Metabolic organization of the spotted ratfish, Hydrolagus colliei (Holocephali: Chimaeriformes): insight into the evolution of energy metabolism in the chondrichthyan fishes

The metabolic organization of a holocephalan, the spotted ratfish (Hydrolagus colliei), was assessed using measurements of key enzymes of several metabolic pathways in four tissues and plasma concentrations of free amino acids (FAA) and non-esterified fatty acids (NEFA) to ascertain if the Holocephali differ metabolically from the Elasmobranchii since these groups diverged ca. 400 Mya. Activities of carnitine palmitoyl transferase indicate that fatty acid oxidation occurs in liver and kidney but not in heart or white muscle. This result mirrors the well-established absence of lipid oxidation in elasmobranch muscle, and more recent studies showing that elasmobranch kidney possesses a capacity for lipid oxidation. High activities in oxidative tissues of enzymes of ketone body metabolism, including D-beta-hydroxybutyrate dehydrogenase, indicate that, like elasmobranchs, ketone bodies are of central importance in spotted ratfish. Like many carnivorous fishes, enzyme activities demonstrate that amino acids are metabolically important, although the concentration of plasma FAA was relatively low. NEFA concentrations are lower than in teleosts, but higher than in most elasmobranchs and similar to that in some "primitive" ray-finned fishes. NEFA composition is comparable to other marine temperate fishes, including high levels of n-6 and especially n-3 polyunsaturated fatty acids. The metabolic organization of the spotted ratfish is similar to that of elasmobranchs: a reduced capacity for lipid oxidation in muscle, lower plasma NEFA levels, and an emphasis on ketone bodies as oxidative fuel. This metabolic strategy was likely present in the common chondrichthyan ancestor, and may be similar to the ancestral metabolic state of fishes.     

Energy metabolism of fish brain

This review focuses on recent research on the metabolic function of fish brain. Fish brain is isolated from the systemic circulation by a blood-brain barrier that allows the transport of glucose, monocarboxylates and amino acids. The limited information available in fishes suggests that oxidation of exogenous glucose and oxidative phosphorylation provide most of the ATP required for brain function in teleosts, whereas oxidation of ketones and amino acids occurs preferentially in elasmobranchs. In several agnathans and benthic teleosts brain glycogen levels rather than exogenous glucose may be the proximate glucose source for oxidation. In situations when glucose is in limited supply, teleost brains utilize other fuels such as lactate or ketones. Information on use of lipids and amino acids as fuels in fish brain is scarce. The main pathways of brain energy metabolism are changed by several effectors. Thus, several parameters of brain energy metabolism have been demonstrated to change post-prandially in teleostean fishes. The absence of food in teleosts elicits profound changes in brain energy metabolism (increased glycogenolysis and use of ketones) in a way similar to that demonstrated in mammals though delayed in time. Environmental factors induce changes in brain energy parameters in teleosts such as the enhancement of glycogenolysis elicited by pollutants, increased capacity for anaerobic glycolysis under hypoxia/anoxia or changes in substrate utilization elicited by adaptation to cold. Furthermore, several studies demonstrate effects of melatonin, insulin, glucagon, GLP-1, cortisol or catecholamines on energy parameters of teleost brain, although in most cases the results are quite preliminary being difficult to relate the effects of those hormones to physiological situations. The few studies performed with the different cell types available in the nervous system of fish allow us to hypothesize few functional relationships among those cells. Future research perspectives are also outlined.     

Antioxidant potential is positively correlated with mitochondrial enzyme activity in Antarctic and non-Antarctic notothenioid fishes

Antarctic notothenioid fishes possess high oxidative capacities, large amounts of intracellular lipid combined with biological membranes enriched in polyunsaturated fatty acids, all of which could make these animals susceptible to oxidative injury, particularly in the form of lipid peroxidation. The central objective in this study was to examine capacities for oxidative metabolism and total antioxidant defense in Antarctic and non-Antarctic notothenioids in order to test the hypothesis that the cold-bodied Antarctic fishes possess elevated activities of citrate synthase (CS), matched by a more robust antioxidant (AOX) defense, than non-Antarctic species. CS activities and total AOX capacities were measured in brain and heart of 4 Antarctic species and 2 non-Antarctic species collected on the 2004 ICEFISH cruise. While no statistical differences are found among Antarctic and non-Antarctic fishes in either CS or AOX capacities, AOX capacity in both tissues expands with CS activity among individuals measured when all species are combined. There is also a 4.5-fold greater AOX capacity, when normalized to CS activity, in brain than in heart indicating the requirement for extra AOX defense in a tissue well known for its particularly high levels of phospholipids more prone to lipid peroxidation.     

