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1. Omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) are essential micronutrients for optimal functioning of cellular metabolism and for somatic growth of all vertebrates including fishes. In addition, n-3 LC-PUFA could also play a key role in response of fishes and other ectothermic vertebrates to changing temperatures. 2. An important, but largely overlooked, consequence of climate change is the reduced availability of dietary n-3 LC-PUFA in aquatic food webs. Changes in availability of dietary n-3 LC-PUFA have recently been proposed as a major driver of novel adaptations and diversification of consumers. Yet, there is only limited knowledge about how n-3 LC-PUFA depletion in aquatic food-webs will affect the performance of wild fishes. 3. Here we combine biochemistry and physiology at the cellular level with physiological and cognitive processes at the whole-animal level to test how ecologically relevant deprivation of n-3 LC-PUFA affects performance of wild juvenile Atlantic salmon (Salmo salar). 4. We found that juvenile salmon had a limited capacity to maintain the fatty acid profile of both muscle and brain under a n-3 LC-PUFA-deficient diet. Despite these findings, brain tissues showed remarkable functional stability in mitochondrial metabolism, and we found no effect of diet on learning ability. However, we found that mitochondrial efficiency in muscles and the somatic growth were reduced under a n-3 LC-PUFA-deficient diet. Importantly, we discovered that the somatic growth of juvenile salmon within both treatments decreased with increasing rate of DHA synthesis and retention. 5. Since DHA is essential for functioning of cellular metabolism, which together with body size are traits closely related to fitness of wild fishes, we suggest that the trade-off between growth rate and accumulation of DHA could play a critical role in resilience of juvenile salmon to the ongoing rapid environmental change.

Endotherms in cold regions improve heat-producing capacity when preparing for winter. We know comparatively little about how this change is fueled by seasonal adaptation in cellular respiration. Thus, we studied the changes of mitochondrial function in red blood cells in sympatric Coal (Periparus ater), Blue (Cyanistes caeruleus), and Great (Parus major) tits between autumn and winter. These species differ more than twofold in body mass and in several aspects of their foraging ecology and social dominance, which could require differential seasonal adaptation of energy expenditure. Coal and Great tits in particular upregulated the mitochondrial respiration rate and mitochondrial volume in winter. This was not directed toward ATP synthesis, instead reflecting increased uncoupling of electron transport from ATP production. Because uncoupling is exothermic, this increased heat-producing capacity at the sub-cellular level in winter. This previously unexplored the route of thermogenesis in birds should be addressed in future work.

Astrocytes, glial cells within the brain, work to protect neurons during high levels of activity by maintaining oxidative homeostasis via regulation of energy supply and antioxidant systems. In recent years, mitochondrial dysfunction has been highlighted as an underlying factor of pathology in many neurological disorders. In animal studies of Fragile X Syndrome (FXS), the leading genetic cause of autism, higher levels of reactive oxygen species, lipid peroxidation, and protein oxidation within the brain indicates that mitochondria function is also altered in FXS. Despite their integral contribution to redox homeostasis within the CNS, the role of astrocytes on the occurrence or progression of neurodevelopmental disorders in this way is rarely considered. This study specifically examines changes to astrocyte mitochondrial function and antioxidant expression that may occur in FXS. Using the Fmr1 knockout (KO) mouse model, mitochondrial respiration and reactive oxygen species (ROS) emission were analyzed in primary cortical astrocytes. While mitochondrial respiration was similar between genotypes, ROS emission was significantly elevated in Fmr1 KO astrocytes. Notably, NADPH-oxidase 2 expression in Fmr1 KO astrocytes was also enhanced but only changes in catalase antioxidant enzyme expression were noted. Characterization of astrocyte factors involved in redox imbalance is invaluable to uncovering potential sources of oxidative stress in neurodevelopmental disorders and more specifically, the intercellular mechanisms that contribute to dysfunction in FXS.

There was an error in Journal of Experimental Biology (2017) 220, [1079-1089][1] ([doi: 10.1242/jeb.151191][2]). In Fig. 2C,D, some of the symbols for the species were incorrect (although the colours were correct). The original and corrected figures are shown below. This does not change the

High-altitude environments require that animals meet the metabolic O2 demands for locomotion and thermogenesis in O2-thin air, but the degree to which convergent metabolic changes have arisen across independent high-altitude lineages or the speed at which such changes arise is unclear. We examined seven high-altitude waterfowl that have inhabited the Andes (3812–4806 m elevation) over varying evolutionary time scales, to elucidate changes in biochemical pathways of energy metabolism in flight muscle relative to low-altitude sister taxa. Convergent changes across high-altitude taxa included increased hydroxyacyl-coA dehydrogenase and succinate dehydrogenase activities, decreased lactate dehydrogenase, pyruvate kinase, creatine kinase, and cytochrome c oxidase activities, and increased myoglobin content. ATP synthase activity increased in only the longest established high-altitude taxa, whereas hexokinase activity increased in only newly established taxa. Therefore, changes in pathways of lipid oxidation, glycolysis, and mitochondrial oxidative phosphorylation are common strategies to cope with high-altitude hypoxia, but some changes require longer evolutionary time to arise.

