James S. Ballantyne

Reciprocal expression of gill Na+/K+-ATPase alpha-subunit isoforms alpha1a and alpha1b during seawater acclimation of three salmonid fishes that vary in their salinity tolerance.

SUMMARY The upregulation of gill Na+/K+-ATPase activity is considered critical for the successful acclimation of salmonid fishes to seawater. The present study examines the mRNA expression of two recently discovered α-subunit isoforms of Na+/K+-ATPase (α1a and α1b) in gill during the seawater acclimation of three species of anadromous salmonids, which vary in their salinity tolerance. Levels of these Na+/K+-ATPase isoforms were compared with Na+/K+-ATPase activity and protein abundance and related to the seawater tolerance of each species. Atlantic salmon (Salmo salar) quickly regulated plasma Na+, Cl– and osmolality levels within 10 days of seawater exposure, whereas rainbow trout (Oncorhynchus mykiss) and Arctic char (Salvelinus alpinus) struggled to ionoregulate, and experienced greater perturbations in plasma ion levels for a longer period of time. In all three species, mRNA levels for theα 1a isoform quickly decreased following seawater exposure whereasα 1b levels increased significantly. All three species displayed similar increases in gill Na+/K+-ATPase activity during seawater acclimation, with levels rising after 10 and 30 days. Freshwater Atlantic salmon gill Na+/K+-ATPase activity and protein content was threefold higher than those of Arctic char and rainbow trout, which may explain their superior seawater tolerance. The role of the α1b isoform may be of particular importance during seawater acclimation of salmonid fishes. The reciprocal expression of Na+/K+-ATPase isoforms α1a and α1b during seawater acclimation suggests they may have different roles in the gills of freshwater and marine fishes; ion uptake in freshwater fish and ion secretion in marine fishes.

Jaws: The Inside Story. The Metabolism of Elasmobranch Fishes

Abstract Elasmobranchs are of metabolic interest for several reasons, including their primitive evolutionary position, their osmotic strategy and their low incidence of neoplasia. Some aspects of the metabolism of elasmobranch fishes are unique when compared with those of the other vertebrates. Although many features of their metabolism can be attributed to their primitive evolutionary position (e.g., fewer isoforms of enzymes and other proteins), some unique features appear to be related to the unusual solute system (urea and methylamines) used by elasmobranchs. The solute system exerts widespread effects, which has an impact on the metabolism of lipids, ketone bodies and amino acids and the structure of proteins and membranes. Effects of urea on the transport of lipid may influence aspects of lipid metabolism, reducing extrahepatic lipid catabolism via effects on nonesterified fatty acid transport and enhancing a need for reliance on ketone bodies. Amino acid metabolism of elasmobranchs is also heavily influenced by the need for continuous synthesis of urea with glutamine as the nitrogen donor. These effects, in turn, may play a role in their low incidence of cancer. Specifically, the reduced availability of glutamine (an important nutrient for rapidly growing cells) coupled with the low levels of nonesterified fatty acids in the blood reduces the availability of molecules essential for tumor growth. This metabolic design may thus provide marine elasmobranchs with a “systemic” resistance to cancer.

High stocking density alters the energy metabolism of brook charr, Salvelinus fontinalis

Brook charr reared for 30 days at a high stocking density (120 kg/m3) had significantly lower final body weight and reduced food consumption when compared to fish reared at a lower stocking density (30 kg/m3). There was no effect of stocking density on plasma cortisol and T3 concentrations, while plasma T4 concentration was lowered in fish held at high stocking density. Plasma glucose levels, liver glycogen content and hepatosomatic index were significantly lower in fish reared at high stocking density, while plasma protein and free fatty acid (FFA) levels were not significantly different between groups. There were significantly higher activities of hepatic phosphofructokinase, fructose biphosphatase, 3-hydroxyacyl CoA dehydrogenase, glycerol kinase and glycerol-3-phosphate dehydrogenase in fish held at high stocking density compared with those at low stocking density. Hepatic hexokinase and glucose-6-phosphate dehydrogenase activities were significantly lower in fish at high stocking density compared with those at low stocking density, while there were no differences in pyruvate kinase, glucose-6-phosphatase, phosphoenolpyruvate carboxykinase, glutamate dehydrogenase, glutamate pyruvate transaminase and glutamate oxaloacetate transaminase between the groups. These results suggest that high stocking density has the effect of mobilizing triglyceride sources, promoting gluconeogenesis from glycerol, but has little effect on protein metabolism.

