The physiological consequences of a very large natural meal in a voracious marine fish, the staghorn sculpin (Leptocottus armatus).

Little information exists on physiological consequences when wild fish eat natural food. Staghorn sculpins at 10-13°C voluntarily consumed 15.8% of their body mass in anchovies. Gastric clearance was slow with >60% of the meal retained in the stomach at 48 h, and was not complete until 84 h. At 14-24 h post-feeding, pH was depressed by 3 units and Cl- concentration was elevated 2-fold in gastric chyme, reflecting HCl secretion, while in all sections of the intestine, pH declined by 1 pH unit but Cl- concentration remained unchanged. PCO2 and total ammonia concentration were greatly elevated throughout the tract, whereas PNH3 and HCO3- concentration were depressed. Intestinal HCO3- secretion rates, measured in gut sacs in vitro, were also lower in fed fish. Whole-animal O2 consumption rate was elevated approximately 2-fold for 72 h post-feeding, reflecting 'specific dynamic action', whereas ammonia and urea-N excretion rates were elevated about 5-fold. Arterial blood exhibited a modest 'alkaline tide' for about 48 h, but there was negligible excretion of metabolic base to the external seawater. PaCO2 and PaO2 remained unchanged. Plasma total amino acid concentration and total lipid concentration were elevated about 1.5-fold for at least 48 h, whereas small increases in plasma total ammonia concentration, PNH3 and urea-N concentration were quickly attenuated. Plasma glucose concentration remained unchanged. We conclude that despite the very large meal, slow processing with high efficiency minimizes internal physiological disturbances. This differs greatly from the picture provided by previous studies on aquacultured species using synthetic diets and/or force-feeding. Questions remain about the role of the gastro-intestinal microbiome in nitrogen and acid-base metabolism.

Temperature and pressure dependency of oxygen consumption during long-term sustained swimming of European eels.

Many aspects of the typically 5000-10,000 km spawning migration of the European eel (Anguilla anguilla) remain unknown. As part of this migration, eels undertake extensive diurnal vertical migrations to depths below 1000 m, being exposed to a wide range of temperatures and hydrostatic pressures. In this experimental study, we exposed eels to different combinations of temperature (12-20°C) and pressure (100--800 kPa) during long-term sustained swimming (32-47 days). Both temperature and pressure affected oxygen consumption rate, such that there was a significant increase of metabolic rate with temperature, whereas pressure reduced oxygen consumption, albeit only at higher temperatures. Average oxygen consumption rates ranged between 15 mg kg-1 h-1 (12°C, 100 kPa) and 30.2 mg kg-1 h-1 (20°C, 100 kPa), highlighting the remarkably high swimming efficiency of this species and, more importantly, indicating that past evaluations of the cost of transport are potentially overestimates as they are often based on experiments conducted at atmospheric pressure at higher temperatures.

The rete mirabile: a possible control site for swimbladder function.

In a recent study, a large number of transport proteins was detected in the transcriptome and proteome of saline perfused rete mirabile tissue of the European eel. In this study, the data set was reanalyzed for the presence of receptor proteins and proteins involved in intracellular signaling pathways. A large number of expressed receptor proteins and proteins involved in intracellular signal transduction was detected. Several G-protein-coupled receptor signal pathways were significantly enriched in their expression level, in particular receptors and signaling pathways involved in the control of blood flow. The enriched signaling pathways also include pathways involved in trafficking of crucial transport proteins like, monocarboxylate transporters, V-ATPase, and aquaporin. The data, therefore, suggest that the rete mirabile has the capacity to control swimbladder function by regulating blood flow and by modifying countercurrent multiplication.

Oxygen consumption and acid secretion in isolated gas gland cells of the European eel Anguilla anguilla.

