic control. This change was rapid, occurring within minutes of drug perfusion, and reversed equally rapidly upon washout. Conversely, apocynin had no effect on 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) fluorescence. DPI also had no effect on either fluorescence signal, suggesting that the effect of apocynin is due to indirect actions of the drug and not due to modulation of NOX. Taken together, our results highlight the pitfalls of pharmacological neuroscience and add to the body of evidence suggesting that apocynin is not a useful compound for targeting NOX.

Short communication: Acute hypoxia does not alter mitochondrial abundance in naked mole-rats.

Hypoxia poses a significant energetic challenge and most species exhibit metabolic remodelling when exposed to prolonged hypoxia. One component of this remodelling is mitochondrial biogenesis/mitophagy, which alter mitochondrial abundance and helps to adjust metabolic throughput to match changes in energy demands in hypoxia. However, how acute hypoxia impacts mitochondrial abundance in hypoxia-tolerant species is poorly understood. To help address this gap, we exposed hypoxia-tolerant naked mole-rats to 3 h of normoxia or acute hypoxia (5% O2) and measured changes in mitochondrial abundance using two well-established markers: citrate synthase (CS) enzyme activity and mitochondrial DNA (mtDNA) abundance. We found that neither marker changed with hypoxia in brain, liver, or kidney, suggesting that mitochondrial biogenesis is not initiated during acute hypoxia in these tissues. Conversely in skeletal muscle, the ratio of CS activity to total protein decreased 50% with hypoxia. However, this change was likely driven by an increase in soluble protein density in hypoxia because CS activity was unchanged relative to wet tissue weight and the mtDNA copy number was unchanged. To confirm this, we examined skeletal muscle mitochondria using transmission electron microscopy and found no change in mitochondrial volume density. Taken together with previous studies of mitochondrial respiratory function, our present findings suggest that naked mole-rats primarily rely on tissue-specific functional remodelling of metabolic pathways and mitochondrial respiratory throughput, and not physical changes in mitochondrial number or volume, to adjust to short-term hypoxic exposure.

Adaptations to a hypoxic lifestyle in naked mole-rats.

Hypoxia is one of the strongest environmental drivers of cellular and physiological adaptation. Although most mammals are largely intolerant of hypoxia, some specialized species have evolved mitigative strategies to tolerate hypoxic niches. Among the most hypoxia-tolerant mammals are naked mole-rats (Heterocephalus glaber), a eusocial species of subterranean rodent native to eastern Africa. In hypoxia, naked mole-rats maintain consciousness and remain active despite a robust and rapid suppression of metabolic rate, which is mediated by numerous behavioural, physiological and cellular strategies. Conversely, hypoxia-intolerant mammals and most other hypoxia-tolerant mammals cannot achieve the same degree of metabolic savings while staying active in hypoxia and must also increase oxygen supply to tissues, and/or enter torpor. Intriguingly, recent studies suggest that naked mole-rats share many cellular strategies with non-mammalian vertebrate champions of anoxia tolerance, including the use of alternative metabolic end-products and potent pH buffering mechanisms to mitigate cellular acidification due to upregulation of anaerobic metabolic pathways, rapid mitochondrial remodelling to favour increased respiratory efficiency, and systemic shifts in energy prioritization to maintain brain function over that of other tissues. Herein, I discuss what is known regarding adaptations of naked mole-rats to a hypoxic lifestyle, and contrast strategies employed by this species to those of hypoxia-intolerant mammals, closely related African mole-rats, other well-studied hypoxia-tolerant mammals, and non-mammalian vertebrate champions of anoxia tolerance. I also discuss the neotenic theory of hypoxia tolerance - a leading theory that may explain the evolutionary origins of hypoxia tolerance in mammals - and highlight promising but underexplored avenues of hypoxia-related research in this fascinating model organism.

Different patterns of chronic hypoxia lead to hierarchical adaptive mechanisms in goldfish metabolism.

