Comparative iTRAQ‐Based Quantitative Proteomic Analysis of Pelteobagrus vachelli Liver under Acute Hypoxia: Implications in Metabolic Responses

More and more frequently these days, aquatic ecosystems are being stressed by nutrient enrichment, pollutants, and global warming, leading to a serious depletion in oxygen concentrations. Although a sudden, significant lack of oxygen will result in mortality, fishes can have an acute behavior (e.g., an increase in breathing rate, reduction in swimming frequency) and physiology responses (e.g., increase in oxygen delivery, and reduction in oxygen consumption) to hypoxia, which allows them to maintain normal physical activity. Therefore, in order to shed further light on the molecular mechanisms of hypoxia adaptation in fishes, the authors conduct comparative quantitative proteomics on Pelteobagrus vachelli livers using iTRAQ. The research identifies 511 acute hypoxia‐responsive proteins in P. vachelli. Furthermore, comparison of several of the diverse key pathways studied (e.g., peroxisome pathway, PPAR signaling pathway, lipid metabolism, glycolysis/gluco‐neogenesis, and amino acid metabolism) help to articulate the different mechanisms involved in the hypoxia response of P. vachelli. Data from proteome analysis shows that P. vachelli can have an acute reaction to hypoxia, including detoxification of metabolic by‐products and oxidative stress in light of continued metabolic activity (e.g., peroxisomes), an activation in the capacity of catabolism to get more energy (e.g., lipolysis and amino acid catabolism), a depression in the capacity of biosynthesis to reduce energy consumption (e.g., biosynthesis of amino acids and lipids), and a shift in the aerobic and anaerobic contributions to total metabolism. The observed hypoxia‐related changes in the liver proteome of the fish can help to understand or can be related to the hypoxia‐related response that takes place in similar conditions in the liver or other proteomes of mammals.     

Perinatal asphyxia pathophysiology in pig and human: a review

In utero fetuses are evidently exposed to several factors that cause an interruption of the oxygen flow through the umbilical cord causing asphyxia leading to hypoxia and metabolic acidosis. These conditions are important causes of intra-partum and neonatal mortality. The main objective of this review is to provide current information regarding the pathophysiology of asphyxia in piglets around parturition; the physiological mechanisms invoked by affected piglets to compensate perinatal hypoxemia are discussed. This review also addresses some similarities and differences of asphyxia between piglets and other mammals, including human neonates. Metabolic acidosis and hypoxia are sequela to asphyxia and can cause profound health effects in postnatal performance because of an abnormal suckling, a reduced absorption of colostrum and inadequate passive transfer of neonatal immunity. Acidosis also cause hypothermia, increased mortality and reduced survival in neonates. One of the first deleterious effects of intrauterine hypoxia is the expulsion of meconium into the amniotic sac leading to meconium staining of the skin, and in severe cases, meconium aspiration into the lungs. Even though there have been technological changes and improvements in husbandry, piglet mortality due to asphyxia remains a major problem. One potential alternative to reduce neonatal mortality in pigs is the monitoring of fetal stress during birth and the implemention of strategies such as the Apgar score, that is often used in human pediatrics. It is also important to consider the physiological, behavioral and biochemical changes that take place during parturition which subsequently impact the vitality, maturity and development of neonatal pigs. Understanding the pathophysiology of fetal hypoxia should help practitioners and farmers implement more effective delivery techniques aimed at reducing neonatal mortality and improving postnatal performance.     

Effect of transient hypoxia on oxygenation of the developing rat brain: relationships among haemoglobin saturation, autoregulation of blood flow and mitochondrial redox state

Multi-wavelength, differential spectroscopy was used to examine the effects of transient hypoxia on oxygen delivery and intracellular utilization in the brain of developing rats. The in vivo redox status of cytochrome a,a3 was compared simultaneously with changes in relative haemoglobin saturation and blood volume in the cerebral cortex during lowered FiO2. During hypoxia, neonates maintained their intracellular cytochrome a,a3 redox state as well as did adults, but did so through unusual characteristics, including: (1) maintenance of haemoglobin oxygenation at lower FiO2; (2) regulation of cerebral blood volume at blood pressures below the point at which autoregulation would fail in the adult; and (3) the capacity to tolerate a greater reduction of cytochrome a,a3 relative to haemoglobin desaturation at lowered FiO2. These data suggest that mechanisms which protect the neonate from hypoxic insult involve preservation of oxygen delivery, increased respiratory compensation for metabolic acidosis, and maintenance of cellular energy requirements predominantly through anaerobic metabolism.     