Swimbladder gas gland cells are known to produce lactic acid required for the acidification of swimbladder blood and decreasing the oxygen carrying capacity of swimbladder blood, i.e., the onset of the Root effect. Gas gland cells have also been shown to metabolize glucose via the pentose phosphate shunt, but the role of the pentose phosphate shunt for acid secretion has not yet been evaluated. Similarly, aerobic metabolism of gas gland cells has been largely neglected so far. In the present study, we therefore simultaneously assessed the role of glycolysis and of the pentose phosphate shunt for acid secretion and recorded oxygen consumption of isolated swimbladder gas gland cells of the European eel. Presence of glucose was essential for acid secretion, and at glucose concentrations of about 1.5 mmol l-1 acid secretion of gas gland cells reached a maximum, indicating that glucose concentrations in swimbladder blood should not be limiting acid production and secretion under physiological conditions. The data revealed that most of the acid was produced in the glycolytic pathway, but a significant fraction was also contributed by the pentose phosphate shunt. Addition of glucose to gas gland cells incubated in a glucose-free medium resulted in a reduction of oxygen uptake. Inhibition of mitochondrial respiration significantly reduced oxygen consumption, but a fraction of mitochondria-independent respiration remained in presence of rotenone and antimycin A. In the presence of glucose, application of either iodo-acetate inhibiting glycolysis or 6-AN inhibiting the pentose phosphate shunt did not significantly affect oxygen uptake, indicating an independent regulation of oxidative phosphorylation and of acid production. Inhibition of the muscarinic acetylcholine receptor caused a slight elevation in acid secretion, while forskolin caused a concentration-dependent reduction in acid secretion, indicating muscarinic and c-AMP-dependent control of acid secretion in gas gland cells.

Aquaporin expression and cholesterol content in eel swimbladder tissue.

Leakiness of the swimbladder wall of teleost fishes must be prevented to avoid diffusional loss of gases out of the swimbladder. Guanine incrustation as well as high concentrations of cholesterol in swimbladder membranes in midwater and deep-sea fish has been connected to a reduced gas permeability of the swimbladder wall. On the contrary, the swimbladder is filled by diffusion of gases, mainly oxygen and CO2 , from the blood and the gas gland cells into the swimbladder lumen. In swimbladder tissue of the zebrafish and the Japanese eel, aquaporin mRNA has been detected, and the aquaporin protein has been considered important for the diffusion of water, which may accidentally be gulped by physostome fish when taking an air breath. In the present study, the expression of two aquaporin 1 genes (Aqp1aa and Aqp1ab) in the swimbladder tissue of the European eel, a functional physoclist fish, was assessed using immunohistochemistry, and the expression of both genes was detected in endothelial cells of swimbladder capillaries as well as in basolateral membranes of gas gland cells. In addition, Aqp1ab was present in apical membranes of swimbladder gas gland cells. The authors also found high concentrations of cholesterol in these membranes, which were several fold higher than in muscle tissue membranes. In yellow eels the cholesterol concentration exceeded the concentration detected in silver eel swimbladder membranes. The authors suggest that aquaporin 1 in swimbladder gas gland cells and endothelial cells facilitates CO2 diffusion into the blood, enhancing the switch-on of the Root effect, which is essential for the secretion of oxygen into the swimbladder. It may also facilitate CO2 diffusion into the swimbladder lumen along the partial gradient established by CO2 production in gas gland cells. Cholesterol has been shown to reduce the gas permeability of membranes and thus could contribute to the gas tightness of swimbladder membranes, which is essential to avoid diffusional loss of gas out of the swimbladder.

A novel hyperbaric swimming respirometer allows the simulation of varying swimming depths in fish respirometry studies.

The understanding of swimming physiology and knowledge on the metabolic costs of swimming are important for assessing effects of environmental factors on migratory behavior. Swim tunnels are the most common experimental setups for measuring swimming performance and oxygen uptake rates in fishes; however, few can realistically simulate depth and the changes in hydrostatic pressure that many fishes experience, e.g. during diel vertical migrations. Here, we present a new hyperbaric swimming respirometer (HSR) that can simulate depths of up to 80 m. The system consists of three separate, identical swimming tunnels, each with a volume of 205 L, a control board and a storage tank with water treatment. The swimming chamber of each tunnel has a length of 1.40 m and a diameter of 20 cm. The HSR uses the principle of intermittent-flow respirometry and has here been tested with female European eels (Anguilla anguilla). Various pressure, temperature and flow velocity profiles can be programmed, and the effect on metabolic activity and oxygen consumption can be assessed. Thus, the HSR provides opportunities to study the physiology of fish during swimming in a simulated depth range that corresponds to many inland, coastal and shelf waters.

