Protein synthesis is lowered by 4EBP1 and eIF2-α signaling while protein degradation may be maintained in fasting, hypoxic Amazonian cichlids Astronotus ocellatus.

The Amazonian cichlid Astronotus ocellatus is highly tolerant to hypoxia, and is known to reduce its metabolic rate by reducing the activity of energetically expensive metabolic processes when oxygen is lacking in its environment. Our objectives were to determine how protein metabolism is regulated in A. ocellatus during hypoxia. Fish were exposed to a stepwise decrease in air saturation (100%, 20%, 10% and 5%) for 2 h at each level, and sampled throughout the experiment. A flooding dose technique using a stable isotope allowed us to observe an overall decrease in protein synthesis during hypoxia in liver, muscle, gill and heart. We estimate that this decrease in rates of protein synthesis accounts for a 20 to 36% decrease in metabolic rate, which would enable oscars to maintain stable levels of ATP and prolong survival. It was also determined for the first time in fish that a decrease in protein synthesis during hypoxia is likely controlled by signaling molecules (4EBP1 and eIF2-α), and not simply due to a lack of ATP. We could not detect any effects of hypoxia on protein degradation as the levels of NH4 excretion, indicators of the ubiquitin proteasome pathway, and enzymatic activities of lysosomal and non-lysosomal proteolytic enzymes were maintained throughout the experiment.

White muscle 20S proteasome activity is negatively correlated to growth rate at low temperature in the spotted wolffish Anarhichas minor.

The effect of temperature and mass on specific growth rate (G) was examined in spotted wolffish Anarhichas minor of different size classes (ranging from 60 to 1500 g) acclimated at different temperatures (4, 8 and 12 degrees C). The relationship between G and 20S proteasome activity in heart ventricle, liver and white muscle tissue was then assessed in fish acclimated at 4 and 12 degrees C to determine if protein degradation via the proteasome pathway could be imposing a limitation on somatic growth. Cardiac 20S proteasome activity was not affected by acclimation temperature nor fish mass and had no correlation with G. Hepatic 20S proteasome activity was higher at 12 degrees C but did not show any relationship with G. Partial correlation analysis showed that white muscle 20S proteasome activity was negatively correlated to G (partial Pearson's r = -0.609) but only at cold acclimation temperature (4 degrees C). It is suggested that acclimation to cold temperature involves compensation of the mitochondrial oxidative capacity which would in turn lead to increased production of oxidatively damaged proteins that are degraded by the proteasome pathway and ultimately negatively affects G at cold temperature.

Rainbow smelt (Osmerus mordax) genomic library and EST resources.

Genomic resources in rainbow smelt (Osmerus mordax) enable us to examine the genome duplication process in salmonids and test hypotheses relating to the fate of duplicated genes. They further enable us to pursue physiological and ecological studies in smelt. A bacterial artificial chromosome library containing 52,410 clones with an average insert size of 146 kb was constructed. This library represents an 11-fold average coverage of the rainbow smelt (O. mordax) genome. In addition, several complementary deoxyribonucleic acid libraries were constructed, and 36,758 sequences were obtained and combined into 12,159 transcripts. Over half of these transcripts have been identified, several of which have been associated with cold adaptation. These basic resources show high levels of similarity (86%) to salmonid genes and provide initial support for genome duplication in the salmonid ancestor. They also facilitate identification of genes important to fish and direct us toward new technologies for other studies in fish biology.

Taurine depresses cardiac contractility and enhances systemic heart glucose utilization in the cuttlefish, Sepia officinalis.

Taurine is the most abundant amino acid in the blood of the cuttlefish, Sepia officinalis, where levels can exceed 200 mmol L(-1). In mammals, intracellular taurine modulates cardiac Ca(2+) handling and carbohydrate metabolism at much lower concentrations but it is not clear if it exerts similar actions in cephalopods. Blood Ca(2+) levels are high in cephalopods and we hypothesized that taurine would depress cardiac Ca(2+) flux and modulate contractility in systemic and branchial hearts of cuttlefish. Heart performance was assessed with an in situ perfused systemic heart preparation and contractility was evaluated using isometrically contracting systemic and branchial heart muscle rings. Stroke volume, cardiac output, and Ca(2+) sensitivity were significantly lower in systemic hearts perfused with supplemental taurine (100 mmol L(-1)) than in controls. In muscle ring preparations, taurine impaired relaxation at high contraction frequencies, an effect abolished by supra-physiological Ca(2+) levels. Taurine did not affect oxygen consumption in non-contracting systemic heart muscle, but extracellular glucose utilization was twice that of control preparations. Collectively, our results suggest that extracellular taurine depresses cardiac Ca(2+) flux and potentiates glucose utilization in cuttlefish. Variations in taurine levels may represent an important mechanism for regulating cardiovascular function and metabolism in cephalopods.

