Article

Hormetic response triggers multifaceted anti-oxidant strategies in immature king penguins (Aptenodytes patagonicus)

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  • Université Claude Bernard Lyon1 - CNRS -
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Abstract

Repeated deep dives are highly pro-oxidative events for air-breathing aquatic foragers such as penguins. At fledging, the transition from a strictly terrestrial to a marine lifestyle may therefore trigger a complex set of anti-oxidant responses to prevent chronic oxidative stress in immature penguins but these processes are still undefined. By combining in vivo and in vitro approaches with transcriptome analysis, we investigated the adaptive responses of sea-acclimatized (SA) immature king penguins (Aptenodytes patagonicus) compared with pre-fledging never-immersed (NI) birds. In vivo, experimental immersion into cold water stimulated a higher thermogenic response in SA penguins than in NI birds, but both groups exhibited hypothermia, a condition favouring oxidative stress. In vitro, the pectoralis muscles of SA birds displayed increased oxidative capacity and mitochondrial protein abundance but unchanged reactive oxygen species (ROS) generation per g tissue because ROS production per mitochondria was reduced. The genes encoding oxidant-generating proteins were down-regulated in SA birds while mRNA abundance and activity of the main antioxidant enzymes were up-regulated. Genes encoding proteins involved in repair mechanisms of oxidized DNA or proteins and in degradation processes were also up-regulated in SA birds. Sea life also increased the degree of fatty acid unsaturation in muscle mitochondrial membranes resulting in higher intrinsic susceptibility to ROS. Oxidative damages to protein or DNA were reduced in SA birds. Repeated experimental immersions of NI penguins in cold-water partially mimicked the effects of acclimatization to marine life, modified the expression of fewer genes related to oxidative stress but in a similar way as in SA birds and increased oxidative damages to DNA. It is concluded that the multifaceted plasticity observed after marine life may be crucial to maintain redox homeostasis in active tissues subjected to high pro-oxidative pressure in diving birds. Initial immersions in cold-water may initiate an hormetic response triggering essential changes in the adaptive antioxidant response to marine life.

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... Once at sea, penguins' daily metabolic cost of thermoregulation is reduced by their ability to develop powerful peripheral vasoconstriction, regional hypothermia and a thick subcutaneous fat layer (Dumonteil et al., 1994;Handrich et al., 1997;Enstipp et al., 2017;Lewden et al., 2017a). Sea acclimatization is also characterized by the fuel selection of skeletal muscle towards lipid transport and oxidation, concomitant with an increase in the content and oxidative capacity of mitochondria in skeletal muscle (Teulier et al., 2012;Rey et al., 2016). These biochemical remodeling processes are associated with an increased thermogenic effect of circulating lipids on whole-body metabolism in vivo and in mitochondrial skeletal muscle in vitro (Talbot et al., 2004;Teulier et al., 2012;Rey et al., 2017). ...
... These biochemical remodeling processes are associated with an increased thermogenic effect of circulating lipids on whole-body metabolism in vivo and in mitochondrial skeletal muscle in vitro (Talbot et al., 2004;Teulier et al., 2012;Rey et al., 2017). Finally, sea acclimatization improves the diving capacity of penguins, by increasing the transport of oxygen and its storage in skeletal muscles, and allowing the maintenance of muscle enzymes involved in glycolytic pathways (Weber et al., 1974;Badwin, 1988;Ponganis et al., 1999Ponganis et al., , 2010Noren et al., 2001;Teulier et al., 2012;Rey et al., 2016). ...
... A total of 25 birds were included in this protocol (13 NI juveniles and 12 SA immatures). Superficial pectoralis muscle was surgically biopsied under general isoflurane anesthesia as described previously (Talbot et al., 2004;Teulier et al., 2012;Rey et al., 2016). The biopsy (100 mg) was freshly used for muscle fiber preparation and bioenergetics analysis. ...
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At fledging, juvenile king penguins (Aptenodytes patagonicus) must overcome the tremendous energetic constraints imposed by their marine habitat, including during sustained extensive swimming activity and deep dives in cold seawater. Both endurance swimming and skeletal muscle thermogenesis require high mitochondrial respiratory capacity while the submerged part of dive cycles repeatedly and greatly reduce oxygen availability imposing a need for solutions to conserve oxygen. The aim of the present study was to determine in vitro whether skeletal muscle mitochondria become more "thermogenic" to sustain heat production or more "economical" to conserve oxygen in sea-acclimatized immature penguins as compared with terrestrial juveniles. Rates of mitochondrial oxidative phosphorylation were measured in permeabilized fibers and mitochondria from the pectoralis muscle. Mitochondrial ATP synthesis and coupling efficiency were measured in isolated muscle mitochondria. The mitochondrial activities of respiratory chain complexes and citrate synthase were also assessed. The results showed that respiration, ATP synthesis and respiratory chain complex activities in pectoralis muscles were increased by sea acclimatization. Further, muscle mitochondria were on average 30% to 45% more energy efficient in sea-acclimatized immatures than in pre-fledging juveniles, depending on the respiratory substrate used (pyruvate; palmitoyl-carnitine). Hence, sea acclimatization favors the development of economical management of oxygen, decreasing the oxygen needed to produce a given amount of ATP. This mitochondrial phenotype may improve dive performance during the early marine life of king penguins, by extending their aerobic dive limit.
... Protein carbonyl quantification was carried using the OxiSelect™ protein carbonyl fluorometric [46] assay kit (Cell Biolabs, San Diego, CA, USA) as described by the manufacturer, except with minor modifications to the washing and dilution steps. Briefly, 50 µL of each sample was mixed with the supplied fluorophore reagent and incubated for~16H in darkness at 22°C. ...
... Briefly, 50 µL of each sample was mixed with the supplied fluorophore reagent and incubated for~16H in darkness at 22°C. The samples were then precipitated with 400 µL of cold trichloroacetic acid (TCA) and unbound fluorophore removed by three washes with 1 mL cold acetone, each followed by [38][39][40][41][42][43][44][45][46][47][48][49][50] centrifugation at 10,000 g for 15 min at 4°C. After evaporating residual acetone, the pellets were solubilized using 50 µL of the guanidinium chloride (GuHCl) provided with the kit and incubated at 22°C. ...
... M.K. Tiwari et al.Free Radical Biology and Medicine 121 (2018)[38][39][40][41][42][43][44][45][46][47][48][49][50] ...
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... Therefore, the larger in vitro fatty acid-stimulated increase in mitochondrial proton conductance reported in sea-acclimatized king penguins has been related to the increased muscle expression of both avian uncoupling protein (avUCP) and avian adenine nucleotide translocase (avANT) (Talbot et al., 2004). Since increases in muscle mitochondrial content and respiratory activity have also been observed in sea-acclimatized king penguins (Rey et al., 2016a;Teulier et al., 2012), it is reasonable to expect that the high metabolic effect of lipid infusion reported in sea-acclimatized penguins may be related in part to an increased expression and activity of avANT/avUCP and the subsequent stimulation of proton conductance by fatty acids (Talbot et al., 2004). However, since only one fatty acid concentration was used in previous in vitro experiments (Talbot et al., 2004), it is not known whether the higher fatty acid-induced stimulation of mitochondrial activity in sea-acclimatized penguins is related to a higher response capacity or an increased sensitivity of mitochondria to the uncoupling effect of fatty acids. ...
... Penguins were kept outside in an open-top enclosure near the laboratory and thus were subjected to natural weather condition until they had completed their moult and constituted the "never-immersed" (NI, n = 4) group. A second group of NI penguins that had completed their moult in the outside enclosure, as described above, was subjected to ten experimental immersions in cold water (8°C) for 5 h per immersion, every second day and constituted the "immersed" (IM, n = 5) group (for further details, see Rey et al., 2016a;Teulier et al., 2016). A third group of fledged penguins of both sexes was caught on the colony soon after returning from a foraging trip, thus ensuring that they were fully acclimatized to marine life and constituted the "sea-acclimatized" (SA, n = 6) group. ...
... Superficial pectoralis muscle (~1-2 g) was surgically biopsied under isoflurane-induced general anaesthesia 36 h after the last feed or water immersion and immediately used for mitochondrial extraction (Rey et al., 2016a;Talbot et al., 2004;Teulier et al., 2016). Surgery and mitochondrial extraction and analyses were conducted from early to late January for NI and IM penguins and during the first week of February for SA penguins. ...
