Regulation of muscle pyruvate dehydrogenase activity and exercise fuel use in high-altitude deer mice.

Adult, lab-reared, highland deer mice acclimate to hypoxia by increasing reliance on carbohydrates to fuel exercise. Yet neither the underlying mechanisms for this shift in fuel use pattern, nor the impact of lifetime hypoxia exposure experienced in the high alpine, are fully understood. Thus, we assessed exercise fuel use in wild highland deer mice running in their native environment. We examined a key step in muscle carbohydrate oxidation, pyruvate dehydrogenase (PDH) and its regulation with exercise at altitude in wild highlanders and using first generation (G1) lab born and raised highlanders acclimated to normoxia or hypoxia. PDH activity was also determined in the gastrocnemius of G1 highlanders using an in situ muscle preparation. We found wild highlanders had a high reliance on carbohydrates while running in their native environment, consistent with data from hypoxia acclimated G1 highlanders. PDH activity in the gastrocnemius was similar post exercise between G1 and wild highlanders. However, when the gastrocnemius was stimulated at a light work rate in situ, PDH activity was higher in hypoxia acclimated G1 highlanders, and associated with a lower intramuscular lactate. These findings were supported by lower PDH kinase 2 protein expression in hypoxia acclimated G-1 mice. Our findings indicate that adult phenotypic plasticity in response to low oxygen is sufficient to increase exercise carbohydrate reliance in highland deer mice. Additionally, variation in PDH regulation with hypoxia acclimation contributes to shifts in whole-animal fuel use patterns and are likely to improved exercise performance via elevated energy yield per mole O2.

Metabolic recovery from submaximal exercise in hypoxia acclimated high altitude deer mice (Peromyscus maniculatus).

Animals living at high-altitude are faced with unremitting low oxygen availability. This can make it difficult to perform daily tasks that require increases in aerobic metabolism. An activity important for survival is aerobic locomotion, and the rapid recovery of muscle metabolism post exercise. Past work shows that hypoxia acclimated high-altitude mice (Peromyscus maniculatus) have a greater reliance on carbohydrates to power exercise than low altitude mice. However, it is unclear how quickly after aerobic exercise these mice can recovery and replenish muscle glycogen stores. The gastrocnemius muscle of high-altitude deer mice has a more aerobic phenotype and a greater capacity to oxidize lipids than low altitude deer mice. This suggests that high altitude mice may recover more rapidly from exercise than their lowland counterparts due to a greater capacity to support glycogen replenishment using intramuscular triglycerides (IMTG). To explore this possibility, we used low- and high-altitude native deer mice born and raised in common lab conditions and acclimated to chronic hypoxia. We determined changes in oxygen consumption following 15 min of aerobic exercise in 12% O2 and sampled skeletal muscles and liver at various time points during recovery to examine changes in key metabolites, including glycogen and IMTG. We found depletion in glycogen stores during exercise only in lowlanders, which returned to resting levels following 90 min of recovery. In contrast, IMTG did not change significantly with exercise or during recovery in either population. These data suggest that exercise recovery is influenced by altitude ancestry in deer mice.

Highland deer mice support increased thermogenesis in response to chronic cold hypoxia by shifting uptake of circulating fatty acids from muscles to brown adipose tissue.