A rapid and convenient method for measuring the fractional rate of protein synthesis in ectothermic animal tissues using a stable isotope tracer.

A method was devised to measure the fractional rate of protein synthesis in fish using a stable isotope labelled tracer (ring-D5-phenylalanine) instead of radioactive phenylalanine. This modified flooding dose technique utilizes gas chromatography with mass spectrometry detection (GC-MS). The technique was validated by measuring the fractional rate of protein synthesis in the liver and white muscle of Arctic charr (Salvelinus alpinus) and then tested by comparing the fractional rate of protein synthesis of fed and starved Arctic charr. The modified technique met the assumptions of the flooding dose technique and was successfully used to detect alterations in the rate of protein synthesis in fed and starved fish. This modified technique allows for studies on protein metabolism to be carried out in situations where the use of radioactivity is difficult, if not impossible.

Effects of chronic food restriction and exercise training on the recovery of cardiac function following ischemia.

Clinical and experimental data suggest that exercise training (ET) and food restriction (FR) improve cardiovascular function. However, the effects of long-term FR or FR in combination with ET on the recovery of cardiac function following ischemia have not been determined. Male Wistar rats were assigned to ad libitum-fed, FR, ad libitum-exercise, and FR-exercise groups. Mechanical function of isolated working hearts was assessed in response to increases in afterload resistance and following global no-flow ischemia. At low workload, there was a significant FR effect on aortic flow as well as an interaction between FR and ET on systolic pressure. These effects remained when hearts were subjected to increases in aortic afterload resistance. During reperfusion of ischemic hearts, there was a significant FR effect on aortic flow and systolic pressure and a significant ET effect on diastolic pressure. An interaction between FR and ET on heart rate was also seen during reperfusion. In terms of percent recovery of heart function following ischemia, FR continued to affect aortic flow, and we observed an interaction between FR and ET on aortic flow. Our results clearly indicate that the myocardium from the FR animal or the FR, exercise-trained rat is more resistant to ischemia.

Effects of propionyl-L-carnitine on isolated mitochondrial function in the reperfused diabetic rat heart.

The effects of propionyl-L-carnitine (PLC) on isolated mitochondrial respiration in the ischemic reperfused diabetic heart were studied. Oral PLC treatment of STZ-diabetic rats was initiated for a period of 6 weeks. After treatment, isolated working hearts from diabetic rats were perfused under aerobic conditions then subjected to 25 min of no-flow ischemia followed by 15 min of aerobic reperfusion. At the end of reperfusion, heart mitochondria was isolated using differential centrifugation and respiration measured in the presence of pyruvate, glutamate, and palmitoylcarnitine. Our results indicate that diabetes was characterized by a pronounced decrease in heart function under aerobic conditions as well as during reperfusion following ischemia. Treatment with PLC resulted in a significant improvement in heart function under these conditions. The depressions in state 3 mitochondrial respiration with both pyruvate and glutamate seen in reperfused hearts from diabetic rats were prevented by PLC. State 3 respiration in the presence of palmitoylcarnitine was also improved in the ischemic reperfused diabetic rat heart. Our results show that PLC improves recovery of mechanical function following ischemia in the diabetic rat heart. The beneficial effects of PLC are associated with enhanced mitochondrial oxidation of fuels.

Cloning of glycerol-3-phosphate dehydrogenase cDNAs from two fish species and effect of temperature on enzyme expression in rainbow smelt (Osmerus mordax).