A first attempt at a holistic analysis of various influencing factors on the fish fauna in the Eastern European Alps.

Fish are some of the most threatened vertebrates in the world due to their often-sensitive response to environmental changes. Major land-use changes in the European Alps have direct and indirect impacts on fish communities, and these impacts are expected to increase in the future. Therefore, the identification of factors that are associated with the distribution of fish communities is of great importance to develop guidelines for management, precautions and sustainable use of running waters. In this study, the relationship of various factors - landscape structure and land use, topography, morphology, hydrology, physical and chemical water characteristics, hormonally active substances, pesticides, food availability, fisheries and piscivores birds - with fish assemblages are analysed. Field data from 81 stream sections from 2001 metres above sea level (m.a.s.l.) down to 219 m.a.s.l. are used in the study. The results reveal that the number of fish species has a strong association with topographic characteristics in the catchment area as well as with landscape configuration. Fish abundance and biomass are associated mostly with land-use type, hydrology, morphology as well as topography. In addition, there are indirect connections between fish abundance and biomass through land-use type, topography, water properties and hydromorphology. The results clearly indicate that not a single factor, but a multitude of factors are associated with the fish communities in the Eastern European Alps.

Expression of transport proteins in the rete mirabile of european silver and yellow eel.

BACKGROUND: In physoclist fishes filling of the swimbladder requires acid secretion of gas gland cells to switch on the Root effect and subsequent countercurrent concentration of the initial gas partial pressure increase by back-diffusion of gas molecules in the rete mirabile. It is generally assumed that the rete mirabile functions as a passive exchanger, but a detailed analysis of lactate and water movements in the rete mirabile of the eel revealed that lactate is diffusing back in the rete. In the present study we therefore test the hypothesis that expression of transport proteins in rete capillaries allows for back-diffusion of ions and metabolites, which would support the countercurrent concentrating capacity of the rete mirabile. It is also assumed that in silver eels, the migratory stage of the eel, the expression of transport proteins would be enhanced. RESULTS: Analysis of the transcriptome and of the proteome of rete mirabile tissue of the European eel revealed the expression of a large number of membrane ion and metabolite transport proteins, including monocarboxylate and glucose transport proteins. In addition, ion channel proteins, Ca2+-ATPase, Na+/K+-ATPase and also F1F0-ATP synthase were detected. In contrast to our expectation in silver eels the expression of these transport proteins was not elevated as compared to yellow eels. A remarkable number of enzymes degrading reactive oxygen species (ROS) was detected in rete capillaries. CONCLUSIONS: Our results reveal the expression of a large number of transport proteins in rete capillaries, so that the back diffusion of ions and metabolites, in particular lactate, may significantly enhance the countercurrent concentrating ability of the rete. Metabolic pathways allowing for aerobic generation of ATP supporting secondary active transport mechanisms are established. Rete tissue appears to be equipped with a high ROS defense capacity, preventing damage of the tissue due to the high oxygen partial pressures generated in the countercurrent system.

Using the swimbladder as a respiratory organ and/or a buoyancy structure-Benefits and consequences.

A swimbladder is a special organ present in several orders of Actinopterygians. As a gas-filled cavity it contributes to a reduction in overall density, but on descend from the water surface its contribution as a buoyancy device is very limited because the swimbladder is compressed by increasing hydrostatic pressure. It serves, however, as a very efficient organ for aerial gas exchange. To avoid the loss of oxygen to hypoxic water at the gills many air-breathing fish show a reduced gill surface area. This, in turn, also reduces surface area available for other functions, so that breathing air is connected to a number of physiological adjustments with respect to ion homeostasis, acid-base regulation and nitrogen excretion. Using the swimbladder as a buoyancy structure resulted in the loss of its function as an air-breathing organ and required the development of a gas secreting mechanism. This was achieved via the Root effect and a countercurrent arrangement of the blood supply to the swimbladder. In addition, a detachable air space with separated blood supply was necessary to allow the resorption of gas from the swimbladder. Gas secretion as well as gas resorption are slow phenomena, so that rapid changes in depth cannot instantaneously be compensated by appropriate volume changes. As gas-filled cavities the respiratory swimbladder and the buoyancy device require surfactant. Due to high oxygen partial pressures inside the bladder air-exposed tissues need an effective reactive oxygen species defense system, which is particularly important for a swimbladder at depth.