Change in plasma amino acid concentrations during breath-hold diving at high altitude

We studied the plasma concentration of various amino acids in 6 Italian sport divers in Italy and at approximately 4,500 m altitude in Peru; 6 Peruvian inhabitants were examined for comparison. We attempted to create a situation of pronounced hypoxia in muscles by breath-hold diving at altitude. The diving reflex diverts blood away from muscles while diving increases central oxygen tension and prevents loss of consciousness. Differences in certain amino acids, probably related to diet, were noted between Italy and Peru. Increases in concentration of plasma alanine and some branched-chain amino acids occurred after breath-hold diving. These changes were similar to those seen after prolonged hard exercise, even though physical work was low. Hypoxia in muscles, common during hard work and during breath-hold diving at altitude, might thus be the stimulus for amino acid release from working muscles.     

Comparison between the antioxidant status of terrestrial and diving mammals

Many diving mammals are known for their ability to deal with nitrogen supersaturation and to tolerate apnea for extended periods. They are all characterized by high oxygen-carrying capacity in blood together with high oxygen storage in their muscle mass due to large myoglobin concentrations. The above properties theoretically also imply a high tissue antioxidant defenses (AD) to counteract reactive oxygen species (ROS) generation associated with the rapid transition from apnea to reoxygenation. Different enzymatic (superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, and glutathione S-transferase), and non-enzymatic (levels of glutathione) AD as well as cellular damage (thiobarbituric acid-reactive substances contents, as a measure of lipoperoxidation) were measured in blood samples obtained from anesthetized animals, and also in blood obtained from recently dead diving mammals, and compared to some terrestrial mammals (n=5 in both groups). The results confirmed that diving mammals have, in general, higher antioxidant status compared to non-diving mammals. Apparently, to avoid exposure of tissues to changing high oxygen levels, and therefore to avoid an oxidative stress condition related to antioxidant consumption and increased ROS generation, diving mammals possess constitutive high levels of antioxidants in tissues. These data are in agreement with short-term AD adaptations related to torpor and to animals that experience large daily changes in oxygen consumption. These data are similar to the long-term adaptations of animals that undergo hibernation, estivation, freezing-thawing and dehydration-rehydration processes. In summary, animals that routinely face high changes in oxygen availability and/or consumption seem to show a general strategy to prevent oxidative damage by having either appropriate high constitutive AD and/or the ability to undergo arrested states, where depressed metabolic rates minimize the oxidative challenge.     

Hypoxic Preconditioning and Hypoxic-Ischemic Brain Damage in the Immature Rat: Pathologic and Metabolic Correlates

Abstract: It has been reported that immature rats subjected to cerebral hypoxia-ischemia sustain less brain damage if they are previously exposed to systemic hypoxia compared with animals not exposed to prior hypoxia. Accordingly, neuropathologic and metabolic experiments were conducted to confirm and extend the observation that hypoxic preconditioning protects the perinatal brain from subsequent hypoxic-ischemic brain damage. Six-day postnatal rats were subjected to systemic hypoxia with 8% oxygen at 37°C for 2.5 h. Twenty-four hours later, they were exposed to unilateral cerebral hypoxia-ischemia for 2.5 h, produced by unilateral common carotid artery ligation and systemic hypoxia with 8% oxygen. Neuropathologic analysis, conducted at 30 days of postnatal age, indicated a substantial reduction in the severity of brain damage in the preconditioned rats, such that only 6 of 14 such animals exhibited cystic infarction, but all 13 animals without prior preconditioning exhibited infarction ( p < 0.001). Measurement of cerebral glycolytic and tricarboxylic acid intermediates and high-energy phosphate reserves at the terminus of and at 4 and 24 h following hypoxia-ischemia showed no differences in the extent of alterations in the preconditioned and nonpreconditioned immature rats. A difference was seen in the restitution of high-energy stores during the first 24 h of recovery from hypoxia-ischemia, with a more optimal preservation of these metabolites in the preconditioned animals, reflecting the less severe ultimate brain damage. Accordingly, the neuroprotection afforded to the preconditioned animals was not the result of any differences in the extent of anaerobic glycolysis, tissue acidosis, or depletion in high-energy reserves during hypoxia-ischemia but rather the result of other mechanisms that improved the metabolic status of the immature brain during the early hours of reperfusion following hypoxia-ischemia.     