Some hypoxia-tolerant species, such as goldfish, experience intermittent and severe hypoxia in their natural habitat, causing them to develop multiple physiological adaptations. However, in fish, the metabolic impact of regular hypoxic exposure on swimming performance in normoxia is less well understood. Therefore, we experimentally tested whether chronic exposure to constant (30 days at 10% air saturation) or intermittent hypoxia (3 h in normoxia and 21 h in hypoxia, 5 days a week) would result in similar metabolic and swimming performance benefits after reoxygenation. Moreover, half of the normoxic and intermittent hypoxic fish were put on a 20-day normoxic training regime. After these treatments, metabolic rate (standard and maximum metabolic rates: SMR and MMR) and swimming performance [critical swimming speed (Ucrit) and cost of transport (COT)] were assessed. In addition, enzyme activities [citrate synthase (CS), cytochrome c oxidase (COX) and lactate dehydrogenase (LDH)] and mitochondrial respiration were examined in red muscle fibres. We found that acclimation to constant hypoxia resulted in (1) metabolic suppression (-45% SMR and -27% MMR), (2) increased anaerobic capacity (+117% LDH), (3) improved swimming performance (+80% Ucrit, -71% COT) and (4) no changes at the mitochondrial level. Conversely, the enhancement of swimming performance was reduced following acclimation to intermittent hypoxia (+45% Ucrit, -41% COT), with a 55% decrease in aerobic scope, despite a significant increase in oxidative metabolism (+201% COX, +49% CS). This study demonstrates that constant hypoxia leads to the greatest benefit in swimming performance and that mitochondrial metabolic adjustments only provide minor help in coping with hypoxia.

What to do with low O2: Redox adaptations in vertebrates native to hypoxic environments.

Reactive oxygen species (ROS) are important cellular signalling molecules but sudden changes in redox balance can be deleterious to cells and lethal to the whole organism. ROS production is inherently linked to environmental oxygen availability and many species live in variable oxygen environments that can range in both severity and duration of hypoxic exposure. Given the importance of redox homeostasis to cell and animal viability, it is not surprising that early studies in species adapted to various hypoxic niches have revealed diverse strategies to limit or mitigate deleterious ROS changes. Although research in this area is in its infancy, patterns are beginning to emerge in the suites of adaptations to different hypoxic environments. This review focuses on redox adaptations (i.e., modifications of ROS production and scavenging, and mitigation of oxidative damage) in hypoxia-tolerant vertebrates across a range of hypoxic environments. In general, evidence suggests that animals adapted to chronic lifelong hypoxia are in homeostasis, and do not encounter major oxidative challenges in their homeostatic environment, whereas animals exposed to seasonal chronic anoxia or hypoxia rapidly downregulate redox balance to match a hypometabolic state and employ robust scavenging pathways during seasonal reoxygenation. Conversely, animals adapted to intermittent hypoxia exposure face the greatest degree of ROS imbalance and likely exhibit enhanced ROS-mitigation strategies. Although some progress has been made, research in this field is patchy and further elucidation of mechanisms that are protective against environmental redox challenges is imperative for a more holistic understanding of how animals survive hypoxic environments.

Supermole-rat to the rescue: Does the naked mole-rat offer a panacea for all that ails us?

Over the previous several decades, many non-traditional research models have offered new avenues of exploration for biomedical research. The promise of these animals is primarily derived from adaptations to unique or challenging environments that share key factors with a disease or pathology of interest (e.g., hypoxemia or hypercarbia are clinically relevant and are also in vivo consequences of environmental hypoxia and hypercapnia, respectively). Animals adapted to such environments allow us to ask the question: how has nature solved a particular problem and what can we learn to inform novel translational research into the treatment of related diseases and pathologies? One of the most promising mammalian models that have garnered increasing attention from researchers and the public are naked mole-rats (NMRs). The NMR is a small and eusocial subterranean rodent species that live in a putatively hypoxic and hypercapnic burrow environment. Intriguingly, whereas most non-traditional biomedical models offer insight into one or only a few diseases related to a common physiological stress, NMRs in contrast have proven to be resistant to a very wide range of ailments, including aging, cancer, and hypoxia- and hypercapnia-related disorders, among many others. In the present commentary, we discuss progress made in understanding how NMRs overcome these challenges and speculate on the origins of their remarkable abilities.

Acute pH alterations do not impact cardiac mitochondrial respiration in naked mole-rats or mice.

Energetically demanding conditions such as hypoxia and exercise favour anaerobic metabolism (glycolysis), which leads to acidification of the cellular milieu from ATP hydrolysis and accumulation of the anaerobic end-product, lactate. Cellular acidification may damage mitochondrial proteins and/or alter the H+ gradient across the mitochondrial inner membrane, which may in turn impact mitochondrial respiration and thus aerobic ATP production. Naked mole-rats are among the most hypoxia-tolerant mammals, and putatively experience intermittent environmental and systemic hypoxia while resting and exercising in their underground burrows. Previous studies in naked mole-rat brain, heart, and skeletal muscle mitochondria have demonstrated adaptations that favour improved efficiency in hypoxic conditions; however, the impact of cellular acidification on mitochondrial function has not been explored. We hypothesized that, relative to hypoxia-intolerant mice, naked mole-rat cardiac mitochondrial respiration is less sensitive to cellular pH changes. To test this, we used high-resolution respirometry to measure mitochondrial respiration by permeabilized cardiac muscle fibres from naked mole-rats and mice exposed in vitro to a pH range from 6.6 to 7.6. Surprisingly, we found that acute pH changes do not impact cardiac mitochondrial respiration or compromise mitochondrial integrity in either species. Our results suggest that acute alterations of cellular pH have minimal impact on cardiac mitochondrial respiration.