Rainbow smelt (Osmerus mordax) can accumulate extreme levels of glycerol in their blood during winter. Low temperatures are required for glycerol accumulation in smelt blood and the enzyme glycerol-3-phosphate dehydrogenase (GPDH) has been suggested to play a role in glycerol production/concentration in this species. In the present study, cDNA sequences encoding glycerol-3-phosphate dehydrogenase (GPDH) from rainbow smelt and Atlantic salmon (Salmo salar) were cloned. The encoded GPDH protein sequences were very similar to one another (88% identity). Using RT-PCR, GPDH mRNA was detected in skin, gill, heart, head kidney, brain and liver from both salmon and smelt obtained in December. However, GPDH was not detected in salmon intestine and spleen or in smelt intestine. Examination of GPDH expression in smelt liver during February by Northern blotting revealed temperature regulation. Elevation of the temperature resulted in a significant decrease in liver GPDH transcript level. Serum glycerol levels decreased concomitantly. These findings suggest a role for GPDH in the accumulation of glycerol in smelt at low temperatures.

Expression levels of genes associated with oxygen utilization, glucose transport and glucose phosphorylation in hypoxia exposed Atlantic cod (Gadus morhua).

Expression level of genes associated with oxygen [cytochrome oxidase 1 (COX1) and myoglobin (Mb)] and glucose utilization [glucose transporters (GLUTs) and hexokinases (HKs)] along with metabolic indices were determined in Atlantic cod (Gadus morhua) subjected to an hypoxic challenge of <45% oxygen saturation for 24 days. There were two closely related HKs considered to be homologues of mammalian HKIs. HKIa and HKIb share 86% sequence identity and are both ubiquitously expressed. Mb was also expressed in many tissues with highest levels occurring in heart. Over the first 15 days of hypoxia there were transient increases in plasma lactate in hypoxic relative to normoxic fish associated with a significant decrease in liver glycogen. Over days 1-6, there were in ten of eleven cases, increased average (with a number of conditions being statistically significant) expression levels of GLUTs (1, 2, 4) and HKs (1a and b) in gill, heart, liver, and white muscle in hypoxic relative to normoxic fish. There were significant increases in COX1 and Mb expression levels in gill by day 24 but no changes in these aerobic indicators in heart or liver. Overall the data suggest a transient increase in genes associated with glucose utilization during the early part of the hypoxic challenge followed by alterations in gene expression in gill.

Responses to hypoxia and recovery: repayment of oxygen debt is not associated with compensatory protein synthesis in the Amazonian cichlid, Astronotus ocellatus.

Oxygen consumption, as an indicator of routine metabolic rate (RoMR), and tissue-specific changes in protein synthesis, as measured by (3)H-labelled phenylalanine incorporation rates, were determined in Astronotus ocellatus to investigate the cellular mechanisms behind hypoxia-induced metabolic depression and recovery. RoMR was significantly depressed, by approximately 50%, when dissolved oxygen levels reached 10% saturation (0.67+/-0.01 mg l(-1) at 28+/-1 degrees C). This depression in RoMR was accompanied by a 50-60% decrease in liver, heart and gill protein synthesis, but only a 30% decrease in brain protein synthesis. During recovery from hypoxia, an overshoot in RoMR to 270% of the normoxic rate was observed, indicating the accumulation of an oxygen debt during hypoxia. This conclusion was consistent with significant increase in plasma lactate levels during the hypoxic exposure, and the fact that lactate levels rapidly returned to pre-hypoxic levels. In contrast, a hyperactivation of protein synthesis did not occur, suggesting the overshoot in oxygen consumption during recovery is attributed to an increase in cellular processes other than protein synthesis.

Cardiorespiratory modifications, and limitations, in post-smolt growth hormone transgenic Atlantic salmon Salmo salar.