Swimming under elevated hydrostatic pressure increases glycolytic activity in gas gland cells of the European eel.

In spite of many decades of research, the spawning migration of the European eel Anguilla anguilla from the European coast to the Sargasso Sea remains a mystery. In particular, the role of the swimbladder as a buoyancy regulating structure is not yet understood. In this study, we exercised silver eels in a swim tunnel under elevated hydrostatic pressure. The transcriptome of gas gland tissue of these exercised eels was then compared to the known transcriptome of not exercised (control) silver eel gas gland cells. Due to the high infection rate of the eel population with the swimbladder parasite Anguillicola crassus, the comparison also included an exercised group of silver eels with a heavily damaged swimbladder, and we compared the previously published transcriptome of not exercised silver eels with a highly damaged swimbladder with the exercised group of silver eels with a heavily damaged swimbladder. The comparisons of unexercised (control) silver eels with exercised silver eels with functional swimbladder (EF), as well as with exercised silver eels with damaged swimbladder (ED), both showed a significant elevation in transcripts related to glycolytic enzymes. This could also be observed within the comparison of unexercised silver eels with a highly infected swimbladder with exercised eels with a damaged swimbladder (DED). In contrast to EF, in ED a significant elevation in transcript numbers of mitochondrial NADH dehydrogenase was observed. While in EF the transcriptional changes suggested that acid production and secretion was enhanced, in ED these changes appeared to be related to thickened tissue and thus elevated diffusion distances. The remarkable number of differentially expressed transcripts coding for proteins connected to cAMP-dependent signaling pathways indicated that metabolic control in gas gland cells includes cAMP-dependent pathways. In contrast to ED, in EF significant transcriptional changes could be related to the reconstruction of the extracellular matrix, while in ED tissue repair and inflammation was more pronounced. Surprisingly, in exercised eels hypoxia inducible transcription factor expression was elevated. In EF, a large number of genes related to the circadian clock were transcriptionally modified, which may be connected to the circadian vertical migrations observed during the spawning migration.

Gills versus kidney for ionoregulation in the obligate air-breathing Arapaima gigas, a fish with a kidney in its air-breathing organ.

In Arapaima gigas, an obligate air-breather endemic to ion-poor Amazonian waters, a large complex kidney runs through the air-breathing organ (ABO). Previous indirect evidence suggested that the kidney, relative to the small gills, may be exceptionally important in ionoregulation and nitrogen (N) waste excretion, with support of kidney function by direct O2 supply from the airspace. We tested these ideas by continuous urine collection and gill flux measurements in ∼700 g fish. ATPase activities were many-fold greater in kidney than gills. In normoxia, gill Na+ influx and efflux were in balance, with net losses of Cl- and K+ Urine flow rate (UFR, ∼11 ml kg-1 h-1) and urinary ions (< 0.2 mmol l-1) were exceptional, with [urine]:[plasma] ratios of 0.02-0.002 for K+, Na+, and Cl-, indicating strong reabsorption with negligible urinary ion losses. Urinary [ammonia] was very high (10 mmol l-1, [urine]:[plasma] ∼17) indicating strong secretion. The kidney accounted for 21-24% of N excretion, with ammonia dominating (95%) over urea-N through both routes. High urinary [ammonia] was coupled to high urinary [HCO3-]. Aerial hypoxia (15.3 kPa) and aerial hyperoxia (>40.9 kPa) had no effects on UFR, but both inhibited branchial Na+ influx, revealing novel aspects of the osmorespiratory compromise. Aquatic hypoxia (4.1 kPa), but not aquatic hyperoxia (>40.9 kPa), inhibited gill Na+ influx, UFR and branchial and urinary ammonia excretion. We conclude that the kidney is more important than gills in ionoregulation, and is significant in N excretion. Although not definitive, our results do not indicate direct O2 supply from the ABO for kidney function.

Cellular oxygen consumption, ROS production and ROS defense in two different size-classes of an Amazonian obligate air-breathing fish (Arapaima gigas).