Naked mole-rats resist the accumulation of hypoxia-induced oxidative damage.

Naked mole-rats are among the few mammals with the ability to endure severe hypoxia. These unique rodents use metabolic rate depression along with various molecular mechanisms to successfully overcome the challenges of oxygen-limitation, which they experience in their underground borrows. While studies have reported that naked mole-rats exhibit inherently higher levels of oxidative damage across their lifespan as compared to mice, it has yet to be determined whether naked mole-rats are vulnerable to oxidative damage during periods of low oxygen exposure. To investigate this phenomenon, we examined cellular oxidative damage markers of macromolecules: DNA oxidation determined as 8-oxo-2'deoxyguanosine (8-OHdG8) levels, RNA oxidation as 8-hydroxyguanosine (8-OHG), protein carbonylation, and lipid peroxidation in normoxic (control), acute (4 h at 7% O2), and chronic (24 h at 7% O2) hypoxia-exposed naked mole-rats. Brain appears to be the most resilient to hypoxia-induced oxidative damage, with both brain and heart exhibiting enhanced antioxidant capacity during hypoxia. Levels of DNA and RNA oxidation were minimally changed in all tissues and no changes were observed in protein carbonylation. Most tissues experienced lipid peroxidation, with liver displaying a 9.6-fold increase during hypoxia. Concomitantly, levels of DNA damage repair proteins were dynamically regulated in a tissue-specific manner, with white adipose displaying a significant reduction during hypoxia. Our findings show that naked mole-rats largely avoid hypoxia-induced oxidative damage, possibly due to their high tolerance to redox stress, or to reduced oxidative requirements made possible during their hypometabolic response when oxygen supply is limited.

Acute Hypoxia Alters Extracellular Vesicle Signatures and the Brain Citrullinome of Naked Mole-Rats (Heterocephalus glaber).

Peptidylarginine deiminases (PADs) and extracellular vesicles (EVs) may be indicative biomarkers of physiological and pathological status and adaptive responses, including to diseases and disorders of the central nervous system (CNS) and related to hypoxia. While these markers have been studied in hypoxia-intolerant mammals, in vivo investigations in hypoxia-tolerant species are lacking. Naked mole-rats (NMR) are among the most hypoxia-tolerant mammals and are thus a good model organism for understanding natural and beneficial adaptations to hypoxia. Thus, we aimed to reveal CNS related roles for PADs in hypoxia tolerance and identify whether circulating EV signatures may reveal a fingerprint for adaptive whole-body hypoxia responses in this species. We found that following in vivo acute hypoxia, NMR: (1) plasma-EVs were remodelled, (2) whole proteome EV cargo contained more protein hits (including citrullinated proteins) and a higher number of associated KEGG pathways relating to the total proteome of plasma-EVs Also, (3) brains had a trend for elevation in PAD1, PAD3 and PAD6 protein expression, while PAD2 and PAD4 were reduced, while (4) the brain citrullinome had a considerable increase in deiminated protein hits with hypoxia (1222 vs. 852 hits in normoxia). Our findings indicate that circulating EV signatures are modified and proteomic content is reduced in hypoxic conditions in naked mole-rats, including the circulating EV citrullinome, while the brain citrullinome is elevated and modulated in response to hypoxia. This was further reflected in elevation of some PADs in the brain tissue following acute hypoxia treatment. These findings indicate a possible selective role for PAD-isozymes in hypoxia response and tolerance.

Naked mole-rat brain mitochondria tolerate in vitro ischaemia.