The diving paradox: new insights into the role of the dive response in air-breathing vertebrates

When aquatic reptiles, birds and mammals submerge, they typically exhibit a dive response in which breathing ceases, heart rate slows, and blood flow to peripheral tissues is reduced. The profound dive response that occurs during forced submergence sequesters blood oxygen for the brain and heart while allowing peripheral tissues to become anaerobic, thus protecting the animal from immediate asphyxiation. However, the decrease in peripheral blood flow is in direct conflict with the exercise response necessary for supporting muscle metabolism during submerged swimming. In free diving animals, a dive response still occurs, but it is less intense than during forced submergence, and whole-body metabolism remains aerobic. If blood oxygen is not sequestered for brain and heart metabolism during normal diving, then what is the purpose of the dive response? Here, we show that its primary role may be to regulate the degree of hypoxia in skeletal muscle so that blood and muscle oxygen stores can be efficiently used. Paradoxically, the muscles of diving vertebrates must become hypoxic to maximize aerobic dive duration. At the same time, morphological and enzymatic adaptations enhance intracellular oxygen diffusion at low partial pressures of oxygen. Optimizing the use of blood and muscle oxygen stores allows aquatic, air-breathing vertebrates to exercise for prolonged periods while holding their breath.     

MYSTERIES OF ADAPTATION TO HYPOXIA AND PRESSURE IN MARINE MAMMALSThe Kenneth S. Norris Lifetime Achievement Award Lecture

This paper reviews past and current work on diving behavior, effects of pressure, and the aerobic diving limit from the perspective of the Ken Norris Lifetime Achievement Award. Because of the influence of Norris to marine mammalogy in general, and to my career in particular, I want to emphasize the important tradition of mentors and colleagues as keystones to a successful career in science, and ultimately to the success of science itself. These two related activities are illustrated by studies on marine mammals that were conducted in an endeavor to understand: (1) the behavioral traits associated with deep diving, (2) the mechanical and physiological effects of pressure during routine dives to great depth, and (3) the degree of oxygen depletion that they routinely endure while diving. The search for answers has resulted in numerous physiological and ecological experiments, along with accompanying theoretical analyses. Currently it appears that some deep-diving mammals may suffer from bends, and some may resort more often than what seems physiologically possible to anaerobic metabolism while diving. Above all, the way divers manage their nitrogen and oxygen stores remains a mystery.     

Does size matter for hypoxia tolerance in fish?

Fish cover a large size range, from milligrams to tonnes, and many of them are regularly exposed to large variations in ambient oxygen levels. For more than half a century, there have been various, often divergent, claims regarding the effect of body size on hypoxia tolerance in fish. Here, we attempt to link old and new empirical data with the current understanding of the physiological mechanisms behind hypoxia tolerance. Three main conclusions are drawn: (1) body size per se has little or no impact on the ability to take up oxygen during hypoxic conditions, primarily because the respiratory surface area matches metabolic rate over a wide size range. If size-related differences are seen in the ability for oxygen uptake in a species, these are likely to reflect adaptation to different life-styles or habitat choice. (2) During severe hypoxia and anoxia, where fish have to rely on anaerobic ATP production (glycolysis) for survival, large individuals have a clear advantage over smaller ones, because small fish will run out of glycogen or reach lethal levels of anaerobic end-products (lactate and H(+)) much faster due to their higher mass-specific metabolic rate. (3) Those fish species that have evolved extreme adaptations to hypoxia, including haemoglobins with exceptionally high oxygen affinities and an alternative anaerobic end-product (ethanol), reveal that natural selection can be a much more powerful determinant of hypoxia tolerance than scaling of physiological functions.     

Balancing conflicting metabolic demands of exercise and diving

During enforced diving, aquatic animals activate a set of physiological reflexes (apnea, bradycardia, peripheral vasoconstriction), which are termed the diving response and are in effect the first line of defense against hypoxia. At least in the Weddell seal, this strategy is now known also to be used in voluntary diving at sea, but the response is necessarily modified to accommodate potentially conflicting demands of diving and swimming exercise. The main modification appears to involve skeletal muscles used in swimming, which, because of their high energy requirements, must be powered by aerobic metabolism. Thus they must remain perfused at rates porportional to swimming velocity (which is why heart rates are adjusted to swimming velocity). The required regulation of O2 delivery is achieved at least in part by a well-paced release of oxygenated red blood cells, stored at the beginning of the dive apparently in the spleen. The main metabolic difference between laboratory and voluntary diving is that, in the latter, working muscles serve as a sink for lactate and thus the entry rates of lactate into the plasma can be balanced by exit rates from the plasma; the maintenance of this balance means that no excess lactate remains for a lactate washout in postdiving exercise except under long, exploratory diving. Even in the latter long dives, however, the amount of lactate formed is far less than would be expected if the energetic shortfall caused by hypoperfusion and O2 lack were made up by anaerobic glycolysis (Pasteur effect). Consequently, during diving, hypoperfused tissues necessarily sustain a metabolic arrest of variable degrees as a mechanism of defense against hypoxia.     