During maximal cold challenge (cold-induced V̇O2,max) in hypoxia, highland deer mice (Peromyscus maniculatus) show higher rates of circulatory fatty acid delivery compared with lowland deer mice. Fatty acid delivery also increases with acclimation to cold hypoxia (CH) and probably plays a major role in supporting the high rates of thermogenesis observed in highland deer mice. However, it is unknown which tissues take up these fatty acids and their relative contribution to thermogenesis. The goal of this study was to determine the uptake of circulating fatty acids into 24 different tissues during hypoxic cold-induced V̇O2,max, by using [1-14C]2-bromopalmitic acid. To uncover evolved and environment-induced changes in fatty acid uptake, we compared lab-born and -raised highland and lowland deer mice, acclimated to either thermoneutral (30°C, 21 kPa O2) or CH (5°C, 12 kPa O2) conditions. During hypoxic cold-induced V̇O2,max, CH-acclimated highlanders decreased muscle fatty acid uptake and increased uptake into brown adipose tissue (BAT) relative to thermoneutral highlanders, a response that was absent in lowlanders. CH acclimation was also associated with increased activities of enzymes citrate synthase and ß-hydroxyacyl-CoA dehydrogenase in the BAT of highlanders, and higher levels of fatty acid translocase CD36 (FAT/CD36) in both populations. This is the first study to show that cold-induced fatty acid uptake is distributed across a wide range of tissues. Highland deer mice show plasticity in this fatty acid distribution in response to chronic cold hypoxia, and combined with higher rates of tissue delivery, this contributes to their survival in the cold high alpine environment.

Evolved changes in phenotype across skeletal muscles in deer mice native to high altitude.

The cold and hypoxic conditions at high altitude necessitate high metabolic O2 demands to support thermogenesis while hypoxia reduces O2 availability. Skeletal muscles play key roles in thermogenesis, but our appreciation of muscle plasticity and adaptation at high altitude has been hindered by past emphasis on only a small number of muscles. We examined this issue in deer mice (Peromyscus maniculatus). Mice derived from both high-altitude and low-altitude populations were born and raised in captivity and then acclimated as adults to normoxia or hypobaric hypoxia (12 kPa O2 for 6-8 wk). Maximal activities of citrate synthase (CS), cytochrome c oxidase (COX), ß-hydroxyacyl-CoA dehydrogenase (HOAD), hexokinase (HK), pyruvate kinase (PK), and lactate dehydrogenase (LDH) were measured in 20 muscles involved in shivering, locomotion, body posture, ventilation, and mastication. Principal components analysis revealed an overall difference in muscle phenotype between populations but no effect of hypoxia acclimation. High-altitude mice had greater activities of mitochondrial enzymes and/or lower activities of PK or LDH across many (but not all) respiratory, limb, core and mastication muscles compared with low-altitude mice. In contrast, chronic hypoxia had very few effects across muscles. Further examination of CS in the gastrocnemius showed that population differences in enzyme activity stemmed from differences in protein abundance and mRNA expression but not from population differences in CS amino acid sequence. Overall, our results suggest that evolved increases in oxidative capacity across many skeletal muscles, at least partially driven by differences in transcriptional regulation, may contribute to high-altitude adaptation in deer mice.NEW & NOTEWORTHY Most previous studies of muscle plasticity and adaptation in high-altitude environments have focused on a very limited number of skeletal muscles. Comparing high-altitude versus low-altitude populations of deer mice, we show that a large number of muscles involved in shivering, locomotion, body posture, ventilation, and mastication exhibit greater mitochondrial enzyme activities in the high-altitude population. Therefore, evolved increases in mitochondrial oxidative capacity across skeletal muscles contribute to high-altitude adaptation.

Function of left ventricle mitochondria in highland deer mice and lowland mice.