Enzyme activities support the use of liver lipid-derived ketone bodies as aerobic fuels in muscle tissues of active sharks

Few data exist to test the hypothesis that elasmobranchs utilize ketone bodies rather than fatty acids for aerobic metabolism in muscle, especially in continuously swimming, pelagic sharks, which are expected to be more reliant on lipid fuel stores during periods between feeding bouts and due to their high aerobic metabolic rates. Therefore, to provide support for this hypothesis, biochemical indices of lipid metabolism were measured in the slow-twitch, oxidative (red) myotomal muscle, heart, and liver of several active shark species, including the endothermic shortfin mako, Isurus oxyrinchus. Tissues were assayed spectrophotometrically for indicator enzymes of fatty acid oxidation (3-hydroxy-o-acyl-CoA dehydrogenase), ketone-body catabolism (3-oxoacid-CoA transferase), and ketogenesis (hydroxy-methylglutaryl-CoA synthase). Red muscle and heart had high capacities for ketone utilization, low capacities for fatty acid oxidation, and undetectable levels of ketogenic enzymes. Liver demonstrated undetectable activities of ketone catabolic enzymes but high capacities for fatty acid oxidation and ketogenesis. Serum concentrations of the ketone beta-hydroxybutyrate varied interspecifically (means of 0.128-0.978 micromol mL(-1)) but were higher than levels previously reported for teleosts. These results are consistent with the hypothesis that aerobic metabolism in muscle tissue of active sharks utilizes ketone bodies, and not fatty acids, derived from liver lipid stores.     

The unusual energy metabolism of elasmobranch fishes

The unusual energy metabolism of elasmobranchs is characterized by limited or absent fatty acid oxidation in cardiac and skeletal muscle and a great reliance on ketone bodies and amino acids as oxidative fuels in these tissues. Other extrahepatic tissues in elasmobranchs rely on ketone bodies and amino acids for aerobic energy production but, unlike muscle, also appear to possess a significant capacity to oxidize fatty acids. This organization of energy metabolism is reflected by relatively low plasma levels of non-esterified fatty acids (NEFA) and by plasma levels of the ketone body ß-hydroxybutyrate that are as high as those seen in fasted mammals. The preference for ketone body oxidation rather than fatty acid oxidation in muscle of elasmobranchs under routine conditions is opposite to the situation in teleosts and mammals. Carbohydrates appear to be utilized as a fuel source in elasmobranchs, similar to other vertebrates. Amino acid- and lipid-fueled ketogenesis in the liver, the lipid storage site in elasmobranchs, sustains the demand for ketone bodies as oxidative fuels. The liver also appears to export NEFA and serves a buoyancy role. The regulation of energy metabolism in elasmobranchs and the effects of environmental factors remain poorly understood. The metabolic organization of elasmobranchs was likely present in the common ancestor of the Chondrichthyes ca. 400 million years ago and, speculatively, it may reflect the ancestral metabolism of jawed vertebrates. We assess hypotheses for the evolution of the unusual energy metabolism of elasmobranchs and propose that the need to synthesize urea has influenced the utilization of ketone bodies and amino acids as oxidative fuels.     

Inhibition of fatty acid oxidation by etomoxir impairs NADPH production and increases reactive oxygen species resulting in ATP depletion and cell death in human glioblastoma cells

Normal differentiated cells rely primarily on mitochondrial oxidative phosphorylation to produce adenosine triphosphate (ATP) to maintain their viability and functions by using three major bioenergetic fuels: glucose, glutamine and fatty acids. Many cancer cells, however, rely on aerobic glycolysis for their growth and survival, and recent studies indicate that some cancer cells depend on glutamine as well. This altered metabolism in cancers occurs through oncogene activation or loss of tumor suppressor genes in multiple signaling pathways, including the phosphoinositide 3-kinase and Myc pathways. Relatively little is known, however, about the role of fatty acids as a bioenergetic fuel in growth and survival of cancer cells. Here, we report that human glioblastoma SF188 cells oxidize fatty acids and that inhibition of fatty acid β-oxidation by etomoxir, a carnitine palmitoyltransferase 1 inhibitor, markedly reduces cellular ATP levels and viability. We also found that inhibition of fatty acid oxidation controls the NADPH level. In the presence of reactive oxygen species scavenger tiron, however, ATP depletion is prevented without restoring fatty acid oxidation. This suggests that oxidative stress may lead to bioenergetic failure and cell death. Our work provides evidence that mitochondrial fatty acid oxidation may provide NADPH for defense against oxidative stress and prevent ATP loss and cell death.     