Animals native to high altitude must overcome the constraining effects of hypoxia on tissue O2 supply to support routine metabolism, thermoregulation in the cold, and exercise. Deer mice (Peromyscus maniculatus) native to high altitude have evolved an enhanced aerobic capacity in hypoxia, along with increased capillarity and oxidative capacity of locomotory muscle. Here, we examined whether exposure to chronic hypoxia during development or adulthood affects muscle phenotype. Deer mice from a highland population were bred in captivity at sea level, and exposed to normoxia or one of four treatments of hypobaric hypoxia (12kPa O2, simulating hypoxia at ~4300m): adult hypoxia (6-8weeks), post-natal hypoxia (birth to adulthood), pre-natal hypoxia (before conception to adulthood), and parental hypoxia (in which mice were conceived and raised in normoxia, but their parents were previously exposed to hypoxia). Litter size was similar across treatments, and pups survived the hypoxia exposures and grew to similar body masses at ~6-8months of age. Hypoxia had no effect on the masses of gastrocnemius and soleus muscles. There was a strong concordance between two distinct histological methods for staining capillaries in the gastrocnemius - alkaline phosphatase activity and binding of Griffonia simplicifolia lectin I - each of which showed that capillarity and muscle fibre size were largely unaffected by hypoxia. Maximal activities of several metabolic enzymes (cytochrome c oxidase, citrate synthase, isocitrate dehydrogenase, and lactate dehydrogenase) in the gastrocnemius were also largely unaffected by hypoxia. Therefore, the evolved muscle phenotype of high-altitude deer mice is relatively insensitive to hypoxia across life stages.

Protein instability is a major obstacle in the production and delivery of monoclonal antibody–based therapies for cancer. This study presents real-time isothermal differential scanning fluorimetry as an emerging method to evaluate the stability of human immunoglobulin G protein with high sensitivity. The stability of polyclonal human immunoglobulin G against urea-induced denaturation was assessed following: (1) oxidation by the free-radical generator 2,2-Azobis[2-amidinopropane]dihydrochloride and (2) in selected storage buffers. Significant differences in immunoglobulin G stability were detected by real-time isothermal differential scanning fluorimetry when the immunoglobulin G was stored in 1,4-Piperazinediethanesulfonic acid buffer compared to phosphate-buffered saline, with half-maximal rate of denaturation occurring at a higher urea concentration in 1,4-Piperazinediethanesulfonic acid than phosphate-buffered saline (K nd;PIPES = 3.56 ± 0.09 M, K nd;PBS = 2.94 ± 0.08 M; P < .01), but differential scanning fluorimetry did not detect differences in unfolding temperature (T m;PIPES = 70.5 ± 0.3°C, T m;PBS = 69.7 ± 0.2°C). The effects of 2,2-Azobis[2-amidinopropane]dihydrochloride-induced oxidation on immunoglobulin G stability were analyzed by real-time isothermal differential scanning fluorimetry; the oxidized protein showed greater sensitivity to urea (K nd;CNTRL = 3.96 ± 0.19 M, K nd;AAPH = 3.49 ± 0.07 M; P < .05). Similarly, differential scanning fluorimetry indicated greater thermal sensitivity of oxidized immunoglobulin G (T m;CNTRL = 70.5 ± 0.3°C, T m;AAPH = 62.9 ± 0.1°C; P < .001). However, a third method for assessing protein stability, pulse proteolysis, proved to be substantially less sensitive and did not detect significant effects of 2,2-Azobis[2-amidinopropane]dihydrochloride on the half-maximal concentration of urea needed to denature immunoglobulin G (C m;CNTRL= 6.8 ± 0.1 M; C m;AAPH = 6.4 ± 0.7 M). Overall these results demonstrate the merit of using real-time isothermal differential scanning fluorimetry as a rapid and sensitive technique for the evaluation of protein stability in solution using a quantitative real-time thermocycler.

Mitochondrial dysfunction and oxidative stress have been suggested to be possible mechanisms underlying hybrid breakdown, as a result of mito-nuclear incompatibilities in respiratory complexes of the electron transport system. However, it remains unclear whether hybridization increases the production of reactive oxygen species (ROS) by mitochondria. We used high-resolution respirometry and fluorometry on isolated liver mitochondria to examine mitochondrial physiology and ROS emission in naturally occurring hybrids of pumpkinseed (Lepomis gibbosus) and bluegill (L. macrochirus). ROS emission was greater in hybrids than in both parent species when respiration was supported by complex I (but not complex II) substrates, and was associated with increases in lipid peroxidation. However, respiratory capacities for oxidative phosphorylation, phosphorylation efficiency, and O2 kinetics in hybrids were intermediate between those in parental species. Flux control ratios of capacities for electron transport (measured in uncoupled mitochondria) relative to oxidative phosphorylation suggested that the limiting influence of the phosphorylation system is reduced in hybrids. This likely helped offset impairments in electron transport capacity and complex III activity, but contributed to augmenting ROS production. Therefore, hybridization can increase mitochondrial ROS production, in support of previous suggestions that mitochondrial dysfunction can induce oxidative stress and thus contribute to hybrid breakdown. This article is protected by copyright. All rights reserved