To gain insight into the mitochondrial mechanisms of hypoxia tolerance in high-altitude natives, we examined left ventricle mitochondrial function of highland deer mice compared with lowland native deer mice and white-footed mice. Highland and lowland native deer mice (Peromyscus maniculatus) and lowland white-footed mice (P. leucopus) were first-generation born and raised in common lab conditions. Adult mice were acclimated to either normoxia or hypoxia (60 kPa) equivalent to ~ 4300 m for at least 6 weeks. Left ventricle mitochondrial physiology was assessed by determining respiration in permeabilized muscle fibers with carbohydrates, lipids, and lactate as substrates. We also measured the activities of several left ventricle metabolic enzymes. Permeabilized left ventricle muscle fibers of highland deer mice showed greater rates of respiration with lactate than either lowland deer mice or white-footed mice. This was associated with higher activities of lactate dehydrogenase in tissue and isolated mitochondria in highlanders. Normoxia-acclimated highlanders also showed higher respiratory rates with palmitoyl-carnitine than lowland mice. Maximal respiratory capacity through complexes I and II was also greater in highland deer mice but only compared with lowland deer mice. Acclimation to hypoxia had little effect on respiration rates with these substrates. In contrast, left ventricle activities of hexokinase increased in both lowland and highland deer mice after hypoxia acclimation. These data suggest that highland deer mice support an elevated cardiac function in hypoxia, in part, with high ventricle cardiomyocyte respiratory capacities supported by carbohydrates, fatty acids, and lactate.

Thermogenesis is supported by high rates of circulatory fatty acid and triglyceride delivery in highland deer mice.

Highland native deer mice (Peromyscus maniculatus) have greater rates of lipid oxidation during maximal cold challenge in hypoxia (hypoxic cold-induced V̇O2,max) compared with their lowland conspecifics. Lipid oxidation is also increased in deer mice acclimated to simulated high altitude (cold hypoxia), regardless of altitude ancestry. The underlying lipid metabolic pathway traits responsible for sustaining maximal thermogenic demand in deer mice is currently unknown. The objective of this study was to characterize key steps in the lipid oxidation pathway in highland and lowland deer mice acclimated to control (23°C, 21 kPa O2) or cold hypoxic (5°C, 12 kPa O2) conditions. We hypothesized that capacities for lipid delivery and tissue uptake will be greater in highlanders and further increase with cold hypoxia acclimation. With the transition from rest to hypoxic cold-induced V̇O2,max, both highland and lowland deer mice showed increased plasma glycerol concentrations and fatty acid availability. Interestingly, acclimation to cold hypoxia led to increased plasma triglyceride concentrations at cold-induced V̇O2,max, but only in highlanders. Highlanders also had significantly greater delivery rates of circulatory free fatty acids and triglycerides due to higher plasma flow rates at cold-induced V̇O2,max. We found no population or acclimation differences in fatty acid translocase (FAT/CD36) abundance in the gastrocnemius or brown adipose tissue, suggesting that fatty acid uptake across membranes is not limiting during thermogenesis. Our data indicate that circulatory lipid delivery plays a major role in supporting the high thermogenic rates observed in highland versus lowland deer mice.

Phenotypic plasticity to chronic cold exposure in two species of Peromyscus from different environments.

Effective thermoregulation is important for mammals, particularly those that remain winter-active. Adjustments in thermoregulatory capacity in response to chronic cold can improve capacities for metabolic heat production (cold-induced maximal oxygen consumption, [Formula: see text]), minimize rates of heat loss (thermal conductance), or both. This can be challenging for animals living in chronically colder habitats where necessary resources (i.e., food, O2) for metabolic heat production are limited. Here we used lowland native white-footed mice (Peromyscus leucopus) and highland deer mice (P. maniculatus) native to 4300 m, to test the hypothesis that small winter-active mammals have evolved distinct cold acclimation responses to tailor their thermal physiology based on the energetic demands of their environment. We found that both species increased their [Formula: see text] after cold acclimation, associated with increases in brown adipose tissue mass and expression of uncoupling protein 1. They also broadened their thermoneutral zone to include lower ambient temperatures. This was accompanied by an increase in basal metabolic rate but only in white-footed mice, and neither species adjusted thermal conductance. Unique to highland deer mice was a mild hypothermia as ambient temperatures decreased, which reduced the gradient for heat loss, possibly to save energy in the chronically cold high alpine. These results highlight that thermal acclimation involves coordinated plasticity of numerous traits and suggest that small, winter-active mammals may adjust different aspects of their physiology in response to changing temperatures to best suit their energetic and thermoregulatory needs.