In air-breathing fish a reduction of gill surface area reduces the danger of losing oxygen taken up in the air-breathing organ (ABO) to hypoxic water, but it also reduces the surface area available for ion exchange, so that ion regulation may at least in part be transferred to other organs, like the kidney or the gut. In the air-breathing Arapaima gigas, gill lamellae regress as development proceeds, and starting as a water-breathing embryo Arapaima turns into an obligate air-breathing fish with proceeding development, suggesting that ion regulation is shifted away from the gills as the fish grows. In Arapaima the kidney projects medially into the ABO and thus, probably a unique situation among fishes, is in close contact to the gas of the ABO. We therefore hypothesized that the kidney would be predestined to adopt an increased importance for ion homeostasis, because the elevated ATP turnover connected to ion transport can easily be met by aerobic metabolism based on the excellent oxygen supply directly from the ABO. We also hypothesized that in gill tissue the reduced ion regulatory activity should result in a reduced metabolic activity. High metabolic activity and exposure to high oxygen tensions are connected to the production of reactive oxygen species (ROS), therefore the tissues exposed to these conditions should have a high ROS defense capacity. Using in vitro studies, we assessed metabolic activity and ROS production of gill, kidney and ABO tissue, and determined the activity of ROS degrading enzymes in small (~ 5g, 2-3 weeks old) and larger (~ 670 g, 3-4 months old) A. gigas. Comparing the three tissues revealed that kidney tissue oxygen uptake by far exceeded the uptake measured in gill tissue or ABO. ROS production was particularly high in gill tissue, and all three tissues had a high capacity to degrade ROS. Gill tissue was characterized by high activities of enzymes involved in the glutathione pathway to degrade ROS. By contrast, the tissues of the ABO and in particular the kidney were characterized by high catalase activities, revealing different, tissue-specific strategies in ROS defense in this species. Overall the differences in the activity of cells taken from small and larger fish were not as pronounced as expected, while at the tissue level the metabolic activity of kidney cells by far exceeded the activity of ABO and gill cells.

Gills and air-breathing organ in O2 uptake, CO2 excretion, N-waste excretion, and ionoregulation in small and large pirarucu (Arapaima gigas).

In the pirarucu (Arapaima gigas), gill surface area and thus gas exchange capacity of the gills are reduced with proceeding development. It, therefore, is expected that A. gigas, starting as a water breather, progressively turns into an obligate air-breathing fish using an air-breathing organ (ABO) for gas exchange. We assessed the air-breathing activity, O2 and CO2 exchange into air and water, ammonia-N and urea-N excretion, ion flux rates, and activities of ion transport ATPases in large versus small pirarucu. We found that even very young A. gigas (4-6 g, 2-3 weeks post-hatch) with extensive gills are air-breathers (18.1 breaths*h-1) and cover most (63%) of their O2 requirements from the air whereas 600-700-g animals (about 3-4 months post-hatch), with reduced gills, obtain 75% of their O2 from the air (10.8 breaths*h-1). Accordingly, the reduction in gill surface area hardly affected O2 uptake, but development had a significant effect on aerial CO2 excretion, which was very low (3%) in small fish and increased to 12% in larger fish, yielding a hyper-allometric scaling coefficient (1.12) in contrast to 0.82-0.84 for aquatic and total CO2 excretion. Mass-specific ammonia excretion decreased in approximate proportion to mass-specific O2 consumption as the fish grew, but urea-N excretion dropped from 18% (at 4-6 g) to 8% (at 600-700 g) of total N-excretion; scaling coefficients for all these parameters were 0.70-0.80. Mass-specific sodium influx and efflux rates, as well as potassium net loss rates, departed from this pattern, being greater in larger fish; hyper-allometric scaling coefficients were > 1.0. Gill V-type H+ ATPase activities were greater than Na+, K+-ATPase activities, but levels were generally low and comparable in large and small fish, and similar activities were detected in the ABO. A. gigas is a carnivorous fish throughout its lifecycle, and, despite fasting, protein oxidation accounted for the major portion (61-82%) of aerobic metabolism in both large and small animals. ABO PO2 and PCO2 (measured in 600-700-g fish) were quite variable, and aerial hypoxia resulted in lower ABO PO2 values. Under normoxic conditions, a positive correlation between breath volume and ABP PO2 was detected, and on average with a single breath more than 50% of the ABO volume was exchanged. ABO PCO2 values were in the range of 1.95-3.89 kPa, close to previously recorded blood PCO2 levels. Aerial hypoxia (PO2 down to 12.65 kPa) did not increase either air-breathing frequency or breath volume.