Physiological investigations of fish gills have traditionally centred on the two principal functions of the gills: gas exchange and ion regulation. Mitochondrion-rich cells (MRCs) are primarily found within the gill filaments of fish, and are thought to proliferate in order to increase the ionoregulatory capacity of the gill in response to environmentally induced osmotic challenges. However, surprisingly little attention has been paid to the metabolic function of mitochondria within fish gills. Here, we describe and validate a simple protocol for the permeabilization of fish gills and subsequent measurement of mitochondrial respiration rates in vitro. Our protocol requires only small tissue samples (8 mg), exploits the natural structure of fish gills, does not require mechanical separation of the gill tissue (so is relatively quick to perform), and yields accurate and highly reproducible measurements of respiration rates. It offers great potential for the study of mitochondrial function in gills over a wide range of fish sizes and species., Summary: Outline of a simple protocol for permeabilization of fish gills and measurement of mitochondrial physiology; this technique exploits the natural structure of gills and does not require mechanical separation.

The cardiovascular system is critical for delivering O2 to tissues. Here we examine the cardiovascular responses to progressive hypoxia in four high-altitude Andean duck species compared to four related low-altitude populations in North America, tested at their native altitude. Ducks were exposed to stepwise decreases in inspired partial pressure of O2 while we monitored heart rate, O2 consumption rate, blood O2 saturation, haematocrit (Hct), and blood haemoglobin concentration [Hb]. We calculated O2 pulse (the product of stroke volume and the arterial-venous O2 content difference), blood O2 concentration, and heart rate variability. Regardless of altitude, all eight populations maintained O2 consumption rate with minimal change in heart rate or O2 pulse, indicating that O2 consumption was maintained by either a constant arterial-venous O2 content difference (an increase in the relative O2 extracted from arterial blood) or by a combination of changes in stroke volume and the arterial-venous O2 content difference. Three high-altitude taxa (yellow-billed pintails, cinnamon teal, and speckled teal) had higher Hct and [Hb], increasing the O2 content of arterial blood, and potentially providing a greater reserve for enhancing O2 delivery during hypoxia. Hct and [Hb] between low- and high-altitude populations of ruddy duck were similar, representing a potential adaptation to diving life. Heart rate variability was generally lower in high-altitude ducks, concurrent with similar or lower heart rates than low-altitude ducks, suggesting a reduction in vagal and sympathetic tone. These unique features of the Andean ducks differ from previous observations in both Andean geese and bar-headed geese, neither of which exhibit significant elevations in Hct or [Hb] compared to their low-altitude relatives, revealing yet another avian strategy for coping with high altitude.

The effect of protein stability on kinetic function is monitored with many techniques that often require large amounts of expensive substrates and specialized equipment not universally available. We present differential scanning fluorimetry (DSF), a simple high-throughput assay performed in real-time thermocyclers, as a technique for analysis of protein unfolding. Furthermore, we demonstrate a correlation between the half-maximal rate of protein unfolding (Knd), and protein unfolding by urea (I50). This demonstrates that DSF methods can determine the structural stability of an enzyme's active site and can compare the relative structural stability of homologous enzymes with a high degree of sequence similarity.

Aging is typically associated with a decline in whole animal performance that ultimately contributes to death. It is suspected that a decline in ATP production leads to dysfunction in cellular processes, contributing to the decline in performance. Birds require large amounts of ATP to support physiological process, especially flight, which is one of the most energetically expensive forms of locomotion in the animal kingdom to sustain. Since the bulk of ATP production is coordinated through mitochondrial activity, we set out to explore mitochondrial function in young (~8 months) and old (~73 months) zebra finches (Taeniopygia guttata). We exploited the fact that avian red blood cells (RBCs) are nucleated and have functional mitochondria to explore the phenomenon of age-related decline in mitochondrial function without the need for terminal sampling. We found that RBCs from old zebra finches have lower flux control ratios (mitochondrial O2 consumption attributed to ATP production; 0.29–0.36-fold), exhibit higher respiration (1.4-fold), and significantly higher citrate synthase activity (1.4-fold) than young birds. Respiration rates normalized to citrate synthase activity suggest that mitochondrial quality is changing, as leak state is significantly lower (0.39-fold) in old zebra finches in comparison to young animals. Overall, our findings indicate a possible change in the function of mitochondria in older zebra finches, which may be associated with a corresponding increase in mitochondrial quantity, possibly to offset a decline in mitochondrial quality.