Fatty acyl CoA synthetase from Antarctic notothenioid fishes may influence substrate specificity of fat oxidation

Antarctic notothenioid fishes possess large lipid stores that are important fuels for aerobic metabolism. Oxidative muscle tissues of these animals oxidize long-chain mono-unsaturated fatty acids more readily than saturated fatty acids. The mechanistic basis(es) for the substrate specificity of their fatty acid-oxidizing pathway is unknown. We examined the substrate specificity of fatty acyl coenzyme A synthetase (FACS) to determine whether the enzyme contributes to targeting unsaturated fatty acids for preferential transport into mitochondria as fuels for beta-oxidation. Maximal activities of FACS were measured in isolated mitochondria from Notothenia coriiceps and Chaenocephalus aceratus oxidative skeletal muscles in the presence of fatty acids differing in chain lengths and degrees of unsaturation. With the exception of C(22:6), maximal activities were greater with unsaturated substrates than with C(16:0), a saturated fatty acid. Monoenoic fatty acids did not produce the highest activities. Predicted amino acid sequences of FACS from Antarctic C. aceratus, Gobionotothen gibberifrons, and N. coriiceps and sub-Antarctic Notothenia angustata and Eleginops maclovinus were determined to identify amino acid candidates that may be important for determining the substrate specificity of FACS. Substitutions cysteine548 and polar threonine552 within the putative fatty acid binding pocket may contribute to preference for unsaturated fatty acyl substrates compared to saturated fatty acids.     

The metabolic organization of a primitive air-breathing fish, the Florida gar (Lepisosteus platyrhincus)

The metabolic organization of the air-breathing Florida gar, Lepisosteus platyrhincus, was assessed by measuring the maximal activities of key enzymes in several metabolic pathways in selected tissues, concentrations of plasma metabolites including nonesterified fatty acids (NEFA), free amino acids (FAA) and glucose as well as tissue FAA levels. In general, L. platyrhincus has an enhanced capacity for carbohydrate metabolism as indicated by elevated plasma glucose levels and high activities of gluconeogenic and glycolytic enzymes. Based upon these properties, glucose appears to function as the major fuel source in the Florida gar. The capacity for lipid metabolism in L. platyrhincus appears limited as plasma NEFA levels and the activities of enzymes involved in lipid oxidation are low relative to many other fish species. L. platyrhincus is capable of oxidizing both D- and L-beta-hydroxybutyrate, with tissue-specific preferences for each stereoisomer, yet the capacity for ketone body metabolism is low compared with other primitive fishes. Based on enzyme activities, the metabolism of the air-breathing organ more closely resembles that of the mammalian lung than a fish swim bladder. The Florida gar sits phylogenetically and metabolically in an intermediate position between the "primitive" elasmobranchs and the "advanced" teleosts. The apparently unique metabolic organization of the gar may have evolved in the context of a bimodal air-breathing environmental adaptation.     

Plasma non-esterified fatty acids of elasmobranchs: comparisons of temperate and tropical species and effects of environmental salinity

We investigated the influence of environments with different average temperatures and different salinities on plasma NEFA in elasmobranchs by comparing species from tropical vs. cold temperate marine waters, and tropical freshwater vs. tropical marine waters. The influence of the environment on plasma NEFA is significant, especially with regard to essential fatty acids (EFA) and the n-3/n-6 ratio. n-3/n-6 ratios in tropical marine elasmobranchs were lower by two-fold or more compared with temperate marine elasmobranchs, because of higher levels of arachidonic acid (AA, 20:4n-6) and docosatetraenoic acid (22:4n-6), and less docosahexaenoic acid (DHA, 22:6n-3), in the tropical species. These results are similar to those in earlier studies on lipids in teleosts. n-3/n-6 ratios and levels of EFA were similar between tropical freshwater and tropical marine elasmobranchs. This suggests that the observation in temperate waters that marine fishes have higher levels of n-3 fatty acids and n-3/n-6 ratios than freshwater fishes may not hold true in tropical waters, at least in elasmobranchs. It also suggests that plasma NEFA are little affected by freshwater vs. seawater adaptation in elasmobranchs. Likewise, we found that plasma NEFA composition and levels were not markedly affected by salinity acclimation (2 weeks) in the euryhaline stingray Himantura signifer. However, in contrast to our comparisons of freshwater-adapted vs. marine species, the level of n-3 fatty acids and the n-3/n-6 ratio were observed to significantly decrease, indicating a potential role of n-3 fatty acids in salinity acclimation in H. signifer.     