Increased Reliance on Carbohydrates for Aerobic Exercise in Highland Andean Leaf-Eared Mice, but Not in Highland Lima Leaf-Eared Mice.

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 (V˙O2max), and submaximal exercise metabolism. Lowland and highland populations of both species showed no difference in V˙O2max running in either normoxia or hypoxia. When run at 75% of 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.

The effect of marine dissolved organic carbon on nickel accumulation in early life-stages of the sea urchin, Strongylocentrotus purpuratus.

Dissolved organic carbon (DOC) is known to ameliorate the toxicity of the trace metal nickel (Ni) to aquatic animals. In theory, this effect is mediated by the capacity of DOC to bind Ni, rendering it less bioavailable, with the resulting reduction in accumulation limiting toxicological effects. However, there is a lack of experimental data examining Ni accumulation in marine settings with natural sources of DOC. In the current study, radiolabelled Ni was used to examine the time- and concentration-dependence of Ni accumulation, using naturally sourced DOC, on developing larvae of the sea urchin Strongylocentrotus purpuratus. Contrary to prediction, the two tested natural DOC samples (collected from the eastern United States, DOC 2 (Seaview park, Rhode Island (SVP)) and DOC 7 (Aubudon Coastal Center, Connecticut)) which had previously been shown to protect against Ni toxicity, did not limit accumulation. The control (artificial seawater with no added DOC), and the DOC 2 sample could mostly be described as having saturable Ni uptake, whereas Ni uptake in the presence of DOC 7 was mostly linear. These data provide evidence that DOC modifies the bioavailability of Ni, through either indirect effects (e.g. membrane permeability) or by the absorption of DOC-Ni complexes. There was some evidence for regulation of Ni accumulation in later-stage embryos (96-h) where the bioconcentration factor for Ni declined with increasing Ni exposure concentration. These data have implications for predictive modelling approaches that rely on known relationships between Ni speciation, bioavailability and bioreactivity, by suggesting that these relationships may not hold for natural marine DOC samples in the developing sea urchin model system.

Plasticity of non-shivering thermogenesis and brown adipose tissue in high-altitude deer mice.

High altitude environments challenge small mammals with persistent low ambient temperatures that require high rates of aerobic heat production in face of low O2 availability. An important component of thermogenic capacity in rodents is non-shivering thermogenesis (NST) mediated by uncoupled mitochondrial respiration in brown adipose tissue (BAT). NST is plastic, and capacity for heat production increases with cold acclimation. However, in lowland native rodents, hypoxia inhibits NST in BAT. We hypothesize that highland deer mice (Peromyscus maniculatus) overcome the hypoxic inhibition of NST through changes in BAT mitochondrial function. We tested this hypothesis using lab born and raised highland and lowland deer mice, and a lowland congeneric (Peromyscus leucopus), acclimated to either warm normoxia (25°C, 760 mmHg) or cold hypoxia (5°C, 430 mmHg). We determined the effects of acclimation and ancestry on whole-animal rates of NST, the mass of interscapular BAT (iBAT), and uncoupling protein (UCP)-1 protein expression. To identify changes in mitochondrial function, we conducted high-resolution respirometry on isolated iBAT mitochondria using substrates and inhibitors targeted to UCP-1. We found that rates of NST increased with cold hypoxia acclimation but only in highland deer mice. There was no effect of cold hypoxia acclimation on iBAT mass in any group, but highland deer mice showed increases in UCP-1 expression and UCP-1-stimulated mitochondrial respiration in response to these stressors. Our results suggest that highland deer mice have evolved to increase the capacity for NST in response to chronic cold hypoxia, driven in part by changes in iBAT mitochondrial function.

Evolved changes in maternal care in high-altitude native deer mice.