BUFFERING CAPACITY OF THE LOCOMOTOR MUSCLE IN CETACEANS: CORRELATES WITH POSTPARTUM DEVELOPMENT, DIVE DURATION, AND SWIM PERFORMANCE

Skeletal muscles of marine mammals must support the metabolic demands of exercise during periods of reduced blood flow associated with the dive response. Enhanced muscle buffering could support anaerobic metabolic processes during apnea, yet this has not been fully investigated in cetaceans. To assess the importance of this adaptation in the diving and swimming performance of cetaceans, muscle buffering capacity due to non-bicarbonate buffers was measured in the longissimus dorsi of ten species of odontocete and one mysticete. Immature specimens from a subset of these species were studied to assess developmental trends. Fetal and neonatal cetaceans have low buffering capacities (range: 34.8–53.9 slykes) that are within the range measured for terrestrial mammals. A lengthy developmental period, independent of muscle myoglobin postnatal development, is required before adult levels are attained. Adult cetacean buffering capacities (range: 63.7–94.5 slykes) are among the highest values recorded for mammals. Cetacean species that demonstrate extremely long dive durations or high burst speed swimming tend to have greater buffering capacities. However, the wide range of body size across cetaceans may complicate these trends. Enhanced muscle buffering capacity may enable small-bodied species to extend breath-hold beyond short aerobic dive limits for foraging or predator evasion when necessary.     

Shift of anaerobic to aerobic metabolism in the rats acclimatized to hypoxia

1. Metabolic acclimatization by repeated exposure to a simulated altitude of 4000, 5000 and 6000 m for 2 hr per day throughout 2 to 11 days was evaluated by the increased formation of ketone bodies as a marker of fatty acid oxidation and the decreased production of lactate and uric acid, the indicators of anaerobic metabolism in rats exposed to an altitude of 8000 m. 2. Pre-exposure of rats to an altitude of 5000 m and over caused an acclimatization to hypoxia. The rise of the altitude to which rats were pre-exposed reduced the period until the acquisition of metabolic acclimatization. 3. Acclimatized rats showed an increased activity of mitochondrial glutamate dehydrogenase without changes in glycolytic enzyme activity in skeletal muscle, heart and liver. 4. Acclimatization to high altitude hypoxia is concluded to involve a shift of the anaerobic glycolysis to aerobic metabolism by the increase in the oxidative enzymes.     

Brünnich's guillemots (Uria lomvia) maintain high temperature in the body core during dives

A major challenge for diving birds, reptiles, and mammals is regulating body temperature while conserving oxygen through a reduction in metabolic processes. To gain insight into how these needs are met, we measured dive depth and body temperatures at the core or periphery between the skin and abdominal muscles simultaneously in freely diving Brünnich's guillemots (Uria lomvia), an arctic seabird, using an implantable data logger (16-mm diameter, 50-mm length, 14-g mass, Little Leonardo Ltd., Tokyo). Guillemots exhibited increased body core temperatures, but decreased peripheral temperatures, during diving. Heat conservation within the body core appeared to result from the combined effect of peripheral vasoconstriction and a high wing beat frequency that generates heat. Conversely, the observed tissue hypothermia in the periphery should reduce metabolic processes as well as heat loss to the water. These physiological effects are likely one of the key physiological adaptations that makes guillemots to perform as an efficient predator in arctic waters.     

Perinatal adaptation in mammals: the impact of metabolic rate.

