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. Full article

Oxidative metabolism is fine-tuned machinery that combines two tightly coupled fluxes of glucose and glutamine-derived carbons. Hypoxia interrupts the coordination between the metabolism of these two nutrients and leads to a decrease of the system efficacy and may eventually cause cell death. The subterranean blind mole rat, Spalax, is an underexplored, underground, hypoxia-tolerant mammalian group which spends its life under sharply fluctuating oxygen levels. Primary Spalax cells are an exceptional model to study the metabolic strategies that have evolved in mammals inhabiting low-oxygen niches. In this study we explored the metabolic frame of glutamine (Gln) homeostasis in Spalax skin cells under normoxic and hypoxic conditions and their impacts on the metabolism of rat cells. Targeted metabolomics employing liquid chromatography and mass spectrometry (LC-MS) was used to track the fate of heavy glutamine carbons (¹³C₅ Gln) after 24 h under normoxia or hypoxia (1% O₂). Our results indicated that large amounts of glutamine-originated carbons were detected as proline (Pro) and hydroxyproline (HPro) in normoxic Spalax cells with a further increase under hypoxia, suggesting a strategy for reduced Gln carbons storage in proteins. The intensity of the flux and the presence of HPro suggests collagen as a candidate protein that is most abundant in animals, and as the primary source of HPro. An increased conversion of αKG to 2 HG that was indicated in hypoxic Spalax cells prevents the degradation of hypoxia-inducible factor 1α (HIF-1α) and, consequently, maintains cytosolic and mitochondrial carbons fluxes that were uncoupled via inhibition of the pyruvate dehydrogenase complex. A strong antioxidant defense in Spalax cells can be attributed, at least in part, to the massive usage of glutamine-derived glutamate for glutathione (GSH) production. The present study uncovers additional strategies that have evolved in this unique mammal to support its hypoxia tolerance, and probably contribute to its cancer resistance, longevity, and healthy aging. Full article

Exercise is an important performance trait in mammals and variation in aerobic capacity and/or substrate allocation during submaximal exercise may be important for survival at high altitude. Comparisons between lowland and highland populations is a fruitful approach to understanding the mechanisms for altitude differences in exercise performance. However, it has only been applied in very few highland species. The leaf-eared mice (LEM, genus Phyllotis) of South America are a promising taxon to uncover the pervasiveness of hypoxia tolerance mechanisms. Here we use lowland and highland populations of Andean and Lima LEM (P. andium and P. limatus), acclimated to common laboratory conditions, to determine exercise-induced maximal oxygen consumption ($\dot{\mathrm{V}}$O₂max), and submaximal exercise metabolism. Lowland and highland populations of both species showed no difference in $\dot{\mathrm{V}}$O₂max running in either normoxia or hypoxia. When run at 75% of $\dot{\mathrm{V}}$O₂max, highland Andean LEM had a greater reliance on carbohydrate oxidation to power exercise. In contrast, highland Lima LEM showed no difference in exercise fuel use compared to their lowland counterparts. The higher carbohydrate oxidation seen in highland Andean LEM was not explained by maximal activities of glycolytic enzymes in the gastrocnemius muscle, which were equivalent to lowlanders. This result is consistent with data on highland deer mouse populations and suggests changes in metabolic regulation may explain altitude differences in exercise performance. Full article