In recent years, there has been a great deal of interest in how growth hormone (GH) transgenesis affects fish physiology. However, the results of these studies are often difficult to interpret because the transgenic and non-transgenic fish had very different environmental/rearing histories. This study used a stable line of size-matched GH Atlantic salmon (Salmo salar) that were reared in a shared tank with controls (at 10 degrees C, for approximately 9 months) to perform a comprehensive examination of the cardiorespiratory physiology of GH transgenic salmon, and serves as a novel test of the theory of symmorphosis. The GH transgenic salmon had a 3.6x faster growth rate, and 21 and 25% higher values for mass-specific routine and standard oxygen consumption (M(O(2))), respectively. However, there was no concurrent increase in their maximum M(O(2)), which resulted in them having an 18% lower metabolic scope and a 9% reduction in critical swimming speed. This decreased metabolic capacity/performance was surprising given that the transgenics had a 29% larger heart with an 18% greater mass-specific maximum in situ cardiac output, a 14% greater post-stress blood haemoglobin concentration, 5-10% higher red muscle and heart aerobic enzyme (citrate synthase or cytochrome oxidase) activities, and twofold higher resting and 1.7x higher post-stress, catecholamine levels. However, gill surface area was the only cardiorespiratory parameter that was not enhanced, and our data suggest that gill oxygen transfer may have been limiting. Overall, this research: (1) shows that there are significant metabolic costs associated with GH transgenesis in this line of Atlantic salmon; (2) provides the first direct evidence that cardiac function is enhanced by GH transgenesis; (3) shows that a universal upregulation of post-smolt (adult) GH transgenic salmon cardiorespiratory physiology, as suggested by symmorphosis, does not occur; and (4) supports the idea that whereas differences in arterial oxygen transport (i.e. cardiac output and blood oxygen carrying capacity) are important determinants of inter-specific differences in aerobicity, diffusion-limited processes must be enhanced to achieve substantial intra-specific improvements in metabolic and swimming performance.

The fish heart as a model system for the study of myoglobin.

A model is presented for myoglobin study based upon naturally occurring differences in myocardial myoglobin content in fish. The sea raven (Hemitripterus americanus) and the ocean pout (Macrozoarces americanus) have heart myoglobin contents of approx. 65 and 5 nmol/g wet wt, respectively. The maximal activities of enzymes associated with energy metabolism are similar in the two hearts. Isolated perfused hearts performed with similar efficiencies based upon similar rates of work, oxygen consumption and lactate production. Under normoxic perfusion conditions both hearts met 98% of the ATP demand by oxidative mechanisms. Myoglobin-rich sea raven hearts performed significantly better than myoglobin-poor ocean pout hearts under conditions of hypoxia and glycolytic blockage. The performance of sea raven hearts was impaired during hypoxia by decreasing the content of functional myoglobin with hydroxylamine. No effect upon performance was observed with the ocean pout heart. The data provide the first evidence that myoglobin plays a role in the maintenance of contractility in heart under hypoxic conditions.

Perfusion-independent oxygen extraction in myoglobin-rich hearts.

Cardiac myoglobin plays a role in oxygen consumption and has a protective effect during periods of hypoxia, but little is known about the role of myoglobin during periods of ischaemia. Myoglobin-rich sea raven hearts and myoglobin-poor ocean pout hearts were isolated and perfused at varying flow rates and under conditions of low and high oxygen demand to assess the role of myoglobin in oxygen extraction. In the myoglobin-rich hearts, oxygen extraction remained constant over the flow range. In the myoglobin-poor hearts, oxygen extraction was significantly elevated, relative to controls, at the lower flow rates but decreased as the flow rate increased. In hearts where myoglobin was inactivated by an oxidizing agent, oxygen extraction was similar to that observed in myoglobin-poor hearts. Under conditions of high oxygen demand, myoglobin-rich hearts again showed a constant oxygen extraction over the flow range. Myoglobin-inactivated hearts had a significantly elevated oxygen extraction at low flows, and this decreased as flow rate increased. These data suggest that myoglobin renders oxygen extraction by fish hearts independent of the rate of perfusion.

Protein synthesis under conditions of anoxia and changing workload in ventricle strips from turtle heart.

An earlier study determined that protein synthesis in isolated perfused turtle (Trachemys [= Pseudemys] scripta elegans) hearts was three-fold lower under conditions of anoxia than under conditions of normoxia. However, the earlier study did not attempt to define the role of work in the isolated perfused preparation. In this study, the effects of varying workload, as defined by changing frequency of contraction, and anoxia on protein synthesis were examined. The ventricle strip preparation allows for comparison of multiple strips from a single heart, which aids in eliminating the variability found between individuals chosen from wild populations. Ventricle strips forced to contract at 24 contractions.min-1 under anoxic conditions failed more rapidly than strips forced to contract at 24 contractions.min-1 under normoxic conditions. Protein synthesis decreased by 32% when compared to normoxic controls. When stimulation was terminated after 2 hr of contraction, the rate of protein synthesis in strips under anoxic conditions was similar to that in strips under normoxic conditions. Also, returning strips to normoxic conditions after 2 hr of anoxia restored protein synthesis to the level of the normoxic controls. A significant correlation between pacing rate and protein synthesis was found under normoxic conditions but not under anoxic conditions when strips were paced at 12, 18 and 24 contractions.min-1. Protein synthesis increased by 30% at the 18 contractions.min-1 frequency and 45% at the 24 contractions.min-1 frequency over the rate at 12 contractions.min-1 frequency. Force-frequency studies revealed that under normoxic conditions force generation did not change until above 24 contractions.min-1, but under anoxic conditions there was a significant negative inotropic effect (20% decrease in force) at 24 contractions.min-1 and fell to 50% of initial at 36 contractions.min-1. These studies indicate that, in the turtle heart, anoxia per se is not the only determinant of protein synthesis but rather that work plays an important role in protein synthesis, as in the mammalian heart.