At high altitude (HA), unremitting low oxygen and persistent cold push small mammals close to their metabolic ceilings, leaving limited scope for aerobically demanding activities. However, HA breeding seasons are relatively short and endemic rodents compensate with larger litters than low altitude (LA) conspecifics. Rodent mothers are the sole source of heat and nutrition for altricial offspring and lactation is energetically costly. Thus, it is unclear how HA females balance energy allocation during the nursing period. We hypothesized that HA female rodents invest heavily in each litter to ensure postnatal survival. We measured maternal energetic output and behaviour in nursing deer mice (Peromyscus maniculatus) native to LA (400 m a.s.l.) and HA (4350 m a.s.l.) under control (24°C, 760 mmHg) and cold hypoxia conditions, simulating HA (5°C, 430 mmHg). Strikingly, resting metabolic rates of lactating HA and LA females under cold hypoxia were 70-85% of their maximum aerobic capacity. In cold hypoxia, LA mothers increased both nursing time and milk fat content, however their pups were leaner and severely growth restricted at weaning. HA mothers also increased nursing in cold hypoxia but for far less time than LA mothers. Despite receiving less care, HA pups in cold hypoxia only experienced small growth restrictions at weaning and maintained body composition. As adults, HA mice raised in cold hypoxia had increased aerobic capacity compared to controls. These data suggest that HA mothers prioritize their own maintenance costs over investing heavily in their offspring. Pups compensate for this lack of care, likely by reducing their own metabolic costs during development.

Ancestral and developmental cold alter brown adipose tissue function and adult thermal acclimation in Peromyscus.

Small, non-hibernating endotherms increase their thermogenic capacity to survive seasonal cold, through adult phenotypic flexibility. In mammals, this response is primarily driven by remodeling of brown adipose tissue (BAT), which matures postnatally in altricial species. In many regions, ambient temperatures can vary dramatically throughout the breeding season. We used second-generation lab-born Peromyscus leucopus, cold exposed during two critical developmental windows, to test the hypothesis that adult phenotypic flexibility to cold is influenced by rearing temperature. We found that cold exposure during the postnatal period (14 °C, birth to 30 days) accelerated BAT maturation and permanently remodeled this tissue. As adults, these mice had increased BAT activity and thermogenic capacity relative to controls. However, they also had a blunted acclimation response when subsequently cold exposed as adults (5 °C for 6 weeks). Mice born to cold-exposed mothers (14 °C, entire pregnancy) also showed limited capacity for flexibility as adults, demonstrating that maternal cold stress programs the offspring thermal acclimation response. In contrast, for P. maniculatus adapted to the cold high alpine, BAT maturation rate was unaffected by rearing temperature. However, both postnatal and prenatal cold exposure limited the thermal acclimation response in these cold specialists. Our results suggest a complex interaction between developmental and adult environment, influenced strongly by ancestry, drives thermogenic capacity in the wild.

Lipid oxidation during thermogenesis in high-altitude deer mice (Peromyscus maniculatus).

When at their maximum thermogenic capacity (cold-induced V̇o2max), small endotherms reach levels of aerobic metabolism as high, or even higher, than running V̇o2max. How these high rates of thermogenesis are supported by substrate oxidation is currently unclear. The appropriate utilization of metabolic fuels that could sustain thermogenesis over extended periods may be important for survival in cold environments, like high altitude. Previous studies show that high capacities for lipid use in high-altitude deer mice may have evolved in concert with greater thermogenic capacities. The purpose of this study was to determine how lipid utilization at both moderate and maximal thermogenic intensities may differ in high- and low-altitude deer mice, and strictly low-altitude white-footed mice. We also examined the phenotypic plasticity of lipid use after acclimation to cold hypoxia (CH), conditions simulating high altitude. We found that lipids were the primary fuel supporting both moderate and maximal rates of thermogenesis in both species of mice. Lipid oxidation increased threefold in mice from 30°C to 0°C, consistent with increases in oxidation of [13C]palmitic acid. CH acclimation led to an increase in [13C]palmitic acid oxidation at 30°C but did not affect total lipid oxidation. Lipid oxidation rates at cold-induced V̇o2max were two- to fourfold those at 0°C and increased further after CH acclimation, especially in high-altitude deer mice. These are the highest mass-specific lipid oxidation rates observed in any land mammal. Uncovering the mechanisms that allow for these high rates of oxidation will aid our understanding of the regulation of lipid metabolism.