Metabolic interaction of hydrogen peroxide and hypoxia in zebrafish fibroblasts.

Cells require oxygen for aerobic metabolism, which may also result in the production of reactive oxygen species (ROS) as a by-product. Under low oxygen conditions, ROS formation has been reported to either increase or decrease. We addressed this physiological response for the first time in zebrafish embryonic fibroblasts (Z3) and used a hydrogen peroxide (H2O2)-specific fluorescent protein (roGFP2-Orp1) either targeted to the mitochondria or expressed in the cytosol. Microfluidic live-cell imaging measurements showed that oxygen deprivation in Z3 cells results in decreased or stable H2O2 levels within the mitochondria or the cytosol, respectively, and that the reductive shift recorded in the mitochondrial matrix is directly dependent on oxygen concentration. The response was accompanied by a transient increase in extracellular acidification rate (ECAR) and a lower cellular reducing potential as assessed by the viability stain alamarBlue. Complex I and III inhibition with Rotenone and Antimycin A led to H2O2 production under normoxia but these inhibitors were not able to avert the reductive shift under hypoxia. Only by system-wide inhibition of flavin-containing oxidases with Diphenyleneiodonium (DPI) were we able to decrease the reductive shift, while selective inhibition of NADPH oxidases with the inhibitor Apocynin had no effect on the hypoxia response. Since DPI also led to a strong increase in ECAR we found that, in order to keep the cytosolic H2O2 levels stable, glycolytic metabolism was of fundamental importance. According to our experiments with the glucose-6-phosphate dehydrogenase inhibitor 6-Aminonicotinamide, this was attributable to the pentose phosphate pathway producing reducing equivalents required for ROS degradation.

Author Correction: Rapid de novo assembly of the European eel genome from nanopore sequencing reads.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Excessive inflammation impairs heart regeneration in zebrafish breakdance mutant after cryoinjury.

Inflammation plays a crucial role in cardiac regeneration. Numerous advantages, including a robust regenerative ability, make the zebrafish a popular model to study cardiovascular diseases. The zebrafish breakdance (bre) mutant shares several key features with human long QT syndrome that predisposes to ventricular arrhythmias and sudden death. However, how inflammatory response and tissue regeneration following cardiac damage occur in bre mutant is unknown. Here, we have found that inflammatory response related genes were markedly expressed in the injured heart and excessive leukocyte accumulation occurred in the injured area of the bre mutant zebrafish. Furthermore, bre mutant zebrafish exhibited aberrant apoptosis and impaired heart regenerative ability after ventricular cryoinjury. Mild dosages of anti-inflammatory or prokinetic drugs protected regenerative cells from undergoing aberrant apoptosis and promoted heart regeneration in bre mutant zebrafish. We propose that immune or prokinetic therapy could be a potential therapeutic regimen for patients with genetic long QT syndrome who suffers from myocardial infarction.

Ionoregulatory and oxidative stress issues associated with the evolution of air-breathing.

Aquatic areas frequently face hypoxic conditions. In order to get sufficient oxygen to support aerobic metabolism, a number of freshwater fish resort to aerial respiration to supplement gill respiration especially in situations with reduced oxygen availability in the water. In many species a concomitant reduction in gill surface area or in gill perfusion reduces possible loss of aerially acquired oxygen to the water at the gills, but it also compromises the ion regulatory capacity of gill tissue. In consequence, the reduced gill contact area with water requires appropriate compensation to maintain ion and acid-base homeostasis, often with important ramifications for other organs. Associated modifications in the structure and function of the gills themselves, the skin, the gut, the kidney, and the physiology of water exchange and ion-linked acid-base regulation are discussed. In air-breathing fish, the gut may gain particular importance for the uptake of ions. In addition, tissues frequently exposed to environmental air encounter much higher oxygen partial pressures than typically observed in fish tissues. Physostomous fish using the swimbladder for aerial respiration, for example, will encounter aerial oxygen partial pressure at the swimbladder epithelium when frequently gulping air in hypoxic water. Hyperoxic conditions or rapid changes in oxygen partial pressures result in an increase in the production of reactive oxygen species (ROS). Accordingly, in air-breathing fish, strategies of ionoregulation may be greatly modified, and the ROS defense capacity of air-exposed tissues is improved.