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In king penguin juveniles, the environmental transition from a terrestrial to a marine habitat, occurring at fledging, drastically stimulates lipid catabolism and the remodelling of muscle mitochondria to sustain extensive swimming activity and thermoregulation in the cold circumpolar oceans. However, the exact nature of these mechanisms remains only partially resolved. Here we investigated, in vitro, the uncoupling effect of increasing doses of fatty acids in pectoralis muscle intermyofibrillar mitochondria isolated, either from terrestrial never-immersed or experimentally cold water immersed pre-fledging king penguins or from sea-acclimatized fledged penguins. Mitochondria exhibited much greater palmitate-induced uncoupling respiration and higher maximal oxidative capacity after acclimatization to marine life. Such effects were not reproduced experimentally after repeated immersions in cold water, suggesting that the plasticity of mitochondrial characteristics may not be primarily driven by cold exposure per se but by other aspects of sea acclimatization.
... For better clarity, these differentially expressed genes were clustered into six functional groups according to their role in controlling redox homeostasis. The data are related to a comprehensive research study on the ontogeny of antioxidant functions in king penguins, "Hormetic response triggers multifaceted anti-oxidant strategies in immature king penguins (Aptenodytes patagonicus)" (Rey et al., 2016) [2]. The raw ...
... For better clarity, these differentially expressed genes were clustered into six functional groups according to their role in controlling redox homeostasis. The data are related to a comprehensive research study on the ontogeny of antioxidant functions in king penguins, "Hormetic response triggers multifaceted anti-oxidant strategies in immature king penguins (Aptenodytes patagonicus)" (Rey et al., 2016) [2]. The raw ...
... At the end of the procedure, pectoralis muscle of each penguin was surgically biopsied under general anesthesia and the muscle biopsy was frozen at À 80°C. More details of the experimental procedure are given in Rey et al. [2]. ...
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... Peroxiredoxin 4 (PRDX4) protects against oxidative damage by scavenging reactive oxygen species in both the intracellular (especially the endoplasmic reticulum) compartments and the extracellular space [39][40][41]. ...
... Oxidative stress response PRDX6 Peroxiredoxin 6 (PRDX6) plays a role in redox regulation, phospholipid turnover, and protection against oxidative injury [39][40][41]. ...
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... For instance, ROS play a vital role in the regulation of muscle metabolism and adaptation towards exercise (Morales-Alamo and Calbet, 2016). ROS signaling may also be involved in the up-regulation of antioxidant pathways and repair mechanisms of cellular damage reported in diving vertebrates, limiting the oxidative stress associated with repeated prolonged apnea and re-oxygenation cycles (Filho et al., 2002;Rey et al., 2016). Another interesting perspective is the differential oxygen sensitivity of these two mitochondrial processes, i.e. the oxygen respiration and the H 2 O 2 release (Hoffman et al., 2007;Treberg et al., 2018). ...
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... It was observed that this breed has higher levels of unsaturated fatty acids (UFAs). Studies have shown that UFAs are the main factors affecting the fluidity of cell membranes, and are involved in resistance to cold damage in animals [27], plants [28], and microorganisms [29]. As a free-range grazing breed, Tibetan pigs have the opportunity to eat more wild plants, which have higher concentrations of UFAs [30]. ...
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... CORT implant) 63 to reduced oxidative damage have been recently documented. In penguins, part of this resistance to oxidative stress might be related to physiological adaptations to marine life and long-term fasting as recently shown in this species 64 . The role of mitochondrial uncoupling (i.e. ...
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... Mito-hormesis has been documented as a mitochondrial reply to exogenously induced-punctual stress events that triggers a retrograde response compensating for the stress by increasing mitochondrial volume density or biochemical function [20,21] and initiating antioxidant systems [22]. Ultimately, the hormetic effect reduces metabolic levels of ROS. ...
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... the entire transcriptome (e.g., Rey et al. 2016) could reveal unknown signaling molecules involved in the reproductive trade-off, which could open the possibility of manipulations allowing the testing of the proposed hypotheses. ...
... Unfortunately, no work has yet addressed this question. Finally, the study of Overview Articles the entire transcriptome (e.g., Rey et al. 2016) could reveal unknown signaling molecules involved in the reproductive trade-off, which could open the possibility of manipulations allowing the testing of the proposed hypotheses. ...
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... Differentially expressed genes between NI vs. SA or NI vs. AA were determined using the empirical Bayes moderated t-statistics implemented in the Bioconductor package limma [5]. We focused on the genes involved in the redox homeostasis and gathered them into six functional clusters according to GenOntology annotation and literature search [2,6,7] . All analyses were performed using the R statistical software Table 1. ...
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The ability to dive for long periods increases with body size1, but relative to the best human divers, marine birds and mammals of similar or even smaller size are outstanding performers. Most trained human divers can reach a little over 100 m in a single-breath dive lasting for 4 min (ref. 2), but king and emperor penguins (weighing about 12 and 30 kg, respectively) can dive to depths of 304 and 534 m for as long as 7.5 and 15.8 min, respectively3, 4, 5. On the basis of their assumed metabolic rates, up to half of the dive durations were believed to exceed the aerobic dive limit, which is the time of submergence before all the oxygen stored in the body has been used up4, 6, 7. But in penguins and many diving mammals7, 8, the short surface intervals between dives are not consistent with the recovery times associated with a switch to anaerobic metabolism4. We show here that the abdominal temperature of king penguins may fall to as low as 11 °C during sustained deep diving. As these temperatures may be 10 to 20 °C below stomach temperature, cold ingested food cannot be the only cause of abdominal cooling. Thus, the slower metabolism of cooler tissues resulting from physiological adjustments associated with diving per se, could at least partly explain why penguins and possibly marine mammals can dive for such long durations.
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Birds seem to employ powerful physiological strategies to curb the harmful effects of reactive oxygen species (ROS) because they generally live longer than predicted by the free radical theory of aging. However, little is known about the physiological mechanisms that confer protection to birds against excessive ROS generation. Hence, we investigated the ability of birds to control mitochondrial ROS generation during physiologically stressful periods. In our study, we analyzed the relationship between the thyroid status and the function of intermyofibrillar and subsarcolemmal mitochondria located in glycolytic and oxidative muscles of ducklings. We found that the intermyofibrillar mitochondria of both glycolytic and oxidative muscles down regulate ROS production when plasma T(3) levels rise. The intermyofibrillar mitochondria of the gastrocnemius muscle (an oxidative muscle) produced less ROS and were more sensitive than the pectoralis muscle (a glycolytic muscle) to changes in plasma T(3). Such differences in the ROS production by glycolytic and oxidative muscles were associated with differences in the membrane proton permeability and in the rate of free radical leakage within the respiratory chain. This is the first evidence which shows that in birds, the amount of ROS that the mitochondria release is dependent on: (1) their location within the muscle; (2) the type of muscle (glycolytic or oxidative) and (3) on the thyroid status. Reducing muscle mitochondrial ROS generation might be an important mechanism in birds to limit oxidative damage during periods of physiological stress.
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The antioxidant defences in aerobic organisms represent the detoxification pathway against toxicity of reactive oxygen species (ROS). These highly reactive molecules are normally produced during the 4-electrons reduction of molecular oxygen to water coupled with oxidative phosphorylation, and during the activity of several enzymatic systems which produce ROS as intermediates. However, the endogenous generation of oxyradicals may be influenced by different environmental and biological factors, and the basal efficiency of antioxidant systems generally reflects the normal prooxidant pressure to which organisms are exposed. If the antioxidant capacity is exceeded (i.e. as a consequence of enhanced intracellular formation of ROS), a pathological condition, generally termed oxidative stress, may arise. In this preliminary work, susceptibility to oxidative stress has been compared in plasma of Adlie penguin (Pygoscelis adeliae), emperor penguin (Aptenodytes forsteri), south polar skua (Catharacta maccormicki) and snow petrel (Pagodroma nivea). Within the framework of the Italian Research Program in Antarctica, blood samples were collected during the austral summer 1998-1999 and the Total Oxyradical Scavenging Capacity (TOSC) analysed. The TOSC assay, measuring the capability of biological samples to neutralise different oxyradicals, has been recently standardised to provide a quantifiable value of biological resistance to toxicity of ROS. Penguins exhibited higher scavenging capacity towards peroxyl radicals than south polar skua and snow petrel. The greater resistance to toxicity of oxyradicals might suggest that penguins are naturally exposed to a higher basal prooxidant pressure in comparison to other analysed Antarctic birds.