We examined the control of breathing and respiratory gas exchange in six species of high-altitude duck that independently colonized the high Andes. We compared ducks from high-altitude populations in Peru (Lake Titicaca at ∼3800 m above sea level; Chancay River at ∼3000–4100 m) with closely related populations or species from low altitude. Hypoxic ventilatory responses were measured shortly after capture at the native altitude. In general, ducks responded to acute hypoxia with robust increases in total ventilation and pulmonary O2 extraction. O2 consumption rates were maintained or increased slightly in acute hypoxia, despite ∼1–2°C reductions in body temperature in most species. Two high-altitude taxa – yellow-billed pintail and torrent duck – exhibited higher total ventilation than their low-altitude counterparts, and yellow-billed pintail exhibited greater increases in pulmonary O2 extraction in severe hypoxia. In contrast, three other high-altitude taxa – Andean ruddy duck, Andean cinnamon teal and speckled teal – had similar or slightly reduced total ventilation and pulmonary O2 extraction compared with low-altitude relatives. Arterial O2 saturation (SaO2) was elevated in yellow-billed pintails at moderate levels of hypoxia, but there were no differences in SaO2 in other high-altitude taxa compared with their close relatives. This finding suggests that improvements in SaO2 in hypoxia can require increases in both breathing and haemoglobin–O2 affinity, because the yellow-billed pintail was the only high-altitude duck with concurrent increases in both traits compared with its low-altitude relative. Overall, our results suggest that distinct physiological strategies for coping with hypoxia can exist across different high-altitude lineages, even among those inhabiting very similar high-altitude habitats.

We examined the control of breathing and respiratory gas exchange in six species of high-altitude duck that independently colonized the high Andes. We compared ducks from high-altitude populations in Peru (Lake Titicaca at ∼3800 m above sea level; Chancay River at ∼3000-4100 m) with closely related populations or species from low altitude. Hypoxic ventilatory responses were measured shortly after capture at the native altitude. In general, ducks responded to acute hypoxia with robust increases in total ventilation and pulmonary O

Mitochondria play a central role in aerobic performance. Studies aimed at elucidating how evolved variation in mitochondrial physiology contributes to adaptive variation in aerobic performance can therefore provide a unique and powerful lens to understanding the evolution of complex physiological traits. Here, we review our ongoing work on the importance of changes in mitochondrial quantity and quality to adaptive variation in aerobic performance in high-altitude deer mice. Whole-organism aerobic capacity in hypoxia (VO2max) increases in response to hypoxia acclimation in this species, but high-altitude populations have evolved consistently greater VO2max than populations from low altitude. The evolved increase in VO2max in highlanders is associated with an evolved increase in the respiratory capacity of the gastrocnemius muscle. This appears to result from highlanders having more mitochondria in this tissue, attributed to a higher proportional abundance of oxidative fibre-types and a greater mitochondrial volume density within oxidative fibres. The latter is primarily caused by an over-abundance of subsarcolemmal mitochondria in high-altitude mice, which is likely advantageous for mitochondrial O2 supply because more mitochondria are situated adjacent to the cell membrane and close to capillaries. Evolved changes in gastrocnemius phenotype appear to be underpinned by population differences in the expression of genes involved in energy metabolism, muscle development, and vascular development. Hypoxia acclimation has relatively little effect on respiratory capacity of the gastrocnemius, but it increases respiratory capacity of the diaphragm. However, the mechanisms responsible for this increase differ between populations: lowlanders appear to adjust mitochondrial quantity and quality (i.e., increases in citrate synthase [CS] activity, and mitochondrial respiration relative to CS activity) and they exhibit higher rates of mitochondrial release of reactive oxygen species (ROS), whereas highlanders only increase mitochondrial quantity in response to hypoxia acclimation. In contrast to the variation in skeletal muscles, the respiratory capacity of cardiac muscle does not appear to be affected by hypoxia acclimation and varies little between populations. Therefore, evolved changes in mitochondrial quantity and quality make important tissue-specific contributions to adaptive variation in aerobic performance in high-altitude deer mice.

The African clawed frog, Xenopus laevis , can withstand extremely arid conditions through aestivation, resulting in dehydration and urea accumulation. Aestivating X. laevis reduce their metabolic rate, and rely on anaerobic glycolysis to meet reduced ATP demands. The present study investigated how severe dehydration affected the transcript levels, kinetic profile, and phosphorylation state of the key glycolytic enzyme pyruvate kinase (PK) in the liver and skeletal muscle of X. laevis . Compared to control frogs, severely dehydrated frogs showed an increase in the transcript abundance of both liver and muscle isoforms of PK. While the kinetics of muscle PK did not differ between dehydrated and control frogs, PK from the liver of dehydrated frogs had a lower K m for phosphoenolpyruvate (PEP) (38%), a lower K a for fructose-1,6-bisphosphate (F1,6P 2 ) (32%), and a greater activation of PK via F1,6P 2 (1.56-fold). PK from dehydrated frogs also had a lower phosphorylation-state (25%) in comparison to the enzyme from control frogs in the liver. Experimental manipulation of the phosphorylation-state of liver PK taken from control frogs by endogenous protein phosphatases resulted in decreased phosphorylation, and a similar kinetic profile as seen in dehydrated frogs. The physiological consequence of dehydration-induced PK modification appears to adjust PK function to remain active during a metabolically depressed state. This study provides evidence for the maintenance of PK activity through elevated mRNA levels and a dephosphorylation event which activates frog liver PK in the dehydrated state in order to facilitate the production of ATP via anaerobic glycolysis.