Hypoxia-inducible transcription factors in fish: expression, function and interconnection with the circadian clock.

The hypoxia-inducible transcription factors are key regulators for the physiological response to low oxygen availability. In vertebrates, typically three Hif-α isoforms, Hif-1α, Hif-2α and Hif-3α, are expressed, each of which, together with Hif-1ß, may form a functional heterodimer under hypoxic conditions, controlling expression of hundreds of genes. A teleost-specific whole-genome duplication complicates the analysis of isoform-specific functions in fish, but recent studies suggest that the existence of paralogues of a specific isoform opens up the possibility for a subfunctionalization. In contrast to during development inside the uterus, fish eggs are freely accessible and studies analyzing Hif expression in fish embryos during development have revealed that Hif proteins are not only controlling the hypoxic response, but are also crucial for proper development and organ differentiation. Significant advances have been made in our knowledge about tissue-specific functions of Hif proteins, especially with respect to gill or gonadal tissue. The hypoxia signalling pathway is known to be tightly and mutually intertwined with the circadian clock in zebrafish and mammals. Recently, a mechanistic explanation for the hypoxia-induced dampening of the transcriptional clock was detected in zebrafish, including also metabolically induced alterations of cellular redox signalling. In turn, MAP kinase-mediated H2O2 signalling modulates the temporal expression of Hif-1α protein, similar to the redox regulation of the circadian clock itself. Once again, the zebrafish has emerged as an excellent model organism with which to explore these specific functional aspects of basic eukaryotic cell biology.

Metabolic Plasticity Enables Circadian Adaptation to Acute Hypoxia in Zebrafish Cells.

BACKGROUND/AIMS: Reduced oxygen availability, hypoxia, is frequently encountered by organisms, tissues and cells, in aquatic environments as well as in high altitude or under pathological conditions such as infarct, stroke or cancer. The hypoxic signaling pathway was found to be mutually intertwined with circadian timekeeping in vertebrates and, as reported recently, also in mammals. However, the impact of hypoxia on intracellular metabolic oscillations is still unknown. METHODS: For determination of metabolites we used Multilabel Reader based fluorescence and luminescence assays, circadian levels of Hypoxia Inducible Factor 1 alpha and oxidized peroxiredoxins were semi quantified by Western blotting and ratiometric quantification of cytosolic and mitochondrial H2O2 was achieved with stable transfections of a redox sensitive green fluorescent protein sensor into zebrafish fibroblasts. Circadian oscillations of core clock gene mRNA´s were assessed using realtime qPCR with subsequent cosine wave fit analysis. RESULTS: Here we show that under normoxia primary metabolic activity of cells predominately occurs during day time and that after acute hypoxia of two hours, administrated immediately before each sampling point, steady state concentrations of glycolytic key metabolites such as glucose and lactate reveal to be highly rhythmic, following a circadian pattern with highest levels during the night periods and reflecting the circadian variation of the cellular response to hypoxia. Remarkably, rhythms in glycolysis are transferred to cellular energy states under normoxic conditions, so that ADP/ATP ratios oscillate as well, which is the first evidence for cycling ADP/ATP pools in a metazoan cell line to our knowledge. Furthermore, the hypoxia induced alterations in rhythms of glycolysis lead to the alignment of three major cellular redox systems, namely the circadian oscillations of NAD+/NADH and NADP+/NADPH ratios and of increased nocturnal levels of oxidized peroxiredoxins, resulting in a highly oxidized nocturnal cellular environment. Of note, circadian rhythms of cytosolic H2O2 remain unaltered, while the transcriptional clock is already attenuated, as it is known to occur also under chronic hypoxia. CONCLUSION: We therefor propose that the realignment of metabolic redox oscillations might initiate the observed hypoxia induced attenuation of the transcriptional clock, based on the reduced binding affinity of the CLOCK/BMAL complex to the DNA in an oxidized environment.