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We tested the usefulness of the fatty acid signature-method in investigating the diet of seabirds in conjunction with the conventional technique of stomach-content analysis. We compared the fatty acid composition of subcutaneous white adipose tissue (SWAT) of king penguin chicks (Aptenodytes patagonicus) during fattening periods to that of total lipids from their food. In both spring and autumn, the fatty acid composition of chick SWAT was identical to that of the dietary lipids. Because the diet of adult king penguins feeding for self-maintenance (i.e. not for their chicks) was essentially unknown, we subsequently analysed their SWAT fatty acid patterns after premolting and prebreeding foraging trips (during which they build up large energy reserves). The fatty acid composition of SWAT from adults was identical to that of chick adipose tissue and food. King penguin diet and SWAT were characterized by high levels of very long-chain mono-unsaturated fatty acids (20 to 24 carbon atoms, 16 to 23% by mass) and (n-3) poly-unsaturated fatty acids (19 to 27%); these consisted mainly of 20:1n-9 (5 to 8%) and 22:1n-11 (5 to 8%), and 22:6n-3 (10 to 13%) and 20:5n-3 (3 to 9%), respectively. Prey items identified from chick stomach contents indicated that the bulk of the food was oceanic myctophid fishes, mainly Electrona carlbergi, Krefftichthys anderssoni and Protomyctophum tenisoni. The fatty acid composition of four other species of myctophid fishes was similar to that of penguin diet and SWAT, but markedly different from that measured for a squid species and that reported for crustaceans. These findings indicate that adult king penguins prey on myctophid fish not only to feed their chicks but also for their own nutrition. The fatty acid signature-technique is therefore a reliable method to gain information on the food and feeding ecology of seabirds when more conventional techniques are of limited value. Such information is important to the understanding of trophic relationships between key species of the ecosystems, and also to provide insight into the nature of avian adaptations to the marine environment.
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The passage from shore to marine life of juvenile penguins represents a major energetic challenge to fuel intense and prolonged demands for thermoregulation and locomotion. Some functional changes developed at this crucial step were investigated by comparing pre-fledging king penguins with sea-acclimatized (SA) juveniles (Aptenodytes patagonicus). Transcriptomic analysis of pectoralis muscle biopsies revealed that most genes encoding proteins involved in lipid transport or catabolism were upregulated, while genes involved in carbohydrate metabolism were mostly downregulated in SA birds. Determination of muscle enzymatic activities showed no changes in enzymes involved in the glycolytic pathway, but increased 3-hydroxyacyl-CoA dehydrogenase, an enzyme of the β-oxidation pathway. The respiratory rates of isolated muscle mitochondria were much higher with a substrate arising from lipid metabolism (palmitoyl-L-carnitine) in SA juveniles than in terrestrial controls, while no difference emerged with a substrate arising from carbohydrate metabolism (pyruvate). In vivo, perfusion of a lipid emulsion induced a fourfold larger thermogenic effect in SA than in control juveniles. The present integrative study shows that fuel selection towards lipid oxidation characterizes penguin acclimatization to marine life. Such acclimatization may involve thyroid hormones through their nuclear beta receptor and nuclear coactivators.
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In this review, it is our aim 1) to describe the high diversity in molecular and structural antioxidant defenses against oxidative stress in animals, 2) to extend the traditional concept of antioxidant to other structural and functional factors affecting the "whole" organism, 3) to incorporate, when supportable by evidence, mechanisms into models of life-history trade-offs and maternal/epigenetic inheritance, 4) to highlight the importance of studying the biochemical integration of redox systems, and 5) to discuss the link between maximum life span and antioxidant defenses. The traditional concept of antioxidant defenses emphasizes the importance of the chemical nature of molecules with antioxidant properties. Research in the past 20 years shows that animals have also evolved a high diversity in structural defenses that should be incorporated in research on antioxidant responses to reactive species. Although there is a high diversity in antioxidant defenses, many of them are evolutionary conserved across animal taxa. In particular, enzymatic defenses and heat shock response mediated by proteins show a low degree of variation. Importantly, activation of an antioxidant response may be also energetically and nutrient demanding. So knowledge of antioxidant mechanisms could allow us to identify and to quantify any underlying costs, which can help explain life-history trade-offs. Moreover, the study of inheritance mechanisms of antioxidant mechanisms has clear potential to evaluate the contribution of epigenetic mechanisms to stress response phenotype variation.
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Cancer is a leading cause of death worldwide. Tumor cells exploit various signaling pathways to promote their growth and metastasis. To our knowledge, the role of angiopoietin-like 4 protein (ANGPTL4) in cancer remains undefined. Here, we found that elevated ANGPTL4 expression is widespread in tumors, and its suppression impairs tumor growth associated with enhanced apoptosis. Tumor-derived ANGPTL4 interacts with integrins to stimulate NADPH oxidase-dependent production of O(2)(-). A high ratio of O(2)(-):H(2)O(2) oxidizes/activates Src, triggering the PI3K/PKBα and ERK prosurvival pathways to confer anoikis resistance, thus promoting tumor growth. ANGPTL4 deficiency results in diminished O(2)(-) production and a reduced O(2)(-):H(2)O(2) ratio, creating a cellular environment conducive to apoptosis. ANGPTL4 is an important redox player in cancer and a potential therapeutic target.
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Temperature (T) reduction increases lifespan, but the mechanisms are not understood. Because reactive oxygen species (ROS) contribute to aging, we hypothesized that lowering T might decrease mitochondrial ROS production. We measured respiratory response and ROS production in isolated mitochondria at 32, 35, and 37 °C. Lowering T decreased the rates of resting (state 4) and phosphorylating (state 3) respiration phases. Surprisingly, this respiratory slowdown was associated with an increase of ROS production and hydrogen peroxide release and with elevation of the mitochondrial membrane potential, ΔΨm. We also found that at lower T mitochondria produced more carbon-centered lipid radicals, a species known to activate uncoupling proteins. These data indicate that reduced mitochondrial ROS production is not one of the mechanisms mediating lifespan extension at lower T. They suggest instead that increased ROS leakage may mediate mitochondrial responses to hypothermia.
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Recent developments in high-throughput methods of analyzing transcriptomic profiles are promising for many areas of biology, including ecophysiology. However, although commercial microarrays are available for most common laboratory models, transcriptome analysis in non-traditional model species still remains a challenge. Indeed, the signal resulting from heterologous hybridization is low and difficult to interpret because of the weak complementarity between probe and target sequences, especially when no microarray dedicated to a genetically close species is available. We show here that transcriptome analysis in a species genetically distant from laboratory models is made possible by using MAXRS, a new method of analyzing heterologous hybridization on microarrays. This method takes advantage of the design of several commercial microarrays, with different probes targeting the same transcript. To illustrate and test this method, we analyzed the transcriptome of king penguin pectoralis muscle hybridized to Affymetrix chicken microarrays, two organisms separated by an evolutionary distance of approximately 100 million years. The differential gene expression observed between different physiological situations computed by MAXRS was confirmed by real-time PCR on 10 genes out of 11 tested. MAXRS appears to be an appropriate method for gene expression analysis under heterologous hybridization conditions.
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Although identified in several bird species, the biological role of the avian homolog of mammalian uncoupling proteins (avUCP) remains extensively debated. In the present study, the functional properties of isolated mitochondria were examined in physiological or pharmacological situations that induce large changes in avUCP expression in duckling skeletal muscle. The abundance of avUCP mRNA, as detected by RT-PCR in gastrocnemius muscle but not in the liver, was markedly increased by cold acclimation (CA) or pharmacological hyperthyroidism but was down-regulated by hypothyroidism. Activators of UCPs, such as superoxide with low doses of fatty acids, stimulated a GDP-sensitive proton conductance across the inner membrane of muscle mitochondria from CA or hyperthyroid ducklings. The stimulation was much weaker in controls and not observed in hypothyroid ducklings or in any liver mitochondrial preparations. The production of endogenous mitochondrial reactive oxygen species (ROS) was much lower in muscle mitochondria from CA and hyperthyroid ducklings than in the control or hypothyroid groups. The addition of GDP markedly increased the mitochondrial ROS production of CA or hyperthyroid birds up to, or above, the level of control or hypothyroid ducklings. Differences in ROS production among groups could not be attributed to changes in antioxidant enzyme activities (superoxide dismutase or glutathione peroxidase). This work provides the first functional in vitro evidence that avian UCP regulates mitochondrial ROS production in situations of enhanced metabolic activity.