The bioenergetics of the vast majority of terrestrial mammals evolved to consuming glucose (Glc) for energy production under regular atmosphere (about 21% oxygen). However, some vertebrate species, such as aquatic turtles, seals, naked mole rat, and blind mole rat, Spalax, have adjusted their homeostasis to continuous function under severe hypoxic environment. The exploration of hypoxia-tolerant species metabolic strategies provides a better understanding of the adaptation to hypoxia. In this study, we compared Glc homeostasis in primary Spalax and rat skin cells under normoxic and hypoxic conditions. We used the targeted-metabolomics approach, utilizing liquid chromatography and mass spectrometry (LC-MS) to track the fate of heavy Glc carbons (¹³C₆ Glc), as well as other methodologies to assist the interpretation of the metabolic landscape, such as bioenergetics profiling, Western blotting, and gene expression analysis. The metabolic profile was recorded under steady-state (after 24 h) of the experiment. Glc-originated carbons were unequally distributed between the cytosolic and mitochondrial domains in Spalax cells compared to the rat. The cytosolic domain is dominant apparently due to the hypoxia-inducible factor-1 alpha (HIF-1α) mastering, since its level is higher under normoxia and hypoxia in Spalax cells. Consumed Glc in Spalax cells is utilized for the pentose phosphate pathway maintaining the NADPH pool, and is finally harbored as glutathione (GSH) and UDP-GlcNAc. The cytosolic domain in Spalax cells works in the semi-uncoupled mode that limits the consumed Glc-derived carbons flux to the tricarboxylic acid (TCA) cycle and reduces pyruvate delivery; however, it maintains the NAD⁺ pool via lactate dehydrogenase upregulation. Both normoxic and hypoxic mitochondrial homeostasis of Glc-originated carbons in Spalax are characterized by their massive cataplerotic flux along with the axis αKG→Glu→Pro→hydroxyproline (HPro). The product of collagen degradation, HPro, as well as free Pro are apparently involved in the bioenergetics of Spalax under both normoxia and hypoxia. The upregulation of 2-hydroxyglutarate production detected in Spalax cells may be involved in modulating the levels of HIF-1α. Collectively, these data suggest that Spalax cells utilize similar metabolic frame for both normoxia and hypoxia, where glucose metabolism is switched from oxidative pathways (conversion of pyruvate to Acetyl-CoA and further TCA cycle processes) to (i) pentose phosphate pathway, (ii) lactate production, and (iii) cataplerotic pathways leading to hexosamine, GSH, and HPro production. Full article

Oxidative stress has been acknowledged as a major factor in aging, senescence and neurodegenerative conditions. Mammalian models are susceptible to these stresses following the restoration of oxygen after anoxia; however, some organisms including the freshwater turtle Trachemys scripta can withstand repeated anoxia and reoxygenation without apparent pathology. T. scripta thus provides us with an alternate vertebrate model to investigate physiological mechanisms of neuroprotection. The objective of this study was to investigate the antioxidant methionine sulfoxide reductase system (Msr) in turtle neuronal tissue. We examined brain transcript and protein levels of MsrA and MsrB and examined the potential for the transcription factor FOXO3a to regulate the oxygen-responsive changes in Msr in vitro. We found that Msr mRNA and protein levels are differentially upregulated during anoxia and reoxygenation, and when cells were exposed to chemical oxidative stress. However, while MsrA and MsrB3 levels increased when cell cultures were exposed to chemical oxidative stress, this induction was not enhanced by treatment with epigallocatechin gallate (EGCG), which has previously been shown to enhance FOXO3a levels in the turtle. These results suggest that FOXO3a and Msr protect the cells from oxidative stress through different molecular pathways, and that both the Msr pathway and EGCG may be therapeutic targets to treat diseases related to oxidative damage. Full article

The anoxia-tolerant crucian carp (Carassius carassius) has been studied in detail for numerous years, with particular focus on unravelling the underlying physiological mechanisms of anoxia tolerance. However, relatively little work has been focused on what occurs beyond anoxia, and often the focus is a single organ or tissue type. In this study, we quantified more than 100 metabolites by capillary electrophoresis-mass spectrometry (CE-MS) in brain, heart, liver, and blood plasma from four experimental groups, being normoxic (control) fish, anoxia-exposed fish, and two groups that had been exposed to anoxia followed by reoxygenation for either 3 h or 24 h. The heart, which maintains cardiac output during anoxia, unexpectedly, was slower to recover compared to the brain and liver, mainly due to a slower return to control concentrations of the energy-carrying compounds ATP, GTP, and phosphocreatine. Crucian carp accumulated amino acids in most tissues, and also surprisingly high levels of succinate in all tissues investigated during anoxia. Purine catabolism was enhanced, leading to accumulation of uric acid during anoxia and increasing urea formation that continued into 24 h of reoxygenation. These tissue-specific differences in accumulation and distribution of the metabolites may indicate an intricate system of transport between tissues, opening for new avenues of investigation of possible mechanisms aimed at reducing the generation of reactive oxygen species (ROS) and resultant tissue damage during reoxygenation. Full article