Plasma non-esterified fatty acids of elasmobranchs: comparisons of temperate and tropical species and effects of environmental salinity.

We investigated the influence of environments with different average temperatures and different salinities on plasma NEFA in elasmobranchs by comparing species from tropical vs. cold temperate marine waters, and tropical freshwater vs. tropical marine waters. The influence of the environment on plasma NEFA is significant, especially with regard to essential fatty acids (EFA) and the n-3/n-6 ratio. n-3/n-6 ratios in tropical marine elasmobranchs were lower by two-fold or more compared with temperate marine elasmobranchs, because of higher levels of arachidonic acid (AA, 20:4n-6) and docosatetraenoic acid (22:4n-6), and less docosahexaenoic acid (DHA, 22:6n-3), in the tropical species. These results are similar to those in earlier studies on lipids in teleosts. n-3/n-6 ratios and levels of EFA were similar between tropical freshwater and tropical marine elasmobranchs. This suggests that the observation in temperate waters that marine fishes have higher levels of n-3 fatty acids and n-3/n-6 ratios than freshwater fishes may not hold true in tropical waters, at least in elasmobranchs. It also suggests that plasma NEFA are little affected by freshwater vs. seawater adaptation in elasmobranchs. Likewise, we found that plasma NEFA composition and levels were not markedly affected by salinity acclimation (2 weeks) in the euryhaline stingray Himantura signifer. However, in contrast to our comparisons of freshwater-adapted vs. marine species, the level of n-3 fatty acids and the n-3/n-6 ratio were observed to significantly decrease, indicating a potential role of n-3 fatty acids in salinity acclimation in H. signifer.     

Expression of genes participating in regulation of fatty acid and glucose utilization and energy metabolism in developing rat hearts

The heart is a unique organ that can use several fuels for energy production. During development, the heart undergoes changes in fuel supply, and it must be able to respond to these changes. We have examined changes in the expression of several genes that regulate fuel transport and metabolism in rat hearts during early development. At birth, there was increased expression of fatty acid transporters and enzymes of fatty acid metabolism that allow fatty acids to become the major source of energy for cardiac muscle during the first 2 wk of life. At the same time, expression of genes that control glucose transport and oxidation was downregulated. After 2 wk, expression of genes for glucose uptake and oxidation was increased, and expression of genes for fatty acid uptake and utilization was decreased. Expression of carnitine palmitoyltransferase I (CPT I) isoforms during development was different from published data obtained from rabbit hearts. CPT Ialpha and Ibeta isoforms were both highly expressed in hearts before birth, and both increased further at birth. Only after the second week did CPT Ialpha expression decrease appreciably below the level of CPT Ibeta expression. These results represent another example of different expression patterns of CPT I isoforms among various mammalian species. In rats, changes in gene expression followed nutrient availability during development and may render cardiac fatty acid oxidation less sensitive to factors that influence malonyl-CoA content (e.g., fluctuations in glucose concentration) and thereby favor fatty acid oxidation as an energy source for cardiomyocytes in early development.     

Nutrient, fatty acid, amino acid and mineral analysis of natural prey of the Hawaiian monk seal, Monachus schauinslandi.

Proximate nutrients, gross energy content, mineral, amino acid and fatty acid composition were determined for teleost, cephalopod and crustacean prey of the Hawaiian monk seal. Crude protein was highest in the octopus, Octopus cyanea (80.0%), crude fat was highest in the Muraenid teleost, Gymnothorax eurostus (14.1%), whereas crude ash was highest in the lobster, Panulirus marginatus (11.6%). Gross energies ranged from 4.0 +/- 0.01 kcal g-1 in the Labrid teleost Bodianus bilulunatus to 6.0 +/- 0.12 kcal g-1 in the moray eel, Gymnothorax undulatus. Essential amino acids occurred in lower concentrations as a percentage of the total amino acids (35.8 +/- 2.6%) than non-essential amino acids (64.2 +/- 2.6%), but the ratio of individual amino acids to total amino acid concentrations were similar to those required by some monogastric terrestrial species and fingerling salmon. The fatty acid concentrations varied widely among species (range = 1.2-16.5 mg 100 mg-1); however, the teleosts had higher total fatty acids than the non-teleosts. This study indicates that, from a nutritional standpoint, some prey may be more beneficial to the Hawaiian monk seal; however, these prey are not necessarily the most abundant or available to some populations of the monk seal.     

