Hypoxia impairs blood glucose homeostasis in naked mole-rat subordinate adults 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 subordinate adults and juveniles; 2) glucose tolerance is similar between developmental stages in normoxia, but glucose clearance times are 2-3-fold longer in juveniles and subordinates than in queens or pups in hypoxia; 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 signalling is impaired by hypoxia in NMRs, but queens utilize IGF-1 to overcome this limitation and effectively regulate blood glucose in hypoxia. This indicates 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.

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.

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.

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.

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.

Ventilatory responses to hypoxic and hypercapnic environments in naked mole-rats.

Extreme environments are powerful drivers of physiological adaptation. Naked mole-rats offer an informative example of this relationship as they putatively encounter intermittent hypoxia and hypercapnia in their subterranean habitat. This has presumably driven the evolution of a suite of cellular and physiological adaptations that enable life in these conditions. Recently, my laboratory and others have begun to examine physiological responses to environmental hypoxia and hypercapnia in naked mole-rats, and the underlying cellular and molecular mechanisms that differentiate the responses of this species from those of other small mammals. Prominent among these adaptations are a robust hypoxic metabolic response and blunted ventilatory responses to hypoxia and hypercapnia. These responses are mediated in part by modifications to the central nervous system signaling pathways that sense and communicate changes in environmental gas levels and initiate and maintain downstream physiological responses. For example, naked mole-rats retain the signaling architecture necessary for "normal" ventilatory responses to hypoxia and hypercapnia; however, the underlying signaling pathways are muted, resulting in reduced, or even the absence of, sensitivity to otherwise powerful environmental stimuli. Herein, I discuss what we have learned about the manifestation and control of ventilatory and metabolic responses to hypoxia and hypercapnia in naked mole-rats. I also highlight areas where additional work is warranted and consider the implications of what we have learned for the ecophysiology of a species that thrives in conditions that are deleterious or lethal to most adult mammals.

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.

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.

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.

Naked Mole-Rat Cortex Maintains Reactive Oxygen Species Homeostasis During In Vitro Hypoxia or Ischemia and Reperfusion.

Neuronal injury during acute hypoxia, ischemia, and following reperfusion are partially attributable to oxidative damage caused by deleterious fluctuations of reactive oxygen species (ROS). In particular, mitochondrial superoxide (O2•-) production is believed to upsurge during lowoxygen conditions and also following reperfusion, before being dismutated to H2O2 and released into the cell. However, disruptions of redox homeostasis may be beneficially attenuated in the brain of hypoxia-tolerant species, such as the naked mole-rat (NMR, Heterocephalus glaber). As such, we hypothesized that ROS homeostasis is better maintained in the brain of NMRs during severe hypoxic/ ischemic insults and following reperfusion. We predicted that NMR brain would not exhibit substantial fluctuations in ROS during hypoxia or reoxygenation, unlike previous reports from hypoxiaintolerant mouse brain. To test this hypothesis, we measured cortical ROS flux using corrected total cell fluorescence measurements from live brain slices loaded with the MitoSOX red superoxide (O2•-) indicator or chloromethyl 2',7'-dichlorodihydrofluorescein diacetate (CM-H2-DCFDA; which fluoresces with whole-cell hydrogen peroxide (H2O2) production) during various low-oxygen treatments, exogenous oxidative stress, and reperfusion. We found that NMR cortex maintained ROS homeostasis during low-oxygen conditions, while mouse cortex exhibited a ~40% increase and a ~30% decrease in mitochondrial O2•- and cellular H2O2 production, respectively. Mitochondrial ROS homeostasis in NMRs was only disrupted following sodium cyanide application, which was similarly observed in mice. Our results suggest that NMRs have evolved strategies to maintain ROS homeostasis during acute bouts of hypoxia and reoxygenation, potentially as an adaptation to life in an intermittently hypoxic environment.

Small RNA sequencing in hypoxic naked mole-rat hearts suggests microRNA regulation of RNA- and translation-related processes.

The naked mole-rat (Heterocephalus glaber) regularly endures intermittent periods of hypoxia in its burrows, surviving in part due to metabolic rate depression (MRD)-a strategy of conserving cellular resources by downregulating nonessential gene expression and reorganizing cellular processes. miRNA are short, noncoding RNAs already implicated for their roles in numerous models of extreme environmental stress; given their rapid, reversible nature, they are ideal for implementing MRD. We performed small RNA sequencing on cardiac tissue from normoxic versus 24 h hypoxic naked mole-rats, and used bioinformatics to predict 18 miRNAs which may be differentially regulated during hypoxia. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway mapping further suggest these miRNAs play roles in largely translation-related functions, including RNA processing and catabolism.

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.

Carotid body responses to O2 and CO2 in hypoxia-tolerant naked mole rats.