Function of myoglobin in oxygen consumption by isolated perfused fish hearts.

Myoglobin, an intracellular O2-binding protein, plays a protective role in maintaining performance of isolated fish hearts under hypoxic conditions. This study was designed to test the hypothesis that the protein contributes to O2 consumption under conditions of increased O2 demand or hypoxia. Isolated myoglobin-rich sea raven (Hemitripterus americanus) hearts and myoglobin-poor ocean pout (Macrozoarces americanus) hearts were perfused under conditions of changing partial pressure of O2 (PO2) and afterload. Sea raven hearts maintained O2 consumption and cardiac performance at low PO2 and high afterload, whereas ocean pout hearts did not. In other cases sea raven and ocean pout hearts were treated with hydroxylamine, which renders myoglobin incapable of binding O2, and subjected to changing PO2 and afterload. Sea raven hearts could not maintain O2 consumption and cardiac performance, whereas hydroxylamine treatment had no effect on O2 consumption in ocean pout hearts under these conditions. These data provide the first evidence to support the concept that myoglobin plays a role in O2 consumption of hearts.

Cardiorespiratory and tissue adenosine responses to hypoxia and reoxygenation in the short-horned sculpin Myoxocephalus scorpius.

Adenosine is a product of adenylate phosphate breakdown that can exert protective effects on tissues during energy limitation. Accumulation of cardiac adenosine under hypoxia is well documented in mammals but has not been shown in fish. Adenosine content was measured in heart and brain tissue from short-horned sculpin Myoxocephalus scorpius L. exposed to acute hypoxia and to graded hypoxia and reoxygenation at 8 degrees C. Cardiorespiratory parameters were recorded along with plasma lactate, K(+), Ca(2+) and Na(+) levels and their relationship to adenosine levels investigated. Sculpin exhibited a large bradycardia during hypoxia, with a concomitant drop in cardiac output that recovers fully with reoxygenation. Ventilation rate also declined with hypoxia, suggesting a depression of activity. Plasma lactate concentration was significantly elevated after 4 h at 2.0 mg l(-1) dissolved oxygen while K(+) levels increased during acute hypoxia. Adenosine levels were maintained in heart under acute and graded hypoxia. Brain levels fluctuated under hypoxia and showed no change with reoxygenation. It is concluded that a depression of cardiac activity in conjunction with an adequate anaerobic metabolism allow sculpin to avoid excessive adenosine accumulation under conditions of moderate hypoxia. Cardiac adenosine levels decreased and plasma K(+) levels and heart rate increased significantly at reoxygenation.

Myoglobin supported oxygen consumption in isolated rat hearts under dysoxic conditions.