Mitochondrial dysfunction and oxidative stress have been suggested to be possible mechanisms underlying hybrid breakdown, as a result of mito-nuclear incompatibilities in respiratory complexes of the electron transport system. However, it remains unclear whether hybridization increases the production of reactive oxygen species (ROS) by mitochondria. We used high-resolution respirometry and fluorometry on isolated liver mitochondria to examine mitochondrial physiology and ROS emission in naturally occurring hybrids of pumpkinseed (Lepomis gibbosus) and bluegill (L. macrochirus). ROS emission was greater in hybrids than in both parent species when respiration was supported by complex I (but not complex II) substrates, and was associated with increases in lipid peroxidation. However, respiratory capacities for oxidative phosphorylation, phosphorylation efficiency, and O

Wood frogs inhabit a broad range across North America, extending from the southern tip of the Appalachian Mountains to the northern boreal forest. Remarkably, they can survive the winter in a frozen state, where as much as 70% of their body water is converted into ice. Whilst in the frozen state, their hearts cease to pump blood, causing their cells to experience ischemia, which can dramatically increase the production of reactive oxygen species within the cell. To overcome this, wood frogs have elevated levels of glutathione, a primary antioxidant. We examined the regulation of glutathione reductase, the enzyme involved in recycling glutathione, in both the frozen and unfrozen (control) state. Glutathione reductase activity from both the control and frozen state showed a dramatic reduction in substrate specificity (Km) for oxidized glutathione (50%) when measured in the presence of glucose (300 mmol l-1) and a increase (157%) when measured in the presence of levels of urea (75 mmol l-1) encountered in the frozen state. However, when we tested the synergistic effect of urea and glucose simultaneously, we observed a substantial reduction in the Km for oxidized glutathione (43%) to a value similar to that with glucose alone. In fact, we found no observable differences in the kinetic and structural properties of glutathione reductase between the two states. Therefore, a significant increase in the affinity for oxidized glutathione in the presence of endogenous levels of glucose suggests that increased glutathione recycling may occur as a result of passive regulation of glutathione reductase by rising levels of glucose during freezing.

Birds that fly at high altitudes must support vigorous exercise in oxygen-thin environments. Here we discuss the characteristics that help high-fliers sustain the high rates of metabolism needed for flight at elevation. Many traits in the O2 transport pathway distinguish birds in general from other vertebrates. These include enhanced gas-exchange effectiveness in the lungs, maintenance of O2 delivery and oxygenation in the brain during hypoxia, augmented O2 diffusion capacity in peripheral tissues, and a high aerobic capacity. These traits are not high-altitude adaptations, because they are also characteristic of lowland birds, but are nonetheless important for hypoxia tolerance and exercise capacity. However, unique specializations also appear to have arisen, presumably by high-altitude adaptation, at every step in the O2 pathway of some highland species. The distinctive features of high-fliers include an enhanced hypoxic ventilatory response and/or an effective breathing pattern, large lungs, haemoglobin with a high O2 affinity, further augmentation of O2 diffusion capacity in the periphery, and multiple alterations in the metabolic properties of cardiac and skeletal muscle. These unique specializations improve the uptake, circulation, and efficient utilization of O2 during high-altitude hypoxia. High-altitude birds also have larger wings than their lowland relatives, presumably to reduce the metabolic costs of staying aloft in low-density air. High-fliers are therefore unique in many ways, but the relative roles of adaptation and plasticity (acclimatization) in high-altitude flight are still unclear. Disentangling these roles will be instrumental if we are to understand the physiological basis of altitudinal range limits and how they might shift in response to climate change.

The metabolic cost of breathing at rest has never been successfully measured in birds, but has been hypothesized to be higher than in mammals of a similar size because of the rocking motion of the avian sternum being encumbered by the pectoral flight muscles. To measure the cost and work of breathing, and to investigate whether species resident at high altitude exhibit morphological or mechanical changes that alter the work of breathing, we studied 11 species of waterfowl: five from high altitudes (>3000 m) in Perú, and six from low altitudes in Oregon, USA. Birds were anesthetized and mechanically ventilated in sternal recumbency with known tidal volumes and breathing frequencies. The work done by the ventilator was measured, and these values were applied to the combinations of tidal volumes and breathing frequencies used by the birds to breathe at rest. We found the respiratory system of high-altitude species to be of a similar size, but consistently more compliant than that of low-altitude sister taxa, although this did not translate to a significantly reduced work of breathing. The metabolic cost of breathing was estimated to be between 1 and 3% of basal metabolic rate, as low or lower than estimates for other groups of tetrapods.

Torrent ducks inhabit fast-flowing rivers in the Andes from sea level to altitudes up to 4500 m. We examined the mitochondrial physiology that facilitates performance over this altitudinal cline by comparing the respiratory capacities of permeabilized fibers, the activities of 16 key metabolic enzymes and the myoglobin content in muscles between high- and low-altitude populations of this species. Mitochondrial respiratory capacities (assessed using substrates of mitochondrial complexes I, II and/or IV) were higher in highland ducks in the gastrocnemius muscle – the primary muscle used to support swimming and diving – but were similar between populations in the pectoralis muscle and the left ventricle. The heightened respiratory capacity in the gastrocnemius of highland ducks was associated with elevated activities of cytochrome oxidase, phosphofructokinase, pyruvate kinase and malate dehydrogenase (MDH). Although respiratory capacities were similar between populations in the other muscles, highland ducks had elevated activities of ATP synthase, lactate dehydrogenase, MDH, hydroxyacyl CoA dehydrogenase and creatine kinase in the left ventricle, and elevated MDH activity and myoglobin content in the pectoralis. Thus, although there was a significant increase in the oxidative capacity of the gastrocnemius in highland ducks, which correlates with improved performance at high altitudes, the variation in metabolic enzyme activities in other muscles not correlated to respiratory capacity, such as the consistent upregulation of MDH activity, may serve other functions that contribute to success at high altitudes.