Chronic cold exposure induces mitochondrial plasticity in deer mice native to high altitudes.

KEY POINTS: Small mammals native to high altitude must sustain high rates of thermogenesis to cope with cold. Skeletal muscle is a key site of shivering and non-shivering thermogenesis, but the importance of mitochondrial plasticity in cold hypoxic environments remains unresolved. We examined high-altitude deer mice, which have evolved a high capacity for aerobic thermogenesis, to determine the mechanisms of mitochondrial plasticity during chronic exposure to cold and hypoxia, alone and in combination. Cold exposure in normoxia or hypoxia increased mitochondrial leak respiration and decreased phosphorylation efficiency and OXPHOS coupling efficiency, which may serve to augment non-shivering thermogenesis. Cold also increased muscle oxidative capacity, but reduced the capacity for mitochondrial respiration via complex II relative to complexes I and II combined. High-altitude mice had a more oxidative muscle phenotype than low-altitude mice. Therefore, both plasticity and evolved changes in muscle mitochondria contribute to thermogenesis at high altitude. ABSTRACT: Small mammals native to high altitude must sustain high rates of thermogenesis to cope with cold and hypoxic environments. Skeletal muscle is a key site of shivering and non-shivering thermogenesis, but the importance of mitochondrial plasticity in small mammals at high altitude remains unresolved. High-altitude deer mice (Peromyscus maniculatus) and low-altitude white-footed mice (P. leucopus) were born and raised in captivity, and chronically exposed as adults to warm (25°C) normoxia, warm hypoxia (12 kPa O2 ), cold (5°C) normoxia, or cold hypoxia. We then measured oxidative enzyme activities, oxidative fibre density and capillarity in the gastrocnemius, and used a comprehensive substrate titration protocol to examine the function of muscle mitochondria by high-resolution respirometry. Exposure to cold in both normoxia or hypoxia increased the activities of citrate synthase and cytochrome oxidase. In lowlanders, this was associated with increases in capillary density and the proportional abundance of oxidative muscle fibres, but in highlanders, these traits were unchanged at high levels across environments. Environment had some distinct effects on mitochondrial OXPHOS capacity between species, but the capacity of complex II relative to the combined capacity of complexes I and II was consistently reduced in both cold environments. Both cold environments also increased leak respiration and decreased phosphorylation efficiency and OXPHOS coupling efficiency in both species, which may serve to augment non-shivering thermogenesis. These cold-induced changes in mitochondrial function were overlaid upon the generally more oxidative phenotype of highlanders. Therefore, both plasticity and evolved changes in muscle mitochondria contribute to thermogenesis at high altitudes.

Coordinated changes across the O2 transport pathway underlie adaptive increases in thermogenic capacity in high-altitude deer mice.

Animals native to the hypoxic and cold environment at high altitude provide an excellent opportunity to elucidate the integrative mechanisms underlying the adaptive evolution and plasticity of complex traits. The capacity for aerobic thermogenesis can be a critical determinant of survival for small mammals at high altitude, but the physiological mechanisms underlying the evolution of this performance trait remain unresolved. We examined this issue by comparing high-altitude deer mice (Peromyscus maniculatus) with low-altitude deer mice and white-footed mice (P. leucopus). Mice were bred in captivity and adults were acclimated to each of four treatments: warm (25°C) normoxia, warm hypoxia (12 kPa O2), cold (5°C) normoxia or cold hypoxia. Acclimation to hypoxia and/or cold increased thermogenic capacity in deer mice, but hypoxia acclimation led to much greater increases in thermogenic capacity in highlanders than in lowlanders. The high thermogenic capacity of highlanders was associated with increases in pulmonary O2 extraction, arterial O2 saturation, cardiac output and arterial-venous O2 difference. Mechanisms underlying the evolution of enhanced thermogenic capacity in highlanders were partially distinct from those underlying the ancestral acclimation responses of lowlanders. Environmental adaptation has thus enhanced phenotypic plasticity and expanded the physiological toolkit for coping with the challenges at high altitude.