The metabolic syndrome and the hepatic fatty acid drainage hypothesis

Much data indicates that lowering of plasma triglyceride levels by hypolipidemic agents is caused by a shift in the liver metabolism towards activation of peroxisome proliferator activated receptor (PPAR)alpha-regulated fatty acid catabolism in mitochondria. Feeding rats with lipid lowering agents leads to hypolipidemia, possibly by increased channeling of fatty acids to mitochondrial fatty acid oxidation at the expense of triglyceride synthesis. Our hypothesis is that increased hepatic fatty acid oxidation and ketogenesis drain fatty acids from blood and extrahepatic tissues and that this contributes significantly to the beneficial effects on fat mass accumulation and improved peripheral insulin sensitivity. To investigate this theory we employ modified fatty acids that change the plasma profile from atherogenic to cardioprotective. One of these novel agents, tetradecylthioacetic acid (TTA), is of particular interest due to its beneficial effects on lipid transport and utilization. These hypolipidemic effects are associated with increased fatty acid oxidation and altered energy state parameters of the liver. Experiments in PPAR alpha-null mice have demonstrated that the effects hypolipidemic of TTA cannot be explained by altered PPAR alpha regulation alone. TTA also activates the other PPARs (e.g., PPAR delta) and this might compensate for deficiency of PPAR alpha. Altogether, TTA-mediated clearance of blood triglycerides may result from a lowered level of apo C-III, with a subsequently induction of hepatic lipoprotein lipase activity and (re)uptake of fatty acids from very low density lipoprotein (VLDL). This is associated with an increased hepatic capacity for fatty acid oxidation, causing drainage of fatty acids from the blood stream. This can ultimately be linked to hypolipidemia, anti-adiposity, and improved insulin sensitivity.     

Do mice bred selectively for high locomotor activity have a greater reliance on lipids to power submaximal aerobic exercise?

Patterns of fuel use during locomotion are determined by exercise intensity and duration, and are remarkably similar across many mammalian taxa. However, as lipids have a high yield of ATP per mole and are stored in large quantities, their use should be favored in endurance-adapted animals. To examine the capacity for alteration or differential regulation of fuel-use patterns, we studied two lines of mice that had been selectively bred for high voluntary wheel running (HR), including one characterized by small hindlimb muscles (HR(mini)) and one without this phenotype (HR(normal)), as well as a nonselected control line. We evaluated: 1) maximal aerobic capacity (Vo(2 max)); 2) whole body fuel use during exercise by indirect calorimetry; 3) cardiac properties; and 4) many factors involved in regulating lipid use. HR mice achieved an increased Vo(2 max) compared with control mice, potentially in part due to HR cardiac capacities for metabolic fuel oxidation and the larger relative heart size of HR(mini) mice. HR mice also exhibited enhanced whole body lipid oxidation rates at 66% Vo(2 max), but HR(mini), HR(normal), and control mice did not differ in the proportional mix of fuels sustaining exercise (% total Vo(2)). However, HR(mini) gastrocnemius muscle had elevated fatty acid translocase (FAT/CD36) sarcolemmal protein and cellular mRNA, fatty acid binding protein (H-FABP) cytosolic protein, peroxisome proliferator-activated receptor (PPAR) α mRNA, and mass-specific activities of citrate synthase, β-hydroxyacyl-CoA dehydrogenase, and hexokinase. Therefore, high-running mouse lines had whole body fuel oxidation rates commensurate with maximal aerobic capacity, despite notable differences in skeletal muscle metabolic phenotypes.     

The lipid compositions of high-Antarctic notothenioid fish species with different life strategies