Aim We examined the effects of chronic hypoxia on diaphragm function in high- and low-altitude populations of Peromyscus mice. Methods Deer mice (P. maniculatus) native to high altitude and congeneric mice native to low altitude (P. leucopus) were born and raised in captivity to adulthood and were acclimated to normoxia or hypobaric hypoxia (12 or 9 kPa, simulating hypoxia at 4300 and 7000 m) for 6-8 weeks. We then measured indices of mitochondrial respiration capacity, force production, and fatigue resistance in the diaphragm. Results Mitochondrial respiratory capacities (assessed using permeabilized fibres with single or multiple inputs to the electron transport system), citrate synthase activity (a marker of mitochondrial volume), twitch force production, and muscle fatigue resistance increased after exposure to chronic hypoxia in both populations. These changes were not well explained by variation in the fibre-type composition of the muscle. However, there were several differences in diaphragm function in high-altitude mice compared to low-altitude mice. Exposure to a deeper level of hypoxia (9 kPa vs 12 kPa) was needed to elicit increases in mitochondrial respiration rates in highlanders. Chronic hypoxia did not increase the emission of reactive oxygen species from permeabilized fibres in highlanders, in contrast to the pronounced increases that occurred in lowlanders. In general, the diaphragm of high-altitude mice had greater capillary length densities, produced less force in response to stimulation and had shorter relaxation times. The latter was associated with higher activity of sarcoplasmic reticulum Ca2+ -ATPase (SERCA) activity in the diaphragm of high-altitude mice. Conclusion Overall, our work suggests that exposure to chronic hypoxia increases the capacities for mitochondrial respiration, force production and fatigue resistance of the diaphragm. However, many of these effects are opposed by evolved changes in diaphragm function in high-altitude natives, such that highlanders in chronic hypoxia maintain similar diaphragm function to lowlanders in sea level conditions.

Background The North American wood frog, Rana sylvatica , endures whole body freezing while wintering on land and has developed multiple biochemical adaptations to elude cell/tissue damage and optimize its freeze tolerance. Blood flow is halted in the frozen state, imparting both ischemic and oxidative stress on cells. A potential build-up of H 2 O 2 may occur due to increased superoxide dismutase activity previously discovered. The effect of freezing on catalase (CAT), which catalyzes the breakdown of H 2 O 2 into molecular oxygen and water, was investigated as a result. Methods The present study investigated the purification and kinetic profile of CAT in relation to the phosphorylation state of CAT from the skeletal muscle of control and frozen R. sylvatica . Results Catalase from skeletal muscle of frozen wood frogs showed a significantly higher V max (1.48 fold) and significantly lower K m for H 2 O 2 (0.64 fold) in comparison to CAT from control frogs (5 °C acclimated). CAT from frozen frogs also showed higher overall phosphorylation (1.73 fold) and significantly higher levels of phosphoserine (1.60 fold) and phosphotyrosine (1.27 fold) compared to control animals. Phosphorylation via protein kinase A or the AMP-activated protein kinase significantly decreased the K m for H 2 O 2 of CAT, whereas protein phosphatase 2B or 2C action significantly increased the K m . Conclusion The physiological consequence of freeze-induced CAT phosphorylation appears to improve CAT function to alleviate H 2 O 2 build-up in freezing frogs. General significance Augmented CAT activity via reversible phosphorylation may increase the ability of R. sylvatica to overcome oxidative stress associated with ischemia.

Background The North American wood frog, Rana sylvatica, is able to overcome subzero conditions through overwintering in a frozen state. Freezing imposes ischemic and oxidative stress on cells as a result of cessation of blood flow. Superoxide dismutases (SODs) catalyze the redox reaction involving the dismutation of superoxide (O2− ) to molecular oxygen and hydrogen peroxide. Methods The present study investigated the regulation of CuZnSOD and MnSOD kinetics as well as the transcript, protein and phosphorylation levels of purified enzyme from the muscle of control and frozen R. sylvatica. Results CuZnSOD from frozen muscle showed a significantly higher Vmax (1.52 fold) in comparison to CuZnSOD from the muscle of control frogs. MnSOD from frozen muscle showed a significantly lower Km for O2− (0.66 fold) in comparison to CuZnSOD from control frogs. MnSOD from frozen frogs showed higher phosphorylation of serine (2.36 fold) and tyrosine (1.27 fold) residues in comparison to MnSOD from control animals. Susceptibility to digestion via thermolysin after incubation with increasing amount of urea (Cm) was tested, resulting in no significant changes for CuZnSOD, whereas a significant change in MnSOD stability was observed between control (2.53 M urea) and frozen (2.92 M urea) frogs. Expressions of CuZnSOD and MnSOD were quantified at both mRNA and protein levels in frog muscle, but were not significantly different. Conclusion The physiological consequence of freeze-induced SOD modification appears to adjust SOD function in freezing frogs. General significance Augmented SOD activity may increase the ability of R. sylvatica to overcome oxidative stress associated with ischemia.