Adaptive Shifts in Gene Regulation Underlie a Developmental Delay in Thermogenesis in High-Altitude Deer Mice.

Aerobic performance is tied to fitness as it influences an animal's ability to find food, escape predators, or survive extreme conditions. At high altitude, where low O2 availability and persistent cold prevail, maximum metabolic heat production (thermogenesis) is an aerobic performance trait that is closely linked to survival. Understanding how thermogenesis evolves to enhance survival at high altitude will yield insight into the links between physiology, performance, and fitness. Recent work in deer mice (Peromyscus maniculatus) has shown that adult mice native to high altitude have higher thermogenic capacities under hypoxia compared with lowland conspecifics, but that developing high-altitude pups delay the onset of thermogenesis. This finding suggests that natural selection on thermogenic capacity varies across life stages. To determine the mechanistic cause of this ontogenetic delay, we analyzed the transcriptomes of thermoeffector organs-brown adipose tissue and skeletal muscle-in developing deer mice native to low and high altitude. We demonstrate that the developmental delay in thermogenesis is associated with adaptive shifts in the expression of genes involved in nervous system development, fuel/O2 supply, and oxidative metabolism pathways. Our results demonstrate that selection has modified the developmental trajectory of the thermoregulatory system at high altitude and has done so by acting on the regulatory systems that control the maturation of thermoeffector tissues. We suggest that the cold and hypoxic conditions of high altitude force a resource allocation tradeoff, whereby limited energy is allocated to developmental processes such as growth, versus active thermogenesis, during early development.

Ontogenesis of evolved changes in respiratory physiology in deer mice native to high altitude.

High-altitude environments are cold and hypoxic, and many high-altitude natives have evolved changes in respiratory physiology that improve O2 uptake in hypoxia as adults. Altricial mammals undergo a dramatic metabolic transition from ectothermy to endothermy in early post-natal life, which may influence the ontogenetic development of respiratory traits at high altitude. We examined the developmental changes in respiratory and haematological traits in deer mice (Peromyscus maniculatus) native to high altitude, comparing the respiratory responses to progressive hypoxia between highland and lowland deer mice. Among adults, highlanders exhibited higher total ventilation and a more effective breathing pattern (relatively deeper tidal volumes), for mice that were caught and tested at their native altitudes and those lab-raised in normoxia. Lab-raised progeny of each population were also tested at post-natal day (P)7, 14, 21 and 30. Highlanders developed an enhanced hypoxic ventilatory response by P21, concurrent with the full maturation of the carotid bodies, and their more effective breathing pattern arose by P14; these ages correspond to critical benchmarks in the full development of homeothermy in highlanders. However, highlanders exhibited developmental delays in ventilatory sensitivity to hypoxia, hyperplasia of type I cells in the carotid body and increases in blood haemoglobin content compared with lowland mice. Nevertheless, highlanders maintained consistently higher arterial O2 saturation in hypoxia across development, in association with increases in blood-O2 affinity that were apparent from birth. We conclude that evolved changes in respiratory physiology in high-altitude deer mice become expressed in association with the post-natal development of endothermy.

Influence of 96h sub-lethal copper exposure on aerobic scope and recovery from exhaustive exercise in killifish (Fundulus heteroclitus).