Oxygen consumption was assessed in contracting, isolated rat hearts subjected to Langendorff perfusion. Initially, hearts were perfused with Krebs-Henseleit bicarbonate medium (KHB). Some hearts were treated with a 10 min pulse of medium containing 0.05 mM phenylhydrazine to oxidize approximately 78% of myoglobin to a state incapable of binding oxygen. Stepwise reduction in input PO2 resulted in a decline in oxygen consumption (MO2) in control and treated hearts. Phenylhydrazine treatment had no effect upon MO2 in hearts perfused with medium having a PO2 of about 585 mmHg or higher. However, at an input PO2 of approximately 370 mmHg, MO2 was decreased to 60% of the level at an input PO2 of 710 mmHg in untreated hearts and significantly lower to 32% of initial level in myoglobin blocked hearts. In subsequent experiments, hearts were perfused with KHB containing human red blood cells (RBCs) to elevate the oxygen content of the perfusate. The addition of RBCs to medium having a PO2 of approximately 140 mmHg resulted in enhancement of MO2 and maintenance of performance. But the preparations were considered to be dysoxic since MO2 with RBCs in the medium (PO2 approximately 140 mmHg) was lower than under perfusion with KHB (PO2 approximately 710 mmHg). This should however not detract from the utility of the model in elucidating myoglobin function under oxygen limiting conditions. At an input PO2 of 140 mmHg hearts treated with phenylhydrazine to impede myoglobin function had a significantly lower MO2 and viability than untreated hearts.(ABSTRACT TRUNCATED AT 250 WORDS)

Control of energy metabolism in fish white muscle.

Concentrations of key metabolites were determined in carp white muscle before exercise and after maximal activity. It was found that the concentration of ATP decreases by about 65%, ADP decreases slightly, and AMP remains unchanged. Consequently, the level of the free adenylate pool decreases. Simultaneously there is an increase in the concentration of IMP and NH4+. The increase in IMP level and the decrease in adenylate pool are essentially in 1:1 stoichiometry, a result showing that the adenylate pool is decreased by the reaction catalyzed by 5'-AMP deaminase (EC 3.5.4.6.). During exercise there is an increase in levels of glucose-6-phosphate, fructose 6-phosphate, and fructose 1,6-diphosphate that, along with the decrease in ATP levels, can account for the increase in glycolytic flux by activation of phosphofructokinase and pyruvate kinase.

Low temperature acclimation decreases rates of protein synthesis in rainbow trout (Oncorhynchus mykiss) heart.

Protein synthesis was assessed in rainbow trout (Oncorhynchus mykiss) hearts perfused with medium containing (3)H phenylalanine. Isolated hearts from fish acclimated to 5° and 15°C were used as the model system, and were perfused at variable test temperatures and pH. Protein synthesis expressed as nmol PHE mg protein(-1) h(-1) was two fold higher in hearts from fish acclimated to 15°C and tested at 15°C and extracellular pH 7.6 than in hearts from fish acclimated to 5°C and tested at 5°C and extracellular pH 8.0. The prime determinant of the decreased rate of protein synthesis was thermal history. Fish acclimated to 5°C had lower levels of RNA mg protein(-1) than fish held at 15°C. There was a direct linear relationship between the rate of protein synthesis in nmol PHE mg protein(-1) h(-1) and RNA content. RNA activity (nmol PHE µg RNA(-1) h(-1) remained constant regardless of thermal history or perfusion condition. Elevated pH resulted in only a marginal decrease in protein synthesis. Test temperature had no effect on in vitro rates of protein synthesis.

Decreased total ventricular and mitochondrial protein synthesis during extended anoxia in turtle heart.

The turtle heart provides a model system to study the effects of anoxia on protein synthesis without the potentially confounding factor of contractile failure and decreased ATP levels. Protein synthesis, as measured by 3H-labeled phenylalanine incorporation, was studied under conditions of normoxia and anoxia in isolated perfused turtle [Trachemys (= Pseudemys) scripta elegans] hearts at 15 degrees C. Heart rate, cardiac output, and ventricular pressure development were unaffected by 2 or 3 h of anoxia. Despite the anoxia, energy levels in the heart were presumably still high, since contractility was maintained. RNA content of ventricle decreased after anoxic perfusion. Rates of total protein synthesis rates in ventricle were threefold lower under anoxia than under normoxia. These findings suggest that the total level of RNA is one determinant of protein synthesis. Incorporation of label into protein extracted from mitochondria was also assessed. The ratio of mitochondrial to whole ventricular protein synthesis was significantly lower after anoxia, revealing preferential control mechanisms under anoxia between the synthesis of total cellular protein and protein destined for mitochondria. Isolated mitochondria were still coupled after 2 or 3 h of anoxia. In effect, the mitochondria enter into a state of hypometabolism in terms of rates of ATP synthesis and protein synthesis, but functional integrity is maintained. The decrease in protein synthesis in general and mitochondrial protein synthesis in particular may represent an adaptation to allow the partitioning of the available energy resources toward mechanical function during anoxia.