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At hatching, breaking eggshell induces a surge in oxygen availability that is likely to generate oxidative stress in newborn chicks. To investigate the involvement of potential adaptive antioxidant mechanisms, we explored some markers of oxidative stress and the regulation of muscle avian uncoupling protein (avUCP) and adenine nucleotide translocase (ANT) in ducklings in the peri-hatching period. When compared with pre-hatching levels, the amount of peroxidized lipids were increased 24 h after external pipping in gastrocnemius muscle (+37%) and heart (+39%) as well as the muscle avUCP mRNA expression (+60%) but the susceptibility of red blood cells to free radicals (a functional test of oxidative status) was not affected. In order to relate these changes to the oxidative transition of hatching, an imposed hypoxia/re-oxygenation protocol was used. Hatched chicks that had spent the last 24 h of incubation in artificial severe hypoxia showed a rise in muscle (+50%) and heart (+69%) lipid peroxidation, an increased susceptibility of red blood cells to free radicals, a marked over-expression of avUCP mRNA (+105%) and a rise in mitochondrial ANT content (+54%). These results suggest that avian UCP and ANT may contribute to prepare incubating eggs to the oxidative stress generated by the hypoxia/re-oxygenation transition naturally occurring at hatching.
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Dive duration in wild king penguins and the energetic cost of swimming in a 30m long swim channel were determined at Ile de la Possession, Crozet Archipelago, using external data loggers and respirometry, respectively. Calibrated electronic data loggers equipped with a pressure sensor were used to determine dive durations: 95% of dives were less than 6 min long and 66% of dives were less than 4 min long. Dive patterns show that king penguins may intersperse long dive durations (4-6.3 min) with short ones (1.5-3 min) and make surface pauses of variable duration between them (0.5-3.5 min), or dive regularly (for up to 5 h) with long dive durations (5 min) and constant interdive surface intervals (1.5 min). The latter indicates that the aerobic dive limits (ADL) of this species could be higher and oxygen consumption lower than previously reported. Assuming that king penguins dive within their aerobic limit, different approaches to the analysis of the data obtained in the swim channel are discussed to derive the ADL. Swimming speeds observed in the channel ranged from 0.9 to 3.4 m s-1. Transport costs were lowest between 1.8 and 2.2 m s-1. Although at 2.2 m s-1 king penguins used only 10.3 Wkg-1 over a dive+surface cycle (minimal transport costs of 4.7 J kg-1 m-1), we speculate that tisse oxygen consumption during submergence may be as low as 0.23 ml O2 kg-1 s-1 (2.1 times standard metabolic rate, SMR) or perhaps lower (which gives an ADL of 4.2 min). During surface phases, oxygen uptake would be increased to at least 1 ml O2kg-1 s-1 (9.3 times SMR). This implies that at least 70% of all dives are aerobic. Potential physiological mechanisms allowing king penguins to partition O2 consumption between submergence and surface periods remain, however, unclear.
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We measured production of reactive oxygen species by intact mitochondria from rat skeletal muscle, heart, and liver under various experimental conditions. By using different substrates and inhibitors, we determined the sites of production (which complexes in the electron transport chain produced superoxide). By measuring hydrogen peroxide production in the absence and presence of exogenous superoxide dismutase, we established the topology of superoxide production (on which side of the mitochondrial inner membrane superoxide was produced). Mitochondria did not release measurable amounts of superoxide or hydrogen peroxide when respiring on complex I or complex II substrates. Mitochondria from skeletal muscle or heart generated significant amounts of superoxide from complex I when respiring on palmitoyl carnitine. They produced superoxide at considerable rates in the presence of various inhibitors of the electron transport chain. Complex I (and perhaps the fatty acid oxidation electron transfer flavoprotein and its oxidoreductase) released superoxide on the matrix side of the inner membrane, whereas center o of complex III released superoxide on the cytoplasmic side. These results do not support the idea that mitochondria produce considerable amounts of reactive oxygen species under physiological conditions. Our upper estimate of the proportion of electron flow giving rise to hydrogen peroxide with palmitoyl carnitine as substrate (0.15%) is more than an order of magnitude lower than commonly cited values. We observed no difference in the rate of hydrogen peroxide production between rat and pigeon heart mitochondria respiring on complex I substrates. However, when complex I was fully reduced using rotenone, rat mitochondria released significantly more hydrogen peroxide than pigeon mitochondria. This difference was solely due to an elevated concentration of complex I in rat compared with pigeon heart mitochondria.
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During diving, intermittent swim stroke patterns, ranging from burst/coast locomotion to prolonged gliding, represent potential energy conservation mechanisms that could extend the duration of aerobic metabolism and, hence, increase the aerobic dive limit (ADL, dive duration associated with onset of lactate accumulation). A 5.6 min ADL for emperor penguins had been previously determined with lactate measurements after dives of <50 m depth. In order to assess locomotory patterns during such dives, longitudinal acceleration was measured with an attached accelerometer in 44 dives of seven adult birds diving from an isolated dive hole in the sea ice of McMurdo Sound, Antarctica. Detection of wing strokes in processed accelerometer data was verified in selected birds with analysis of simultaneous Crittercam underwater video footage. Mean dive duration of birds equipped with the accelerometer and a time-depth recorder (TDR) was 5.7+/-2.2 min; 48% of these dives were greater than the measured 5.6 min ADL (ADL(M)). Highest stroke frequencies (0.92+/-0.31 Hz, N=981) occurred during the initial descent to 12 m depth. Swimming effort was reduced to a mean stroke frequency <0.70 Hz during other phases of the dive (while traveling below 12 m depth, during foraging ascents/descents to and from the sub-ice surface, and during final ascents to exit). The longest stroke interval (8.6 s) occurred during a feeding excursion to the undersurface of the ice. In dives >ADL(M), mean stroke frequency during travel segments was significantly less than that in dives <ADL(M) (P<0.05). Mean stroke frequency of the entire dive correlated inversely (P<0.05) with diving duration (r=-0.67) and with mean dive depth (r=-0.43). Emperor penguins did not exhibit any significant (>10 s) periods of prolonged gliding during these shallow (<60 m) foraging dives. However, a stroke/glide pattern was evident with more than 50% of strokes associated with a stroke interval >1.6 s, and with lower stroke frequency associated with increased dive duration.
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The main objective of this study was to determine heart rate(fh) and the energetic costs of specific behaviours of king penguins while ashore and while foraging at sea during their breeding period. In particular, an estimate was made of the energetic cost of diving in order to determine the proportion of dives that may exceed the calculated aerobic dive limit (cADL; estimated usable O2 stores/estimated rate of oxygen consumption during diving). An implanted data logger enabled fh and diving behaviour to be monitored from 10 free-ranging king penguins during their breeding period. Using previously determined calibration equations, it was possible to estimate rate of oxygen consumption(V̇O2) when the birds were ashore and during various phases of their foraging trips. Diving behaviour showed a clear diurnal pattern, with a mixture of deep (>40 m),long (>3 min) and shallow (<40 m), short (<3 min) dives from dawn to dusk and shallow, short dives at night. Heart rate during dive bouts and dive cycles (dive + post-dive interval) was 42% greater than that when the birds were ashore. During diving, fh was similar to the `ashore'value (87±4 beats min–1), but it did decline to 76% of the value recorded from king penguins resting in water. During the first hour after a diving bout, fh was significantly higher than the average value during diving (101±4 beats min–1) and for the remainder of the dive bout. Rates of oxygen consumption estimated from these (and other) values of fh indicate that when at sea, metabolic rate (MR) was 83%greater than that when the birds were ashore [3.15 W kg–1(–0.71, +0.93), where the values in parentheses are the computed standard errors of the estimate], while during diving bouts and dive cycles,it was 73% greater than the `ashore' value. Although estimated MR during the total period between dive bouts was not significantly different from that during dive bouts [5.44 W kg–1 (–0.30, +0.32)], MR during the first hour following a dive bout was 52% greater than that during a diving bout. It is suggested that this large increase following diving(foraging) activity is, at least in part, the result of rewarming the body,which occurs at the end of a diving bout. From the measured behaviour and estimated values of V̇O2, it was evident that approximately 35% of the dives were in excess of the cADL. Even if V̇O2 during diving was assumed to be the same as when the birds were resting on water,approximately 20% of dives would exceed the cADL. As V̇O2 during diving is, in fact, that estimated for a complete dive cycle, it is quite feasible that V̇O2 during diving itself is less than that measured for birds resting in water. It is suggested that the regional hypothermia that has been recorded in this species during diving bouts may be at least a contributing factor to such hypometabolism.