Lipid and fatty acid compositions of five notothenioid fishes from the Antarctic Weddell and Lazarev Seas were investigated in detail with regard to their different modes of life. The pelagic Aethotaxis mitopteryx was the lipid-richest species (mean of 61.4% of dry mass, DM) followed by Pleuragramma antarcticum (37.7%DM). The benthopelagic Trematomus lepidorhinus had an intermediate lipid content of 23.2%DM. The benthic Bathydraco marri (20.8%DM) and Dolloidraco longedorsalis (14.5%DM) belonged to the lipid-poorer species. Triacylglycerols were the major lipid class in all species. Important fatty acids were 16:0, 16:1(n-7), 18:1(n-9), 18:1(n-7), 20:5(n-3) and 22:6(n-3). The enhanced proportions of the long-chain monounsaturated fatty acids, 20:1 and 22:1, in the lipid-rich pelagic fishes clearly reflected the ingestion of the two copepod species, Calanoides acutus and Calanus propinquus, which are the only known Antarctic zooplankters rich in these fatty acids. Although wax esters are the major storage lipid in many prey species, they were absent in all notothenioid fishes studied. Thus, wax esters ingested with prey are probably converted to triacylglycerols via fatty acids or metabolised by the fishes. The enhanced lipid accumulation with increasingly pelagic lifestyle has energetic advantages, especially with regard to improved buoyancy. It is still unknown to what extent these lipids are utilised as energy reserves, since it has been suggested that not only the benthic but also the pelagic Antarctic fishes are rather sluggish, with a low scope for activity and hence low metabolic requirements. Accepted: 14 May 2000     

Fat to the fire: the regulation of lipid oxidation with exercise and environmental stress

Lipids are an important fuel for submaximal aerobic exercise. The ways in which lipid oxidation is regulated during locomotion is an area of active investigation. Indeed, the integration between cellular regulation of lipid metabolism and whole-body exercise performance is a fascinating but often overlooked research area. Additionally, the interaction between environmental stress, exercise, and lipid oxidation has not been sufficiently examined. There are many functional and structural steps as fatty acids are mobilized, transported, and oxidized in working muscle, which may serve either as regulatory points for responding to acute or chronic stimuli or as raw material for natural selection. At the whole-animal level, the partitioning of lipids and carbohydrates across exercise intensities is remarkably similar among mammals, which suggests that there is conservation in regulatory mechanisms. Conversely, the proportions of circulatory and intramuscular fuels differ between species and across exercise intensities. Responses to acute and chronic environmental stress likely involve the interaction of genetic and nongenetic changes in the fatty acid pathway. Determining which of these factors help regulate the fatty acid pathway and what impact they have on whole-animal lipid oxidation and performance is an important area of future research. Using an integrative approach to complete the information loop from gene to physiological function provides the most powerful mode of analysis.     

Kinetic Characterization of Myoglobins from Vertebrates with Vastly Different Body Temperatures

Fish myoglobins are structurally distinct from the previously characterized mammalian myoglobins. Teleost fishes express generally lower levels of myoglobin than those found in mammals. Although the oxygen binding affinity is essentially the same as mammalian myoglobins, oxygen dissociation rates and carbon monoxide combination rates of the teleost myoglobins studied are significantly faster. Thus, the kinetic parameters of myoglobin from two Antarctic teleost species, measured close to their body temperature of −1°C, are comparable to those of mammalian myoglobins with higher body temperatures. These data suggest myoglobins from Antarctic teleosts may function at extreme environmental temperatures.     

Effects of fatty acid provision during severe hypoxia on routine and maximal performance of the in situ tilapia heart

The ability to maintain stable cardiac function during environmental hypoxia exposure is crucial for hypoxia tolerance in animals and depends upon the maintenance of cardiac energy balance as well as the state of the heart’s extracellular environment (e.g., availability of metabolic fuels). Hypoxic depression of plasma [non-esterified fatty acids] (NEFA), an important cardiac aerobic fuel, is a common response in many species of hypoxia-tolerant fishes, including tilapia. We tested the hypothesis that decreased plasma [NEFA] is important for maintaining stable cardiac function during and following hypoxia exposure, based on the premise that continued reliance upon cardiac fatty acid metabolism under such conditions could impair cardiac function. We examined the effect of severe hypoxia exposure (PO₂ < 0.2 kPa) on routine and maximum performance of the in situ perfused tilapia heart under conditions of routine (400 μmol L^?1) and low (75 μmol L^?1) [palmitate], which mimicked the in vivo levels of plasma [NEFA] found in normoxic and hypoxic tilapia, respectively. Under both concentrations of palmitate, the in situ tilapia heart showed exceptional hypoxic performance as a result of a high maximum glycolytic potential, confirming our previous results using a perfusate without fatty acids. We additionally provide evidence suggesting that non-contractile ATP demand is depressed in tilapia heart during hypoxia exposure. Cardiac performance during and following severe hypoxia exposure was unaffected by the level of palmitate. Thus, we conclude that hypoxic depression of plasma [NEFA] in fishes does not play a role in cardiac hypoxia tolerance.