AIM: Naked mole rats (NMRs) exhibit blunted hypoxic (HVR) and hypercapnic ventilatory responses (HCVR). The mechanism(s) underlying these responses are largely unknown. We hypothesized that attenuated carotid body (CB) sensitivity to hypoxia and hypercapnia contributes to the near absence of ventilatory responses to hypoxia and CO2 in NMRs. METHODS: We measured ex vivo CB sensory nerve activity, phrenic nerve activity (an estimation of ventilation), and blood gases in urethane-anesthetized NMRs and C57BL/6 mice breathing normoxic, hypoxic, or hypercapnic gases. CB morphology, carbon monoxide, and H2 S levels were also determined. RESULTS: Relative to mice, NMRs had blunted CB and HVR. Morphologically, NMRs have larger CBs, which contained more glomus cells than in mice. Furthermore, NMR glomus cells form a dispersed pattern compared to a clustered pattern in mice. Hemeoxygenase (HO)-1 mRNA was elevated in NMR CBs, and an HO inhibitor increased CB sensitivity to hypoxia in NMRs. This increase was blocked by an H2 S synthesis inhibitor, suggesting that interrupted gas messenger signaling contributes to the blunted CB responses and HVR in NMRs. Regarding hypercapnia, CB and ventilatory responses to CO2 in NMRs were larger than in mice. Carbonic anhydrase (CA)-2 mRNA is elevated in NMR CBs, and a CA inhibitor blocked the augmented CB response to CO2 in NMRs, indicating CA activity regulates augmented CB response to CO2 . CONCLUSIONS: Consistent with our hypothesis, impaired CB responses to hypoxia contribute in part to the blunted HVR in NMRs. Conversely, the HCVR and CB are more sensitive to CO2 in NMRs.

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.

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.

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.

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.

Lactate inhibits naked mole-rat cardiac mitochondrial respiration.

In aerobic conditions, the proton-motive force drives oxidative phosphorylation (OXPHOS) and the conversion of ADP to ATP. In hypoxic environments, OXPHOS is impaired, resulting in energy shortfalls and the accumulation of protons and lactate. This results in cellular acidification, which may impact the activity and/or integrity of mitochondrial enzymes and in turn negatively impact mitochondrial respiration and thus aerobic ATP production. Naked mole-rats (NMRs) are among the most hypoxia-tolerant mammals and putatively experience intermittent hypoxia in their underground burrows. However, if and how NMR cardiac mitochondria are impacted by lactate accumulation in hypoxia is unknown. We predicted that lactate alters mitochondrial respiration in NMR cardiac muscle. To test this, we used high-resolution respirometry to measure mitochondrial respiration in permeabilized cardiac muscle fibres from NMRs exposed to 4 h of in vivo normoxia (21% O2) or hypoxia (7% O2). We found that: (1) cardiac mitochondria cannot directly oxidize lactate, but surprisingly, (2) lactate inhibits mitochondrial respiration, and (3) decreases complex IV maximum respiratory capacity. Finally, (4) in vivo hypoxic exposure decreases the magnitude of lactate-mediated inhibition of mitochondrial respiration. Taken together, our results suggest that lactate may retard electron transport system function in NMR cardiac mitochondria, particularly in normoxia, and that NMR hearts may be primed for anaerobic metabolism.

Metabolomic Analysis of Carbohydrate and Amino Acid Changes Induced by Hypoxia in Naked Mole-Rat Brain and Liver.

Hypoxia poses a major physiological challenge for mammals and has significant impacts on cellular and systemic metabolism. As with many other small rodents, naked mole-rats (NMRs; Heterocephalus glaber), who are among the most hypoxia-tolerant mammals, respond to hypoxia by supressing energy demand (i.e., through a reduction in metabolic rate mediated by a variety of cell- and tissue-level strategies), and altering metabolic fuel use to rely primarily on carbohydrates. However, little is known regarding specific metabolite changes that underlie these responses. We hypothesized that NMR tissues utilize multiple strategies in responding to acute hypoxia, including the modulation of signalling pathways to reduce anabolism and reprogram carbohydrate metabolism. To address this question, we evaluated changes of 64 metabolites in NMR brain and liver following in vivo hypoxia exposure (7% O2, 4 h). We also examined changes in matched tissues from similarly treated hypoxia-intolerant mice. We report that, following exposure to in vivo hypoxia: (1) phenylalanine, tyrosine and tryptophan anabolism are supressed both in NMR brain and liver; (2) carbohydrate metabolism is reprogramed in NMR brain and liver, but in a divergent manner; (3) redox state is significantly altered in NMR brain; and (4) the AMP/ATP ratio is elevated in liver. Overall, our results suggest that hypoxia induces significant metabolic remodelling in NMR brain and liver via alterations of multiple metabolic pathways.