Cortisol-induced changes in some aspects of the intermediary metabolism of Salvelinus fontinalis

Cortisol was administered to brook charr (Salvelinus fontinalis) in the form of slow-release intraperitoneal implants (a) to investigate the effect of chronic cortisol stimulation (up to 60 days) on various aspects of intermediary metabolism, and (b) to determine whether such cortisol-induced changes were comparable to those seen in chronically fasted charr. Except for fish sampled at 1 and 6 h after implantation, there was no consistent increase in the plasma cortisol levels of the cortisol-implanted animals. Nevertheless, there were significant treatment effects (particularly after 60 days) on certain metabolite levels, and key hepatic enzymes, including a lowering of plasma glucose and hepatic glycogen concentrations, increased activities of FBPase, G6PDH, GK, and G3PDH, and reduced activities of PFK. When taken together these changes are indicative of a direct or indirect gluconeogenic action of cortisol, in which metabolites other than amino acids (possibly glycerol) are utilized as substrates. These metabolic changes differed from those found in food-deprived brook charr which appeared to use proteins, as well as lipids, as energy sources.

Influence of salinity on the energetics of gill and kidney of Atlantic salmon (Salmo salar).

The effect of seawater acclimation and adaptation to various salinities on the energetics of gill and kidney of Atlantic salmon (Salmo salar) was examined. Smolts and non-smolts previously reared in fresh water were exposed to a rapid increase in salinity to 30 ppt. Plasma osmolarity, [Na+], [Cl−], [K+] and [Mg++] increased in both groups but were significantly lower in smolts than non-smolts. Gill Na+, K+-ATPase specific activity, initially higher in smolts, increased in both groups after 18 days in seawater. Kidney Na+, K+-ATPase specific activity was not affected by salinity in either group. Gill and kidney citrate synthase specific activity was not affected by seawater exposure in smolts but decreased in non-smolts. In a second experiment, Atlantic salmon smolts reared in fresh water were acclimated to 0, 10 or 30 ppt seawater for 3 months at a temperature of 13–14°C. Gill Na+, K+-ATPase was positively correlated with salinity, displaying 2.5- and 5-fold higher specific activity at 10 and 30 ppt, respectively, than at 0 ppt. Kidney Na+, K+-ATPase specific activity was not significantly affected by environmental salinity. Citrate synthase and cytochrome c oxidase specific activities in gill were slightly (6–13%) lower at 10 ppt than at 0 and 30 ppt, whereas kidney activities were lowest at 30 ppt. Oxygen consumption of isolated gill filaments was significantly higher when incubated in isosmotic saline and at 30 ppt than at 0 ppt, but was not affected by the prior acclimation salinity. The results indicate that although high salinity induces increased gill Na+, K+-ATPase activity, it does not induce substantial increases in metabolic capacity of gill or kidney.

Metabolic responses to salinity acclimation in juvenile shortnose sturgeon Acipenser brevirostrum