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The longevity of birds is surprising since they exhibit high metabolic rates and elevated blood sugar levels compared with mammals of the same body size, which presumably expose them to higher rates of free oxygen radical production, which is implicated in accelerated senescence. Uncoupling proteins (UCPs) are transporters of the inner mitochondrial membrane and their physiological activity is still a subject of debate. Avian UCP was found in birds but data on its activity are scarce. Avian UCP (Gallus gallus) was overexpressed in yeast and we assessed its ability to prevent mitochondrial reactive oxygen species (ROS) production by measuring ROS damage (aconitase activity) and antioxidant defences (MnSOD activity). We show that avian UCP protects yeast mitochondria against the deleterious impact of ROS, but without stimulation of superoxide dismutase activity. Avian UCP protein was specifically immunodetected and retinoic acid, which belongs to the carotenoid family, was found to trigger its activity. These data show that avian UCP basal activity protects against ROS damage. However, when activated by retinoic acid, avian UCP can also operate as the mammalian thermogenic UCP1. The hypothesis that avian UCP activities are state- and species-dependent is further discussed.
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In order to determine the rate and magnitude of respiratory O2 depletion during dives of emperor penguins (Aptenodytes forsteri), air sac O2 partial pressure (PO2) was recorded in 73 dives of four birds at an isolated dive hole. These results were evaluated with respect to hypoxic tolerance, the aerobic dive limit (ADL; dive duration beyond which there is post-dive lactate accumulation) and previously measured field metabolic rates (FMRs). 55% of dives were greater in duration than the previously measured 5.6-min ADL. PO2 and depth profiles revealed compression hyperoxia and gradual O2 depletion during dives. 42% of final PO2s during the dives (recorded during the last 15 s of ascent) were <20 mmHg (<2.7 kPa). Assuming that the measured air sac PO2 is representative of the entire respiratory system, this implies remarkable hypoxic tolerance in emperors. In dives of durations greater than the ADL, the calculated end-of-dive air sac O2 fraction was <4%. The respiratory O2 store depletion rate of an entire dive, based on the change in O2 fraction during a dive and previously measured diving respiratory volume, ranged from 1 to 5 ml O2 kg(-1) min(-1) and decreased exponentially with diving duration. The mean value, 2.1+/-0.8 ml O2 kg(-1) min(-1), was (1) 19-42% of previously measured respiratory O(2) depletion rates during forced submersions and simulated dives, (2) approximately one-third of the predicted total body resting metabolic rate and (3) approximately 10% of the measured FMR. These findings are consistent with a low total body metabolic rate during the dive.
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The existence of hypoxia-induced reactive oxygen species (ROS) production remains controversial. However, numerous observations with a variety of methods and in many cells and tissue types are supportive of this idea. Skeletal muscle appears to behave much like heart in that in the early stages of hypoxia there is a transient elevation in ROS, whereas in chronic exposure to very severe hypoxia there is evidence of ongoing oxidative stress. Important remaining questions that are addressed in this review include the following. Are there levels of PO2 in skeletal muscle, typical of physiological or mildly pathophysiological conditions, that are low enough to induce significant ROS production? Does the ROS associated with muscle contractile activity reflect imbalances in oxygen uptake and demand that drive the cell to a more reduced state? What are the possible molecular mechanisms by which ROS may be elevated in hypoxic skeletal muscle? Is the production of ROS in hypoxia of physiological significance, both with respect to cell signaling pathways promoting cell function and with respect to damaging effects of long-term exposure? Discussion of these and other topics leads to general conclusions that hypoxia-induced ROS may be a normal physiological response to imbalance in oxygen supply and demand or environmental stress and may play a yet undefined role in normal response mechanisms to these stimuli. However, in chronic and extreme hypoxic exposure, muscles may fail to maintain a normal redox homeostasis, resulting in cell injury or dysfunction.
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Mitochondrial respiratory and phosphorylation activities, mitochondrial uncoupling and hydrogen peroxide formation were studied in isolated rat skeletal muscle mitochondria during experimentally induced hypothermia (25°C) and hyperthermia (42°C) compared to the physiological temperature of resting muscle (35°C). For non-phosphorylating mitochondria, increasing the temperature from 25°C to 42°C led to a decrease in membrane potential, hydrogen peroxide production and quinone reduction levels. For phosphorylating mitochondria, no temperature-dependent changes in these mitochondrial functions were observed. However, the efficiency of oxidative phosphorylation decreased, while the oxidation and phosphorylation rates and oxidative capacities of the mitochondria increased with increasing assay temperature. An increase in proton leak, including uncoupling protein-mediated proton leak, was observed with increasing assay temperature, which could explain the reduced oxidative phosphorylation efficiency and reactive oxygen species production. Copyright © 2015. Published by Elsevier Inc.
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Oxidative stress has long been linked to disease development and accelerated aging, prompting professionals in the biomedical field to suggest the use of antioxidants to prevent or even reverse these conditions. But growing clinical evidence is showing that this in fact might not be effective, calling for additional investigation to prove that certain molecular factors involved in oxidation, specifically reactive oxidative species (ROS), are not detrimental. In “Bedside to Bench,” Michael Ristow highlights recent human studies with antioxidant supplementation that have failed to show any improvement in health span. Moreover, other relevant evidence has pointed towards a beneficial role for ROS in lifespan under stress conditions, although how this is mediated and regulated inside the cell is not fully understood. In “Bench to Bedside,” Hiroyuki Kawagishi and Toren Finkel peruse the biological and signaling underpinnings of ROS in living organisms, which suggest different amounts of ROS may explain their dual role in lifespan and disease and the lack of effect of antioxidants in the body. The authors propose targeting pathways and molecules involved in removing cellular damage rather than ROS, which could make therapies to increase lifespan more effective and preclude diseases caused by oxidation and aging.
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Thyroid hormones (TH) are major contributor to oxidative stress in mammals because they (1) stimulate reactive oxygen species generation (ROS), (2) impair antioxidant defenses, and (3) increase the susceptibility to free radicals of most tissues. Unlike mammals, THs seem to diminish mitochondrial ROS while they have limited effect on the antioxidant machinery in birds. However, how THs modify the susceptibility to ROS has never been explored in an avian model, and very little is known about their effect on oxidative balance in birds. Therefore, the objective of our study was to examine the effect of chronic pharmacological hypo- and hyperthyroidism on (i) the susceptibility of mitochondrial membranes to ROS; and (ii) the level of oxidative stress assessed by measuring oxidative damage to lipids, nucleic acids and proteins in the gastrocnemius muscle of ducklings. We show that hypothyroidism had no effect on the susceptibility of mitochondrial membranes to free radicals. Hypothyroid ducklings had lower oxidized lipids (-31%) and DNA (-25%) but a similar level of protein carbonylation relative to controls. Conversely, mitochondrial membranes of hyperthyroid ducklings exhibited higher unsaturation (+12%) and peroxidation (+31%) indexes than in controls indicating a greater susceptibility to free radicals. However, hyperthyroid ducklings exhibited more oxidative damages on proteins (+67%) only, whereas lipid damages remained unchanged, and there was a slight reduction (-15%) in damages to DNA compared to euthyroid controls. Our results indicate that birds and mammals present fundamental differences in their oxidative stress response to thyroid status. J. Exp. Zool. 9999A: XX-XX, 2014. © 2014 Wiley Periodicals, Inc.
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For many years, mitochondria were viewed as semiautonomous organelles, required only for cellular energetics. This view has been largely supplanted by the concept that mitochondria are fully integrated into the cell and that mitochondrial stresses rapidly activate cytosolic signaling pathways that ultimately alter nuclear gene expression. Remarkably, this coordinated response to mild mitochondrial stress appears to leave the cell less susceptible to subsequent perturbations. This response, termed mitohormesis, is being rapidly dissected in many model organisms. A fuller understanding of mitohormesis promises to provide insight into our susceptibility for disease and potentially provide a unifying hypothesis for why we age.