Naked mole-rats (NMRs; Heterocephalus glaber) are among the most hypoxia-tolerant mammals. There is evidence that the NMR brain tolerates in vitro hypoxia and NMR brain mitochondria exhibit functional plasticity following in vivo hypoxia; however, if and how these organelles tolerate ischaemia and how ischaemic stress impacts mitochondrial energetics and redox regulation is entirely unknown. We hypothesized that mitochondria fundamentally contribute to in vitro ischaemia resistance in the NMR brain. To test this, we treated NMR and CD-1 mouse cortical brain sheets with an in vitro ischaemic mimic and evaluated mitochondrial respiration capacity and redox regulation following 15 or 30 min of ischaemia or ischaemia/reperfusion (I/R). We found that, relative to mice, the NMR brain largely retains mitochondrial function and redox balance post-ischaemia and I/R. Specifically: (i) ischaemia reduced complex I and II-linked respiration ∼50-70% in mice, vs. ∼20-40% in NMR brain, (ii) NMR but not mouse brain maintained relatively steady respiration control ratios and robust mitochondrial membrane integrity, (iii) electron leakage post-ischaemia was lesser in NMR than mouse brain and NMR brain retained higher coupling efficiency, and (iv) free radical generation during and following ischaemia and I/R was lower from NMR brains than mice. Taken together, our results indicate that NMR brain mitochondria are more tolerant of ischaemia and I/R than mice and retain respiratory capacity while avoiding redox derangements. Overall, these findings support the hypothesis that hypoxia-tolerant NMR brain is also ischaemia-tolerant and suggest that NMRs may be a natural model of ischaemia tolerance in which to investigate evolutionarily derived solutions to ischaemic pathology. KEY POINTS: Ischaemia is highly deleterious to the mammalian brain and this damage is largely mediated by mitochondrial dysfunction. Naked mole-rats are among the most hypoxia-tolerant mammals and their brain tolerates ischaemia ex vivo, but the impact of ischaemia on mitochondrial function is unknown. Naked mole-rat but not mouse brain mitochondria retain respiratory capacity and membrane integrity following ischaemia or ischaemia/reperfusion. Differences in free radical management and respiratory pathway control between species may mediate this tolerance. These results help us understand how natural models of hypoxia tolerance also tolerate ischaemia in the brain.

Hypoxic naked mole-rat brains use microRNA to coordinate hypometabolic fuels and neuroprotective defenses.

Naked mole-rats are among the mammalian champions of hypoxia tolerance. They evolved adaptations centered around reducing metabolic rate to overcome the challenges experienced in their underground burrows. In this study, we used next-generation sequencing to investigate one of the factors likely supporting hypoxia tolerance in naked mole-rat brains, posttranscriptional microRNAs (miRNAs). Of the 212 conserved miRNAs identified using small RNA sequencing, 18 displayed significant differential expression during hypoxia. Bioinformatic enrichment revealed that hypoxia-mediated miRNAs were suppressing energy expensive processes including de novo protein translation and cellular proliferation. This suppression occurred alongside the activation of neuroprotective and neuroinflammatory pathways, and the induction of central signal transduction pathways including HIF-1α and NFκB via miR-335, miR-101, and miR-155. MiRNAs also coordinated anaerobic glycolytic fuel sources, where hypoxia-upregulated miR-365 likely suppressed protein levels of ketohexokinase, the enzyme responsible for catalyzing the first committed step of fructose catabolism. This was further supported by a hypoxia-mediated reduction in glucose transporter 5 proteins that import fructose into the cell. Yet, messenger RNA and protein levels of lactate dehydrogenase, which converts pyruvate to lactate in the absence of oxygen, were elevated during hypoxia. Together, this demonstrated the induction of anaerobic glycolysis despite a lack of reliance on fructose as the primary fuel source, suggesting that hypoxic brains are metabolically different than anoxic naked mole-rat brains that were previously found to shift to fructose-based glycolysis. Our findings contribute to the growing body of oxygen-responsive miRNAs "OxymiRs" that facilitate natural miRNA-mediated mechanisms for successful hypoxic exposures.

MicroRNA-mediated inhibition of AMPK coordinates tissue-specific downregulation of skeletal muscle metabolism in hypoxic naked mole-rats.

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% O2) or severe hypoxia (3% O2), 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.

Burrowing star-nosed moles (Condylura cristata) are not hypoxia tolerant.

Star-nosed moles (Condylura cristata) have an impressive diving performance and burrowing lifestyle, yet no ventilatory data are available for this or any other talpid mole species. We predicted that, like many other semi-aquatic and fossorial small mammals, star-nosed moles would exhibit: (i) a blunted (i.e. delayed or reduced) hypoxic ventilatory response, (ii) a reduced metabolic rate and (iii) a lowered body temperature (Tb) in hypoxia. We thus non-invasively measured these variables from wild-caught star-nosed moles exposed to normoxia (21% O2) or acute graded hypoxia (21-6% O2). Surprisingly, star-nosed moles did not exhibit a blunted HVR or decreased Tb in hypoxia, and only manifested a significant, albeit small (<8%), depression of metabolic rate at 6% O2 relative to normoxic controls. Unlike small rodents inhabiting similar niches, star-nosed moles are thus intolerant to hypoxia, which may reflect an evolutionary trade-off favouring the extreme sensory biology of this unusual insectivore.

WITHDRAWN: Utilizing comparative models in biomedical research.

The Publisher regrets that this article is an accidental duplication of an article that has already been published in Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, Volume 255, 2021, 110593, https://doi.org/10.1016/j.cbpb.2021.110593. The duplicate article has therefore been withdrawn. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/article-withdrawal.