Mammalian birth is accompanied by profound changes in metabolic rate that can be described in terms of body size relationship (Kleiber's rule). Whereas the fetus, probably as an adaptation to the low intrauterine pO2, exhibits an "inappropriately" low, adult-like specific metabolic rate, the term neonate undergoes a rapid metabolic increase up to the level to be expected from body size. A similar, albeit slowed, "switching-on" of metabolic size allometry is found in human preterm neonates whereas animals that are normally born in a very immature state are able to retard or even suppress the postnatal metabolic increase in favor of weight gain and O2 supply. Moreover, small immature mammalian neonates exhibit a temporary oxyconforming behavior which enhances their hypoxia tolerance, yet is lost to the extent by which the size-adjusted metabolic rate is "locked" by increasing mitochondrial density. Beyond the perinatal period, there are no other deviations from metabolic size allometry among mammals except in hibernation where the temporary "switching-off" of Kleiber's rule is accompanied by a deep reduction in tissue pO2. This gives support to the hypothesis that the postnatal metabolic increase represents an "escape from oxygen" similar to the evolutionary roots of mitochondrial respiration, and that the overall increase in specific metabolic rate with decreasing size might contribute to prevent tissues from O2 toxicity.     

Teaching the physiology of adaptation to hypoxic stress with the aid of a classic paper on high altitude by Houston and Riley

Many pathological conditions exist where tissues exhibit hypoxia or low oxygen tension. Hypoxic hypoxia arises when there is a reduction in the amount of oxygen entering the blood and occurs in healthy people at high altitude. In 1946, research sponsored by the United States Navy led to the collection and subsequent publication of masses of data demonstrating the physiological consequences and adaptations of ascent to high altitude. This article describes how a figure from a 1947 paper from the American Physiological Society Legacy collection (Houston CS, Riley RL. Respiratory and circulatory changes during acclimatization to high altitude. Am J Physiol 149: 565-588) may be used to allow students to review their understanding of some of the generalized effects of hypoxia on the body. In particular, this figure summarizes some of the adaptive responses that take place in the oxygen transport system as a consequence of prolonged hypoxia.     

Metabolic rate does not scale with body mass in cultured mammalian cells

The allometric scaling of metabolic rate with organism body mass can be partially accounted for by differences in cellular metabolic rates. For example, hepatocytes isolated from horses consume almost 10-fold less oxygen per unit time as mouse hepatocytes [Porter and Brand, Am J Physiol Regul Integr Comp Physiol 269: R226-R228, 1995]. This could reflect a genetically programmed, species-specific, intrinsic metabolic rate set point, or simply the adaptation of individual cells to their particular in situ environment (i.e., within the organism). We studied cultured cell lines derived from 10 mammalian species with donor body masses ranging from 5 to 600,000 g to determine whether cells propagated in an identical environment (media) exhibited metabolic rate scaling. Neither metabolic rate nor the maximal activities of key enzymes of oxidative or anaerobic metabolism scaled significantly with donor body mass in cultured cells, indicating the absence of intrinsic, species-specific, cellular metabolic rate set points. Furthermore, we suggest that changes in the metabolic rates of isolated cells probably occur within 24 h and involve a reduction of cellular metabolism toward values observed in lower metabolic rate organisms. The rate of oxygen delivery has been proposed to limit cellular metabolic rates in larger organisms. To examine the effect of oxygen on steady-state cellular respiration rates, we grew cells under a variety of physiologically relevant oxygen regimens. Long-term exposure to higher medium oxygen levels increased respiration rates of all cells, consistent with the hypothesis that higher rates of oxygen delivery in smaller mammals might increase cellular metabolic rates.     

Deer mouse aerobic performance across altitudes: effects of developmental history and temperature acclimation

Aerobic physiology at high altitudes has been studied in many animals. Prior work on laboratory-bred deer mice (a species with a wide altitudinal range) showed depression of aerobic capacity at high altitude, even after acclimation. However, wild deer mice show no reduction in thermogenic performance at high altitude, and performance limits seem to be due to physiological and anatomical adjustments to environmental temperature and not to oxygen availability. We asked whether across-altitude performance differences exist in deer mice after accounting for temperature acclimation (approximately 5 degrees and 20 degrees -25 degrees C) and prenatal and neonatal development altitude (340 vs. 3,800 m). We measured maximal thermogenic oxygen consumption (VO2sum) in cold exposure and ran mice on a treadmill to elicit maximal exercise oxygen consumption (VO2max). We found a 10% reduction in VO2max at 3,800 m compared with that at 340 m; thus, the mice were able to compensate for most of the 37% reduction in oxygen availability at the higher altitude. Development altitude did not affect VO2max. There was no effect of test altitude or development altitude on VO2sum in warm-acclimated animals, but both test and development altitude strongly affected VO2sum in cold-acclimated mice, and compensation for hypoxia at 3,800 m was considerably less than that for exercise.