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Motivation: When running experiments that involve multiple high density oligonucleotide arrays, it is important to remove sources of variation between arrays of non-biological origin. Normalization is a process for reducing this variation. It is common to see non-linear relations between arrays and the standard normalization provided by Affymetrix does not perform well in these situations. Results: We present three methods of performing normalization at the probe intensity level. These methods are called complete data methods because they make use of data from all arrays in an experiment to form the normalizing relation. These algorithms are compared to two methods that make use of a baseline array: a one number scaling based algorithm and a method that uses a non-linear normalizing relation by comparing the variability and bias of an expression measure. Two publicly available datasets are used to carry out the comparisons. The simplest and quickest complete data method is found to perform favorably. Availability: Software implementing all three of the complete data normalization methods is available as part of the R package Affy, which is a part of the Bioconductor project http://www.bioconductor.org. Supplementary information: Additional figures may be found at http://www.stat.berkeley.edu/~bolstad/normalize/index.html
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While diving, seals are exposed to apnea-induced hypoxemia and repetitive cycles of ischemia/reperfusion. While on land, seals experience sleep apnea, as well as prolonged periods of food and water deprivation. Prolonged fasting, sleep apnea, hypoxemia and ischemia/reperfusion increase oxidant production and oxidative stress in terrestrial mammals. In seals, however, neither prolonged fasting nor apnea-induced hypoxemia or ischemia/reperfusion increase systemic or local oxidative damage. The strategies seals evolved to cope with increased oxidant production are reviewed in the present manuscript. Among these strategies, high antioxidant capacity and the oxidant-mediated activation of hormetic responses against hypoxia and oxidative stress are discussed. In addition to expanding our knowledge of the evolution of antioxidant defenses and adaptive responses to oxidative stress, understanding the mechanisms that naturally allow mammals to avoid oxidative damage has the potential to advance our knowledge of oxidative stress-induced pathologies and to enhance the translative value of biomedical therapies in the long term.
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The sirtuins are highly conserved NAD-dependent deacetylases that were shown to regulate lifespan in lower organisms and affect diseases of aging in mammals, such as diabetes, cancer, and inflammation. Most relevant to the amelioration of disease, the SIR2 ortholog SIRT1 has been shown to deacetylate many important transcription factors to exert an overarching influence on numerous metabolic pathways. Here we discuss several diseases of aging for which SIRT1 has been recently shown to confer protection. These findings suggest that manipulating sirtuin activity pharmacologically may be a fruitful area to improve human health.
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Oxygen store depletion and a diving bradycardia in emperor penguins (Aptenodytes forsteri) expose tissues to critical levels of hypoxemia and ischemia. To assess the prevention of re-perfusion injury and reactive oxygen species (ROS) damage in emperor penguins, superoxide radical production, lipid peroxidation (thiobarbituric acid reactive substances (TBARS)), and antioxidant enzyme activity profiles in biopsy samples from muscle and liver were determined and compared to those in the chicken and 8 species of flighted marine birds (non-divers and plunge divers). In muscle of emperor penguins, superoxide production and TBARS levels were not distinctly different from those in the other species; among the antioxidant enzymes, catalase (CAT) and glutathione-S-transferase (GST) activities were significantly elevated above all species. In the liver of emperor penguins, TBARS levels were not significantly different from other species; only CAT activity was significantly elevated, although GST and glutathione peroxidase (GPX) activities were 2-3 times higher than those in other species. The potential for ROS formation and lipid peroxidation is not reduced in the pectoral muscle or liver of the emperor penguin. Scavenging of hydrogen peroxide by CAT and the conjugation of glutathione with reactive intermediates and peroxides by GST and GPX appear to be important in the prevention of ROS damage and re-perfusion injury in these birds.
Article
The scavenger receptors (SRs) comprise structurally and functionally divergent groups of cell surface and secreted proteins that play an important role in innate immune defenses. Searching translated chicken genomic databases revealed many proteins homologous to mammalian SRs. SR mediated immune functions (oxidative burst, degranulation, phagocytosis, nitric oxide (NO) production, and cytokine expression) were evaluated in chicken heterophils, peripheral blood mononuclear cells (PBMC), and a chicken macrophage cell line (HD11) using various SR class A and B ligands. Results showed that the SR-A ligands, fucoidan, poly(I) and poly(G), but not SR-B ligands, phosphatidylserine and LDL, stimulated dose-dependent NO production in HD11 cells. However, SR-A ligands failed to induce NO in chicken monocytes. Quantitative RT-PCR indicated that SR ligands differentially regulated the gene expression of cytokines and chemokine in HD11 cells with a strong up-regulation of the cytokines IL-1 beta and IL-6 and the chemokine MIP-1 beta, but had no effect on IL-4, IL-12, IFN-gamma, and IFN-beta. SR-B ligands did not alter expression of these genes. SR-A ligands had no stimulatory effect on functional response in heterophils. However, LDL, a SR-B ligand stimulated oxidative burst in both heterophils and PBMC. Additionally, results indicate that SRs are involved in bacterial binding in macrophages.
Article
Heat shock proteins (Hsps) have been studied for many years and there is now a large body of evidence that demonstrates the role of Hsp upregulation in tissue and cell protection in a wide variety of stress conditions. Oxidative stress is known to be involved in a number of pathological conditions, including neurodegeneration, cardiovascular disease and stroke, and even plays a role in natural aging. In this review we summarize the current understanding of the role of Hsps and the heat shock response (HSR) in these pathological conditions and discuss the therapeutic potential of an Hsp therapy for these disorders. However, although an Hsp based therapy appears to be a promising approach for the treatment of diseases that involve oxidative damage, there are some significant hurdles that must be overcome before this approach can be successful. For example, to be effective an Hsp based therapy will need to ensure that the upregulation of Hsps occurs in the right place (i.e. be cell specific), at the right time and to a level and specificity that ensures that all the important binding partners, namely the co-chaperones, are also present at the appropriate levels. It is therefore unlikely that strategies that involve genetic modifications that result in overexpression of specific Hsps will achieve such sophisticated and coordinated effects. Similarly, it is likely that some pharmaceutical inducers of Hsps may be too generic to achieve the desired specific effects on Hsp expression, or may simply fail to reach their target cells due to delivery problems. However, if these difficulties can be overcome, it is clear that an effective Hsp based therapy would be of great benefit to the wide range of depilating conditions in which oxidative stress plays a critical role.
Article
Animal heme-containing peroxidases play roles in innate immunity, hormone biosynthesis, and the pathogenesis of inflammatory diseases. Using the peroxidase-like domain of Duox1 as a query, we carried out homology searching of the National Center for Biotechnology Information database. Two novel heme-containing peroxidases were identified in humans and mice. One, termed VPO1 for vascular peroxidase 1, exhibits its highest tissue expression in heart and vascular wall. A second, VPO2, present in humans but not in mice, is 63% identical to VPO1 and is highly expressed in heart. The peroxidase homology region of VPO1 shows 42% identity to myeloperoxidase and 57% identity to the insect peroxidase peroxidasin. A molecular model of the VPO1 peroxidase region reveals a structure very similar to that of known peroxidases, including a conserved heme binding cavity, critical catalytic residues, and a calcium binding site. The absorbance spectra of VPO1 are similar to those of lactoperoxidase, and covalent attachment of the heme to VPO1 protein was demonstrated by chemiluminescent heme staining. VPO1 purified from heart or expressed in HEK cells is catalytically active, with a K(m) for H(2)O(2) of 1.5 mM. When co-expressed in cells, VPO1 can use H(2)O(2) produced by NADPH oxidase enzymes. VPO1 is likely to carry out peroxidative reactions previously attributed exclusively to myeloperoxidase in the vascular system.