Production of industrial effluents have led to increased copper (Cu) pollution of aquatic ecosystems, impacting the physiology of aquatic vertebrates. Past work has shown that Cu exerts its toxicity by disruption ion regulation and/ or increasing oxidative stress. However, it remains unclear how Cu may influence aerobic metabolism and hypoxia tolerance, two possible targets of its toxicity. To address this issue, we exposed freshwater acclimated killifish (F. heteroclitus) to a 96 h Cu exposure at a target concentration of 100 µg L-1. We determined resting oxygen consumption (MO2), MO2max after exhaustive exercise, and followed MO2 for 3 h in post-exercise recovery in water with either no Cu or 100 µg L-1 Cu. We assessed hypoxia tolerance by determining the critical oxygen tension (Pcrit). It was found that killifish exposed to combined 96 h Cu exposure and Cu present during metabolic measurements, showed a significant decrease in MO2max and in aerobic scope (MO2max - MO2rest), compared to control fish. However, changes in blood and muscle lactate and muscle glycogen were not consistent with an upregulation of anaerobic metabolism as compensation for reduced aerobic performance in Cu exposed fish. Hypoxia tolerance was not influenced by the 96 h Cu exposure or by presence or absence of Cu during the Pcrit test. This study suggests that Cu differentially influences responses to changes in oxygen demand and oxygen availability.

Developmental delay in shivering limits thermogenic capacity in juvenile high-altitude deer mice (Peromyscus maniculatus).

Many endotherms native to cold and hypoxic high-altitude (HA) environments have evolved a highly vascularized and aerobic skeletal muscle. This specialized muscle phenotype contributes via shivering to an enhanced capacity for aerobic thermogenesis (cold-induced V̇O2,max). However, it is unclear how selection at HA for shivering thermogenesis acts early in the development of small altricial mammals, which are born with immature skeletal muscles and without the capacity for homeothermic endothermy. We have previously shown that postnatal maturation of brown adipose tissue and non-shivering thermogenesis is delayed in HA native deer mouse pups (Peromyscus maniculatus). To assess whether HA adaptation has also altered the developmental program of skeletal muscle and shivering thermogenesis, we used laboratory-reared descendants of deer mice native to low altitude (LA, 430 m a.s.l.) and HA (4350 m a.s.l.) and a LA congeneric outgroup (P. leucopus). We found that LA juveniles were able to shiver robustly at 2 weeks after birth. However, HA juveniles were unlikely able to shiver at this point, resulting in a 30% lower capacity for thermoregulation compared with lowlanders. It was only at 27 days after birth that HA juveniles had established the aerobic muscle phenotype characteristic of HA adults and a superior cold-induced V̇O2,max compared with LA mice of the same age. The capacity for shivering may be delayed in HA mice to allow energy to be allocated to other important processes such as growth.

Development of homeothermic endothermy is delayed in high-altitude native deer mice (Peromyscus maniculatus).

Altricial mammals begin to independently thermoregulate during the first few weeks of postnatal development. In wild rodent populations, this is also a time of high mortality (50-95%), making the physiological systems that mature during this period potential targets for selection. High altitude (HA) is a particularly challenging environment for small endotherms owing to unremitting low O2 and ambient temperatures. While superior thermogenic capacities have been demonstrated in adults of some HA species, it is unclear if selection has occurred to survive these unique challenges early in development. We used deer mice (Peromyscus maniculatus) native to high and low altitude (LA), and a strictly LA species (Peromyscus leucopus), raised under common garden conditions, to determine if postnatal onset of endothermy and maturation of brown adipose tissue (BAT) is affected by altitude ancestry. We found that the onset of endothermy corresponds with the maturation and activation of BAT at an equivalent age in LA natives, with 10-day-old pups able to thermoregulate in response to acute cold in both species. However, the onset of endothermy in HA pups was substantially delayed (by approx. 2 days), possibly driven by delayed sympathetic regulation of BAT. We suggest that this delay may be part of an evolved cost-saving measure to allow pups to maintain growth rates under the O2-limited conditions at HA.