Polymerization of actin filaments and microtubules constitutes a ubiquitous demand for cellular adenosine-5′-triphosphate (ATP) and guanosine-5′-triphosphate (GTP). In anoxia-tolerant animals, ATP consumption is minimized during overwintering conditions, but little is known about the role of cell structure in anoxia tolerance. Studies of overwintering mammals have revealed that microtubule stability in neurites is reduced at low temperature, resulting in withdrawal of neurites and reduced abundance of excitatory synapses. Literature for turtles is consistent with a similar downregulation of peripheral cytoskeletal activity in brain and liver during anoxic overwintering. Downregulation of actin dynamics, as well as modification to microtubule organization, may play vital roles in facilitating anoxia tolerance. Mitochondrial calcium release occurs during anoxia in turtle neurons, and subsequent activation of calcium-binding proteins likely regulates cytoskeletal stability. Production of reactive oxygen species (ROS) formation can lead to catastrophic cytoskeletal damage during overwintering and ROS production can be regulated by the dynamics of mitochondrial interconnectivity. Therefore, suppression of ROS formation is likely an important aspect of cytoskeletal arrest. Furthermore, gasotransmitters can regulate ROS levels, as well as cytoskeletal contractility and rearrangement. In this review we will explore the energetic costs of cytoskeletal activity, the cellular mechanisms regulating it, and the potential for cytoskeletal arrest being an important mechanism permitting long-term anoxia survival in anoxia-tolerant species, such as the western painted turtle and goldfish. Full article

Metabolic suppression is an essential strategy to cope with chronic hypoxia. This review examines the physiological processes used to survive in low oxygen environments. It proposes a novel mechanism–the remodeling of membrane lipids–to suppress ATP use and production. Temperature (homeoviscous adaptation), diet (natural doping in migrant birds) and body mass (membrane pacemaker of metabolism) have an impact on the lipid composition of membranes, which, in turn, modulates metabolic capacity. Vertebrate champions of hypoxia tolerance show extensive changes in membrane lipids upon in vivo exposure to low oxygen. These changes and those observed in hibernating mammals can promote the downregulation of ion pumps (major ATP consumers), ion channels, mitochondrial respiration capacity (state 3, proton leak, cytochrome c oxidase), and energy metabolism (β-oxidation and glycolysis). A common membrane signal regulating the joint inhibition of ion pumps and channels could be an exquisite way to preserve the balance between ATP supply and demand in hypometabolic states. Membrane remodeling together with more traditional mechanisms could work in concert to cause metabolic suppression. Full article

The utility of measuring whole-animal performance to frame the metabolic response to environmental hypoxia is well established. Progressively reducing ambient oxygen (O₂) will initially limit maximum metabolic rate as a result of a hypoxemic state and ultimately lead to a time-limited, tolerance state supported by substrate-level phosphorylation when the O₂ supply can no longer meet basic needs (standard metabolic rate, SMR). The metabolic consequences of declining ambient O₂ were conceptually framed for fishes initially by Fry’s hypoxic performance curve, which characterizes the hypoxemic state and its consequences to absolute aerobic scope (AAS), and Hochachka’s concept of scope for hypoxic survival, which characterizes time-limited life when SMR cannot be supported by O₂ supply. Yet, despite these two conceptual frameworks, the toolbox to assess whole-animal metabolic performance remains rather limited. Here, we briefly review the ongoing debate concerning the need to standardize the most commonly used assessments of respiratory performance in hypoxic fishes, namely critical O₂ (the ambient O₂ level below which maintenance metabolism cannot be sustained) and the incipient lethal O₂ (the ambient O₂ level at which a fish loses the ability to maintain upright equilibrium), and then we advance the idea that the most useful addition to the toolbox will be the limiting-O₂ concentration (LOC) performance curve. Using Fry & Hart’s (1948) hypoxia performance curve concept, an LOC curve was subsequently developed as an eco-physiological framework by Neil et al. and derived for a group of fish during a progressive hypoxia trial by Claireaux and Lagardère (1999). In the present review, we show how only minor modifications to available respirometry tools and techniques are needed to generate an LOC curve for individual fish. This individual approach to the LOC curve determination then increases its statistical robustness and importantly opens up the possibility of examining individual variability. Moreover, if peak aerobic performance at a given ambient O₂ level of each individual is expressed as a percentage of its AAS, the water dissolved O₂ that supports 50% of the individual’s AAS (DO_AAS-50) can be interpolated much like the P₅₀ for an O₂ hemoglobin dissociation curve (when hemoglobin is 50% saturated with O₂). Thus, critical O₂, incipient lethal O₂, DO_AAS-50 and P₅₀ and can be directly compared within and across species. While an LOC curve for individual fish represents a start to an ongoing need to seamlessly integrate aerobic to anaerobic capacity assessments in a single, multiplexed respirometry trial, we close with a comparative exploration of some of the known whole-organism anaerobic and aerobic capacity traits to examine for correlations among them and guide the next steps. Full article