Article
Publisher Summary Gluthatione peroxidase is assayed by a modification of the method of Paglia and Valentine. Glutathione peroxidase catalyzes the reduction of hydroperoxides with glutathione as the reductant. The simple method is a spectrophotometric assay in which the reduction of GSSG is coupled to the oxidation of NADPH through glutathione reductase. The acetone precipitation serves to partially purify as well as concentrate the sample for application onto chromatography columns. The multiplicity of forms of glutathione peroxidase in vitro may or may not represent true in vivo forms. The hypothesis that glutathione peroxidase protects membranes from damage due to lipid peroxidation requires that the soluble enzyme come in contact with the peroxide substrates in the membrane. Study of the multiple forms may give information about the characteristics of the enzyme and the role it plays in protecting the cell from peroxidative damage. The aggregation studies suggest that the in vivo form of the enzyme may be a large aggregate. The enzyme resists disaggregation by the normal agents used for this purpose.
Article
Juvenile king and macaroni penguins are terrestrial seabirds and must face an intensive and prolonged energetic demand during their passage from shore to marine life in cold subantarctic seawater. Evidence for progressive thermal adaptation was sought by measurement of metabolic rate (MR) and body (Tb) and skin (Tsk) temperatures in unrestrained, fully immersed penguins. Steady-state responses obtained after the 3rd h of immersion in never-immersed (NI) penguins were compared with those of penguins acclimatized to seawater temperature (A). NI macaroni penguins, unlike NI king penguins, showed a fall in Tb on their first immersion but, once acclimatized, were able to maintain their homeothermy due to an increase (greater than 3.2 W/kg) in regulatory thermogenesis. In NI king penguins, during a simulation of seawater adaptation by 10 successive immersions, MR at 7 degrees C water temperature (Tw) rose from 6.0 to 9.4 W/kg (becoming 3-5 times higher than in air), whereas Tb rose from 37.6 to 38.4 degrees C. In both species occurrence of peak MR at much lower Tw, progressive increase in thermogenesis capacity, and lower conductance in water after adaptation to marine life (28 and 36% less in A king and macaroni penguins, respectively) showed that the passage from shore to marine life consisted of a true cold acclimatization.
Article
This review concentrates on the physiological responses, and their control, in freely diving birds and mammals that enable them to remain submerged and sometimes quite active for extended periods of time. Recent developments in technology have provided much detailed information on the behavior of these fascinating animals. Unfortunately, the advances in technology have been insufficient to enable physiologists to obtain anything like the same level of detail on the metabolic rate and physiological adjustments that occur during natural diving. This has led to much speculation and calculations based on many assumptions concerning usable oxygen stores and metabolic rate during diving, in an attempt to explain the observed behavior. Despite their shortcomings, these calculations have provided useful insights into the degree of adaptations of various species of aquatic birds and mammals. Many of them, e.g., ducks, smaller penguins, fur seals, and Weddell seals, seem able to metabolize aerobically, when diving, at approximately the same (if not greater) rate as they do at the surface. Their enhanced oxygen stores are able to support aerobic metabolism, at what would not be considered unusually low levels, for the duration of the dives, although there are probably circulatory readjustments to ensure that the oxygen stores are managed judiciously. For other species, such as the larger penguins, South Georgian shag, and female elephant seals, there is a general consensus that they must either be reducing their aerobic metabolic rate when diving, possibly by way of regional hypothermia, and/or producing ATP, at least partly, by anaerobiosis and metabolizing the lactic acid when at the surface (although this is hardly likely in the case of the female elephant seals). Circulation is the proximate regulator of metabolism during aerobic diving, and heart rate is the best single indicator of circulatory adjustment. During voluntary dives, heart rates range from extreme bradycardia to well above resting, reflecting metabolic performance. Efferent cardiac control is largely parasympathetic. Reflex cardiorespiratory responses are modulated by conditioning and habituation, but reflexes predominate during extended dives and during recovery, when gas exchange is maximized.
Article
Oxidative stress is an unavoidable consequence of life in an oxygen-rich atmosphere. Oxygen radicals and other activated oxygen species are generated as by-products of aerobic metabolism and exposure to various natural and synthetic toxicants. The "Oxygen Paradox" is that oxygen is dangerous to the very life-forms for which it has become an essential component of energy production. The first defense against oxygen toxicity is the sharp gradient of oxygen tension, seen in all mammals, from the environmental level of 20% to a tissue concentration of only 3-4% oxygen. These relatively low tissue levels of oxygen prevent most oxidative damage from ever occurring. Cells, tissues, organs, and organisms utilize multiple layers of antioxidant defenses and damage removal, and replacement or repair systems in order to cope with the remaining stress and damage that oxygen engenders. The enzymes comprising many of these protective systems are inducible under conditions of oxidative stress adaptation, in which the expression of over 40 mammalian genes is upregulated. Mitotic cells have the additional defensive ability of entering a transient growth-arrested state (in the first stages of adaptation) in which DNA is protected by histone proteins, energy is conserved by diminished expression of nonessential genes, and the expression of shock and stress proteins is greatly increased. Failure to fully cope with an oxidative stress can switch mitotic cells into a permanent growth-arrested, senescence-like state in which they may survive for long periods. Faced with even more severe oxidative stress, or the declining protective enzymes and adaptive capacity associated with aging, cells may "sacrifice themselves" by apoptosis, which protects surrounding healthy tissue from further damage. Only under the most severe oxidative stress conditions will cells undergo a necrotic death, which exposes surrounding tissues to the further vicissitudes of an inflammatory immune response. This remarkable array of systems for defense; damage removal, replacement, and repair; adaptation; growth modulation; and apoptosis make it possible for us to enjoy life in an oxygen-rich environment.
Article
Superoxide generated using exogenous xanthine oxidase indirectly activates an uncoupling protein (UCP)-mediated proton conductance of the mitochondrial inner membrane. We investigated whether endogenous mitochondrial superoxide production could also activate proton conductance. When respiring on succinate, rat skeletal muscle mitochondria produced large amounts of matrix superoxide. Addition of GDP to inhibit UCP3 markedly inhibited proton conductance and increased superoxide production. Both superoxide production and the GDP-sensitive proton conductance were suppressed by rotenone plus an antioxidant. Thus, endogenous superoxide can activate the proton conductance of UCP3, which in turn limits mitochondrial superoxide production. These observations provide a departure point for studies under more physiological conditions.
Article
Plasma concentrations of thyroid hormones (TH) were investigated during the extended posthatching developmental period (approximately 11 months) of a semi-altricial bird species, the king penguin (Aptenodytes patagonicus). The first period of growth in summer was marked by a progressive rise in plasma T4 concentration that paralleled rapid increases in body mass and in structural and down growth. By contrast, plasma T3 concentration had already reached adult levels in newly hatched chicks and did not change thereafter. Circulating TH of king penguin chicks thus follow an original pattern when comparing to altricial and precocial species. During the austral winter, the long period of undernutrition of king penguin chicks was characterized by a decrease in circulating TH that can be related to a seasonal stop in growth and energy saving mechanisms. Plasma TH concentrations increased again during the second growth phase in spring, and they reached their highest levels at the end of the fledging period, slightly before juveniles initiated their first foraging trip at sea. As expected, plasma T4 levels were elevated when chicks moulted, developing a true-adult type waterproof plumage. The data also suggest that T4 plays a major role in skeletal development and pectoral muscle maturation in anticipation of marine life. Plasma T3 was at its highest during the period when juveniles improved resistance to cold waters by going back and forth to the sea, suggesting a role for circulating T3 in cold acclimatization occurring at that time.
Article
Juvenile king penguins develop adaptive thermogenesis after repeated immersion in cold water. However, the mechanisms of such metabolic adaptation in birds are unknown, as they lack brown adipose tissue and uncoupling protein-1 (UCP1), which mediate adaptive non-shivering thermogenesis in mammals. We used three different groups of juvenile king penguins to investigate the mitochondrial basis of avian adaptive thermogenesis in vitro. Skeletal muscle mitochondria isolated from penguins that had never been immersed in cold water showed no superoxide-stimulated proton conductance, indicating no functional avian UCP. Skeletal muscle mitochondria from penguins that had been either experimentally immersed or naturally adapted to cold water did possess functional avian UCP, demonstrated by a superoxide-stimulated, GDP-inhibitable proton conductance across their inner membrane. This was associated with a markedly greater abundance of avian UCP mRNA. In the presence (but not the absence) of fatty acids, these mitochondria also showed a greater adenine nucleotide translocase-catalysed proton conductance than those from never-immersed penguins. This was due to an increase in the amount of adenine nucleotide translocase. Therefore, adaptive thermogenesis in juvenile king penguins is linked to two separate mechanisms of uncoupling of oxidative phosphorylation in skeletal muscle mitochondria: increased proton transport activity of avian UCP (dependent on superoxide and inhibited by GDP) and increased proton transport activity of the adenine nucleotide translocase (dependent on fatty acids and inhibited by carboxyatractylate).