Abstract The influence of salinity on the metabolic processes was investigated in the shortnose sturgeon Acipenser brevirostrum . Hatchery-reared juveniles (mean 274 g, 16 months old) were cultured at 18 °C for 10 weeks at salinities of 0‰, 5‰, 10‰ and 20‰. Plasma ions and metabolites were measured to assess the osmoregulatory and metabolic status of the fish through the experimental period. At the conclusion of the trial, metabolic enzymes were measured in several tissues, as were gill and intestinal Na + K + -ATPase in fish cultured at 0‰ and 20‰. In addition, tissue proximate analysis was performed on the white muscle of fish cultured at 0‰ and 20‰. An initial change in plasma osmolality was observed in those fish cultured at the higher salinities (10‰ and 20‰), followed by a return to values observed in fresh water acclimated fish. No significant changes were observed in total plasma free amino acid (FAA) concentrations. Changes were observed in individual amino acid concentrations, some of which may be related to an increased demand for oxidizable substrates in fish exposed to seawater. Total non-esterified fatty acid (NEFA) concentrations were elevated initially in the fish cultured at 20‰ compared with those cultured in fresh water (1732.1±288.1 and 1060.5±119.7 nmol/ml). The proximate analysis revealed a lower white muscle lipid level in fish cultured at 20‰ compared to fish cultured in fresh water (1.1±0.1% and 2.1±0.5%). This suggests that lipids are important in meeting the energetic demands of salinity exposure. An oxidative enzyme (cytochrome- c oxidase) was lower in the gills of fish cultured at 20‰, indicating a decreased aerobic capacity of the gill.

The osmotic response of the Asian freshwater stingray (Himantura signifer) to increased salinity: a comparison with marine (Taeniura lymma) and Amazonian freshwater (Potamotrygon motoro) stingrays

SUMMARY The white-edge freshwater whip ray Himantura signifer can survive in freshwater (0.7‰) indefinitely or in brackish water (20‰) for at least two weeks in the laboratory. In freshwater, the blood plasma was maintained hyperosmotic to that of the external medium. There was approximately 44 mmol l-1 of urea in the plasma, with the rest of the osmolality made up mainly by Na+ and Cl-. In freshwater, it was not completely ureotelic, excreting up to 45% of its nitrogenous waste as urea. Unlike the South American freshwater stingray Potamotrygon motoro, H. signifer has a functional ornithine-urea cycle (OUC) in the liver, with hepatic carbamoylphosphate synthetase III (CPS III) and glutamine synthetase (GS) activities lower than those of the marine blue-spotted fan tail ray Taeniura lymma. More importantly, the stomach of H. signifer also possesses a functional OUC, the capacity (based on CPS III activity) of which was approximately 70% that in the liver. When H. signifer was exposed to a progressive increase in salinity through an 8-day period, there was a continuous decrease in the rate of ammonia excretion. In 20‰ water, urea levels in the muscle, brain and plasma increased significantly. In the plasma, osmolality increased to 571 mosmol kg-1, in which urea contributed 83 mmol l-1. Approximately 59% of the excess urea accumulated in the tissues of the specimens exposed to 20‰ water was equivalent to the deficit in ammonia excretion through the 8-day period, indicating that an increase in the rate of urea synthesis de novo at higher salinities would have occurred. Indeed, there was an induction in the activity of CPS III in both the liver and stomach, and activities of GS, ornithine transcarbamoylase and arginase in the liver. Furthermore, there was a significant decrease in the rate of urea excretion during passage through 5‰, 10‰ and 15‰ water. Although the local T. lymma in full-strength sea water (30‰) had a much greater plasma urea concentration (380 mmol l-1), its urea excretion rate (4.7 μmol day-1 g-1) was comparable with that of H. signifier in 20‰ water. Therefore, H. signifer appears to have reduced its capacity to retain urea in order to survive in the freshwater environment and, consequently, it could not survive well in full-strength seawater.

Compositional correlates of metabolic depression in the mitochondrial membranes of estivating snails