The African clawed frog, Xenopus laevis endures whole body dehydration which can increase its reliance on anaerobic glycolysis for energy production. This makes the regulation of the terminal enzyme of glycolysis, lactate dehydrogenase (LDH), crucial to stress survival. We investigated the enzymatic properties and posttranslational modification state of purified LDH from the skeletal muscle of control and dehydrated (30% total body water loss) X. laevis. LDH from the muscle of dehydrated frogs showed a 93% reduction in phosphorylation on threonine residues and an 80% reduction of protein nitrosylation. LDH from dehydrated muscle also showed a 74% lower Vmax in the pyruvate oxidizing direction and a 78% decrease in Vmax in the lactate reducing direction along with a 33% lower Km for pyruvate and a 40% higher Km for lactate. In the presence of higher levels of urea and molecular crowding by polyethylene glycol, used to mimic conditions in the cells of dehydrated animals, the Km values of control and dehydrated LDH demonstrated opposite responses. In the pyruvate oxidizing direction, control muscle LDH was unaffected by these additives, whereas the affinity for pyruvate dropped further for LDH from dehydrated muscle. The opposite effect was more pronounced in the lactate reducing direction as control LDH showed an increased affinity for lactate, whereas LDH from dehydrated animals showed a further reduction in affinity. The physiological consequences of dehydration-induced LDH regulation appear to poise the enzyme towards lactate production when urea levels are high and lactate catabolism when urea levels are low, perhaps helping to maintain glycolysis under dehydrating conditions whilst providing for the ability to recycle lactate upon rehydration.

One of the most adaptive facultative anaerobes among vertebrates is the freshwater turtle, Trachemys scripta elegans. Upon a decrease in oxygen supply and oxidative phosphorylation, these turtles are able to reduce their metabolic rate and recruit anaerobic glycolysis to meet newly established ATP demands. Within the glycolytic pathway, aldolase enzymes cleave fructose-1,6-bisphosphate to triose phosphates facilitating an increase in anaerobic production of ATP. Importantly, this enzyme exists primarily as tissue-specific homotetramers of aldolase A, B or C located in skeletal muscle, liver and brain tissue, respectively. The present study characterizes aldolase activity and structure in the liver tissue of a turtle whose survival greatly depends on increased glycolytic output during anoxia. Immunoblot and mass spectrometry analysis verified the presence of both aldolase A and B in turtle liver tissue, and results from co-immunoprecipitation experiments suggested that in the turtle aldolase proteins may exist as an uncommon heterotetramer. Expression levels of aldolase A protein increased significantly in liver tissue to 1.59±0.11-fold after 20 h anoxia, when compared to normoxic control values (P

Lactate dehydrogenase (LDH; E.C. 1.1.1.27) is a crucial enzyme involved in energy metabolism in muscle, facilitating the production of ATP via glycolysis during oxygen deprivation by recycling NAD+. The present study investigated purified LDH from the muscle of 20 h anoxic and normoxicT. s. elegans, and LDH from anoxic muscle showed a significantly lower (47%)Kmfor L-lactate and a higherVmaxvalue than the normoxic form. Several lines of evidence indicated that LDH was converted to a low phosphate form under anoxia: (a) stimulation of endogenously present protein phosphatases decreased theKmof L-lactate of control LDH to anoxic levels, whereas (b) stimulation of kinases increased theKmof L-lactate of anoxic LDH to normoxic levels, and (c) dot blot analysis shows significantly less serine (78%) and threonine (58%) phosphorylation in anoxic muscle LDH as compared to normoxic LDH. The physiological consequence of anoxia-induced LDH dephosphorylation appears to be an increase in LDH activity to promote the reduction of pyruvate in muscle tissue, converting the glycolytic end product to lactate to maintain a prolonged glycolytic flux under energy-stressed anoxic conditions.

The intertidal marine snail,Littorina littorea, has evolved to withstand extended bouts of oxygen deprivation brought about by changing tides or other potentially harmful environmental conditions. Survival is dependent on a strong suppression of its metabolic rate and a drastic reorganization of its cellular biochemistry in order to maintain energy balance under fixed fuel reserves. Lactate dehydrogenase (LDH) is a crucial enzyme of anaerobic metabolism as it is typically responsible for the regeneration of NAD+, which allows for the continued functioning of glycolysis in the absence of oxygen. This study compared the kinetic and structural characteristics of the D-lactate specific LDH (E.C. 1.1.1.28) from foot muscle of aerobic control versus 24 h anoxia-exposedL. littorea. Anoxic LDH displayed a near 50% decrease inVmax(pyruvate-reducing direction) as compared to control LDH. These kinetic differences suggest that there may be a stable modification and regulation of LDH during anoxia, and indeed, subsequent dot-blot analyses identified anoxic LDH as being significantly less acetylated than the corresponding control enzyme. Therefore, acetylation may be the regulatory mechanism that is responsible for the suppression of LDH activity during anoxia, which could allow for the production of alternative glycolytic end products that in turn would increase the ATP yield under fixed fuel reserves.