Article
The cellular stress response is a universal mechanism of extraordinary physiological/pathophysiological significance. It represents a defense reaction of cells to damage that environmental forces inflict on macromolecules. Many aspects of the cellular stress response are not stressor specific because cells monitor stress based on macromolecular damage without regard to the type of stress that causes such damage. Cellular mechanisms activated by DNA damage and protein damage are interconnected and share common elements. Other cellular responses directed at re-establishing homeostasis are stressor specific and often activated in parallel to the cellular stress response. All organisms have stress proteins, and universally conserved stress proteins can be regarded as the minimal stress proteome. Functional analysis of the minimal stress proteome yields information about key aspects of the cellular stress response, including physiological mechanisms of sensing membrane lipid, protein, and DNA damage; redox sensing and regulation; cell cycle control; macromolecular stabilization/repair; and control of energy metabolism. In addition, cells can quantify stress and activate a death program (apoptosis) when tolerance limits are exceeded.
Article
Thyroid hormones (THs) have long been known to be involved in the control of thermoregulation in birds and mammals. In particular, they are reported to play a role in the regulation of heat production. The underlying mechanisms could be the stimulation of the nuclear and mitochondrial transcription of several genes involved in energy metabolism and/or a direct action on the activity of components of the mitochondrial respiratory chain. Attention has recently been focussed on a subfamily of mitochondrial anion carriers called uncoupling proteins (UCPs). These proteins are suspected to be involved in a partial dissipation of the mitochondrial proton electrochemical gradient that would uncouple phosphorylations from oxidations and hence produce heat. However, the involvement of uncoupling mechanisms in thermogenesis and particularly in the thermogenic effect of TH is still unclear. The thermogenic role of UCP1, specifically expressed in brown adipose tissue, and its regulation by TH in rodents is quite well recognised, but the involvement in heat production of its mammalian homologues UCP2, ubiquitously expressed, and UCP3, muscle and adipose tissue-specific, as well as the role of the muscular avian UCP (avUCP), are to be further investigated. The expression of the UCP2 and UCP3 genes was shown to be enhanced by TH in muscle of several rodent species, and to be increased in situations where thermogenesis is stimulated, whereas results are more contrasted in pig. There is now increasing evidence that the physiological role of the mammalian UCP3 and UCP2 is rather related to lipid oxidation and/or prevention of reactive oxygen species accumulation than to heat production by uncoupling. The expression of avUCP was also recently demonstrated to be strongly regulated by thyroid status in chicken, and overexpressed in experimental conditions favouring high triiodothyronine concentrations and thermogenesis. However, its real uncoupling activity and contribution to thermogenesis remain to be established.
Article
The precise mechanistic sequence producing the beneficial effects on health and lifespan seen with interventions as diverse as caloric restriction, intermittent fasting, exercise, and consumption of dietary phytonutrients is still under active characterization, with large swaths of the research community kept in relative isolation from one another. Among the explanatory models capable of assisting in the identification of precipitating elements responsible for beneficial influences on physiology seen in these states, the hormesis perspective on biological systems under stress has yielded considerable insight into likely evolutionarily consistent organizing principles functioning in all four conditions. Recent experimental findings provide the tantalizing initial lodestones for an entirely new research front examining molecular substrates of stress resistance. In this novel body of research, a surprising new twist has emerged: Reactive oxygen species, derived from the mitochondrial electron transport system, may be necessary triggering elements for a sequence of events that result in benefits ranging from the transiently cytoprotective to organismal-level longevity. With the recent appreciation that reactive oxygen species and reactive nitrogen species function as signaling elements in a interconnected matrix of signal transduction, the entire basis of many widely accepted theories of aging that predominated in the past may need to be reconsidered to facilitate the formulation of an new perspective more correctly informed by the most contemporaneous experimental findings. This perspective, the mitohormesis theory, can be used in many disparate domains of inquiry to potentially explain previous findings, as well as point to new targets of research. The utility of this perspective for research on aging is significant, but beyond that this perspective emphasizes the pressing need to rigorously characterize the specific contribution of the stoichiometry of reactive oxygen species and reactive nitrogen species in the various compartments of the cell to cytoprotection and vitality. Previous findings regarding the influences of free radical chemistry on cellular physiology may have represented assessments examining the consequences of isolated elevation of signaling elements within a larger signal transductive apparatus, rather than definitive characterizations of the only modality of reactive oxygen species (and reactive nitrogen species) influence. In applying this perspective, it may be necessary for the research community, as well as the practicing clinician, to engender a more sanguine perspective on organelle level physiology, as it is now plausible that such entities have an evolutionarily orchestrated capacity to self-regulate that may be pathologically disturbed by overzealous use of antioxidants, particularly in the healthy.
Article
Diving seals experience heart rate reduction and preferential distribution of the oxygenated blood flow to the heart and brain, widespread peripheral vasoconstriction, and selective ischemia in the most hypoxia-tolerant tissues. The first breath after the dive restores the oxygenated blood flow to all tissues and raises the potential for the production of reactive oxygen species (ROS). We hypothesized that in order to counteract the damaging effects of ROS and to tolerate repetitive cycles of ischemia/reperfusion associated with diving, ringed seal (Phoca hispida) tissues have elevated activities of antioxidant enzymes. Activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) were measured by spectrophotometric techniques in heart, kidney, liver, lung, and muscle extracts of ringed seals and domestic pigs (Sus scrofa). The results suggest that in ringed seal heart SOD, GPx and GST activities are an efficient protective mechanism for counteracting ROS production and its deleterious effects. Apparently CAT activity in seal liver and GPx activity in seal muscle participate in the removal of hydroperoxides, while seal lung appears to be protected from oxidative damage by SOD and GPx activities.
Article
Life is the interplay between structure and energy, yet the role of energy deficiency in human disease has been poorly explored by modern medicine. Since the mitochondria use oxidative phosphorylation (OXPHOS) to convert dietary calories into usable energy, generating reactive oxygen species (ROS) as a toxic by-product, I hypothesize that mitochondrial dysfunction plays a central role in a wide range of age-related disorders and various forms of cancer. Because mitochondrial DNA (mtDNA) is present in thousands of copies per cell and encodes essential genes for energy production, I propose that the delayed-onset and progressive course of the age-related diseases results from the accumulation of somatic mutations in the mtDNAs of post-mitotic tissues. The tissue-specific manifestations of these diseases may result from the varying energetic roles and needs of the different tissues. The variation in the individual and regional predisposition to degenerative diseases and cancer may result from the interaction of modern dietary caloric intake and ancient mitochondrial genetic polymorphisms. Therefore the mitochondria provide a direct link between our environment and our genes and the mtDNA variants that permitted our forbears to energetically adapt to their ancestral homes are influencing our health today.
Article
The problem of identifying differentially expressed genes in designed microarray experiments is considered. Lonnstedt and Speed (2002) derived an expression for the posterior odds of differential expression in a replicated two-color experiment using a simple hierarchical parametric model. The purpose of this paper is to develop the hierarchical model of Lonnstedt and Speed (2002) into a practical approach for general microarray experiments with arbitrary numbers of treatments and RNA samples. The model is reset in the context of general linear models with arbitrary coefficients and contrasts of interest. The approach applies equally well to both single channel and two color microarray experiments. Consistent, closed form estimators are derived for the hyperparameters in the model. The estimators proposed have robust behavior even for small numbers of arrays and allow for incomplete data arising from spot filtering or spot quality weights. The posterior odds statistic is reformulated in terms of a moderated t-statistic in which posterior residual standard deviations are used in place of ordinary standard deviations. The empirical Bayes approach is equivalent to shrinkage of the estimated sample variances towards a pooled estimate, resulting in far more stable inference when the number of arrays is small. The use of moderated t-statistics has the advantage over the posterior odds that the number of hyperparameters which need to estimated is reduced; in particular, knowledge of the non-null prior for the fold changes are not required. The moderated t-statistic is shown to follow a t-distribution with augmented degrees of freedom. The moderated t inferential approach extends to accommodate tests of composite null hypotheses through the use of moderated F-statistics. The performance of the methods is demonstrated in a simulation study. Results are presented for two publicly available data sets.