The phospholipid and protein compositions of mitochondrial membranes from hepatopancreas of active and estivating terrestrial snails (Cepaea nemoralis) were compared. Mitochondria from estivating snails contained 82.7% less cardiolipin, and this was associated with an 83.9% reduction in cytochrome-c oxidase activity. Substantial changes also occurred in the proportional amounts of other individual phospholipid classes and their constituent fatty acids, including a 72% loss of total mitochondrial phospholipids, a 37% increase in monoenes, and 49% fewer n-3 fatty acids in membranes of estivating snails. These changes are consistent with those correlated with lowered metabolic rate and lower rates of proton leak in other animal models. Estivating snail hepatopancreas showed no change in total phospholipid content, indicating that the phospholipids lost from mitochondrial membranes may be sequestered elsewhere within the cell. We suggest that estivating snails remodel mitochondrial membranes as part of a coordinated, reversible suppression of mitochondrial membrane-associated processes, which may include a concomitant reduction in rates of proton pumping and leaking.The phospholipid and protein compositions of mitochondrial membranes from hepatopancreas of active and estivating terrestrial snails ( Cepaea nemoralis) were compared. Mitochondria from estivating snails contained 82.7% less cardiolipin, and this was associated with an 83.9% reduction in cytochrome- c oxidase activity. Substantial changes also occurred in the proportional amounts of other individual phospholipid classes and their constituent fatty acids, including a 72% loss of total mitochondrial phospholipids, a 37% increase in monoenes, and 49% fewer n-3 fatty acids in membranes of estivating snails. These changes are consistent with those correlated with lowered metabolic rate and lower rates of proton leak in other animal models. Estivating snail hepatopancreas showed no change in total phospholipid content, indicating that the phospholipids lost from mitochondrial membranes may be sequestered elsewhere within the cell. We suggest that estivating snails remodel mitochondrial membranes as part of a coordinated, reversible suppression of mitochondrial membrane-associated processes, which may include a concomitant reduction in rates of proton pumping and leaking.

Regulation of a renal urea transporter with reduced salinity in a marine elasmobranch, Raja erinacea.

SUMMARY Marine elasmobranchs retain urea and other osmolytes, e.g. trimethylamine oxide (TMAO), to counterbalance the osmotic pressure of seawater. We investigated whether a renal urea transporter(s) would be regulated in response to dilution of the external environment. A 779 bp cDNA for a putative skate kidney urea transporter (SkUT) was cloned, sequenced and found to display relatively high identity with facilitated urea transporters from other vertebrates. Northern analysis using SkUT as a probe revealed three signals in the kidney at 3.1, 2.8 and 1.6 kb. Upon exposure to 50% seawater, the levels of all three SkUT transcripts were significantly diminished in the kidney (by 1.8- to 3.5-fold). In response to environmental dilution, renal tissue osmolality and urea concentration decreased, whereas water content increased. There were no significant differences in osmolyte and mRNA levels between the dorsal–lateral bundle and ventral sections of the kidney. Taken together, these findings provide evidence that the downregulation of SkUT may play a key role in lowering tissue urea levels in response to external osmolality.

Correlations of plasma lipid metabolites with hibernation and lactation in wild black bears Ursus americanus

Abstract. During the denning period, black bears (Ursus americanus) are capable of enduring several months without food. At the same time, female bears that are pregnant or lactating have an added metabolic stress. Based on laboratory studies, much of the energy required to support metabolism and lactation during denning in black bears comes from lipid reserves. These lipid reserves are mobilized and the most metabolically active lipid fraction in the blood are nonesterified fatty acids (NEFA). Therefore, we hypothesized that plasma NEFAs would be higher in denning relative to active bears and in lactating relative to non-lactating female bears. We further hypothesized that in bears with elevated plasma NEFA levels, other lipid-related parameters (e.g., ketone bodies, albumin, cholesterol, lipase) would also be elevated in the plasma. Denning bears had significantly increased NEFA levels in all classes (saturates, monoenes, and polyenes). A doubling of plasma NEFA levels and a 33% increase in albumin, the plasma fatty acid binding protein, in denning bears, resulted in NEFA/albumin ratios that were higher in denning bears (4:1) compared to those of active bears (3:1). Bears became relatively ketonemic with a 17-fold increase in D-β-hydroxybutyrate levels during the denning period. Plasma cholesterol approximately doubled and lipase was ten-fold lower in denning relative to active bears. These findings indicate a strong correlation between plasma lipid metabolites and the denning period in a wild population of black bears.