Land snails,Otala lactea, survive in seasonally hot and dry environments by entering a state of aerobic torpor called estivation. During estivation, snails must prevent excessive dehydration and reorganize metabolic fuel use so as to endure prolonged periods without food. Glutamate dehydrogenase (GDH) was hypothesized to play a key role during estivation as it shuttles amino acid carbon skeletons into the Krebs cycle for energy production and is very important to urea biosynthesis (a key molecule used for water retention). Analysis of purified foot muscle GDH from control and estivating conditions revealed that estivated GDH was approximately 3-fold more active in catalyzing glutamate deamination as compared to control. This kinetic difference appears to be regulated by reversible protein phosphorylation, as indicated by ProQ Diamond phosphoprotein staining and incubations that stimulate endogenous protein kinases and phosphatases. The increased activity of the high-phosphate form of GDH seen in the estivating land snail foot muscle correlates well with the increased use of amino acids for energy and increased synthesis of urea for water retention during prolonged estivation.

Glutamate dehydrogenase (GDH) (EC 1.4.1.3) is a crucial enzyme involved in bridging two metabolic pathways, gating the use of glutamate for either amino acid metabolism, or carbohydrate metabolism. The present study investigated GDH from tail muscle of the freshwater crayfish Orconectes virilis exploring changes to kinetic properties, phosphorylation levels and structural stability between two forms of the enzyme (aerobic control and 20-h severe hypoxic). Evidence indicated that GDH was converted to a high phosphate form under oxygen limitation. ProQ Diamond phosphoprotein staining showed a 42% higher bound phosphate content on GDH from muscle of severely hypoxic crayfish compared with the aerobic form, and treatment of this GDH with commercial phosphatase (alkaline phosphatase), and treatments that stimulated the activities of different endogenous protein phosphatases (stimulating PP1 + PP2A, PP2B, and PP2C) yielded significant increases in the fold activation by ADP of GDH from both control and severe hypoxic conditions. By contrast, stimulation of the activities of endogenous protein kinases (AMPK, PKA or CaMK) significantly reduced the ADP fold activation from control animals. The physiological consequence of severe hypoxia-induced GDH phosphorylation may be to suppress GDH activity under low oxygen, shutting off this critical bridge point between two metabolic pathways.

Freshwater crayfish, Orconectes virilis, can experience periodic exposures to hypoxia or anoxia due to low water flow (in summer) or ice cover (in winter) in their natural habitat. Hypoxia/anoxia disrupts energy metabolism and triggers mechanisms that to support ATP levels while often also suppressing ATP use. Arginine kinase (AK) (E.C. 2.7.3.3) is a crucial enzyme involved in energy metabolism in muscle, gating the use of phosphagen stores to buffer ATP levels. The present study investigated AK from tail muscle of O. virilis identifying changes to kinetic properties, phosphorylation state and structural stability between the enzyme from aerobic control and 20 h anoxic crayfish. Muscle AK from anoxia-exposed crayfish showed a significantly higher (by 59%) K m for l-arginine and a lower I50 value for urea than the aerobic form. Several lines of evidence indicated that AK was converted to a high phosphate form under anoxia: (a) aerobic and anoxic forms of AK showed well-separated elution peaks on DEAE ion exchange chromatography, (b) ProQ Diamond phosphoprotein staining showed a 64% higher bound phosphate content on anoxic AK compared with the aerobic form, and (c) treatment of anoxic AK with alkaline phosphatase reduced K m l-arginine to aerobic levels whereas incubation of aerobic AK with protein kinase A catalytic subunit raised the K m to anoxic levels. The physiological consequence of anoxia-induced AK phosphorylation may be to suppress AK activity in the phosphagen-synthesizing direction and, together with reduced cellular pH and ATP levels, promote the phosphagen-catabolizing direction under anoxic conditions. This is first time that AK has been shown to be regulated by reversible phosphorylation.

The stability of a protein can be monitored by many different techniques. However, these protocols are often lengthy, consume large amounts of protein, and require expensive and specialized instruments. In this study, we present a new protocol to analyze the kinetics of protein unfolding utilizing a quantitative real-time thermocycler. By using modified qRT-PCR equipment, it is possible to analyze the effects of a wide range of denaturants (and their interactions with temperature change) on protein stability on a microplate platform where multiple samples can be run in parallel under virtually identical conditions and with highly sensitive detection. Using this set-up, researchers can determine the concentration of denaturant that results in a half-maximal rate of protein denaturation (KND), the maximum rate of denaturation (Dmax), and the cooperativity of individual denaturants in protein unfolding (I1/4-coefficient). In the present study we use hexokinase as a model protein and urea as a model denaturant to illustrate this new met hod and the kinetics of protein unfolding that it can supply. This method allows the researcher to explore a large number of denaturants, at either constant or variable temperatures, within the same assay. Overall, this method provides an estimate of denaturation kinetics that was previously inaccessible.