The Relationship between Myoglobin, Aerobic Capacity, Nitric Oxide Synthase Activity and Mitochondrial Function in Fish Hearts.

The dynamic interactions between nitric oxide (NO) and myoglobin (Mb) in the cardiovascular system have received considerable attention. The loss of Mb, the principal O2 carrier and a NO scavenger/producer, in the heart of some red-blooded fishes provides a unique opportunity for assessing this globin's role in NO homeostasis and mitochondrial function. We measured Mb content, activities of enzymes of NO and aerobic metabolism [NO Synthase (NOS) and citrate synthase, respectively] and mitochondrial parameters [Complex-I and -I+II respiration, coupling efficiency, reactive oxygen species production/release rates and mitochondrial sensitivity to inhibition by NO (i.e., NO IC50)] in the heart of three species of red-blooded fish. The expression of Mb correlated positively with NOS activity and NO IC50, with low NOS activity and a reduced NO IC50 in the Mb-lacking lumpfish (Cyclopterus lumpus) as compared to the Mb-expressing Atlantic salmon (Salmo salar) and short-horned sculpin (Myoxocephalus scorpius). Collectively, our data show that NO levels are fine-tuned so that NO homeostasis and mitochondrial function are preserved; indicate that compensatory mechanisms are in place to tightly regulate [NO] and mitochondrial function in a species without Mb; and strongly suggest that the NO IC50 for oxidative phosphorylation is closely related to a fish's hypoxia tolerance.

Contrasting strategies of hypoxic cardiac performance and metabolism in cichlids and armoured catfish.

The heart of tropical fishes is a particularly useful model system in which to investigate mechanisms of hypoxic tolerance. Here we focus on insights gained from two groups of fishes, cichlids and armoured catfishes. Cichlids respond to hypoxia by entering a sustained hypometabolism with decreased heart performance to match whole animal circulatory needs. Heart rate is decreased along with protein turnover to reduce adenosine triphosphate demand. This occurs despite the inherent capacity for high levels of cardiac power development. Although highly hypoxic tolerant at the whole animal level, the heart of cichlids does not have high constitutive activities of glycolytic enzymes compared to other species. Information is conflicting with respect to changes in glycolytic gene expression and enzyme activity following hypoxic exposure with some studies showing increases and others decreases. In contrast to cichlids, species of armoured catfish, that are routinely exposed to water of low oxygen content, do not display hypoxic bradycardia. Under hypoxia there are early changes in glucose trafficking suggestive of activation of glycolysis before lactate accumulation. Thereafter, heart glycogen is mobilized and lactate accumulates in both heart and blood, in some species to very high levels. Heart performance under hypoxia is enhanced by defense of intracellular pH. A functional sarcoplasmic reticulum and binding of hexokinase to the outer mitochondrial membrane may also play a role in cardioprotection. Maintenance of heart performance under hypoxia may relate to a tradeoff between air breathing via a modified stomach and circulatory demands for digestion.

Interrelationship Between Contractility, Protein Synthesis and Metabolism in Mantle of Juvenile Cuttlefish (Sepia officinalis).

Young juvenile cuttlefish (Sepia officinalis) can grow at rates as high as 12% body weight per day. How the metabolic demands of such a massive growth rate impacts muscle performance that competes for ATP is unknown. Here, we integrate aspects of contractility, protein synthesis, and energy metabolism in mantle of specimens weighing 1.1 g to lend insight into the processes. Isolated mantle muscle preparations were electrically stimulated and isometric force development monitored. Preparations were forced to contract at 3 Hz for 30 s to simulate a jetting event. We then measured oxygen consumption, glucose uptake and protein synthesis in the hour following the stimulation. Protein synthesis was inhibited with cycloheximide and glycolysis was inhibited with iodoacetic acid in a subset of samples. Inhibition of protein synthesis impaired contractility and decreased oxygen consumption. An intact protein synthesis is required to maintain contractility possibly due to rapidly turning over proteins. At least, 41% of whole animal MO2 is used to support protein synthesis in mantle, while the cost of protein synthesis (50 µmol O2 mg protein-1) in mantle was in the range reported for other aquatic ectotherms. A single jetting challenge stimulated protein synthesis by approximately 25% (2.51-3.12% day-1) over a 1 h post contractile period, a similar response to that which occurs in mammalian skeletal muscle. Aerobic metabolism was not supported by extracellular glucose leading to the contention that at this life stage either glycogen or amino acids are catabolized. Regardless, an intact glycolysis is required to support contractile performance and protein synthesis in resting muscle. It is proposed that glycolysis is needed to maintain intracellular ionic gradients. Intracellular glucose at approximately 3 mmol L-1 was higher than the 1 mmol L-1 glucose in the bathing medium suggesting an active glucose transport mechanism. Octopine did not accumulate during a single physiologically relevant jetting challenge; however, octopine accumulation increased following a stress that is sufficient to lower Arg-P and increase free arginine.

Reversion to developmental pathways underlies rapid arm regeneration in juvenile European cuttlefish, Sepia officinalis (Linnaeus 1758).

Coleoid cephalopods, including the European cuttlefish (Sepia officinalis), possess the remarkable ability to fully regenerate an amputated arm with no apparent fibrosis or loss of function. In model organisms, regeneration usually occurs as the induction of proliferation in differentiated cells. In rare circumstances, regeneration can be the product of naïve progenitor cells proliferating and differentiating de novo . In any instance, the immune system is an important factor in the induction of the regenerative response. Although the wound response is well-characterized, little is known about the physiological pathways utilized by cuttlefish to reconstruct a lost arm. In this study, the regenerating arms of juvenile cuttlefish, with or without exposure at the time of injury to sterile bacterial lipopolysaccharide extract to provoke an antipathogenic immune response, were assessed for the transcription of early tissue lineage developmental genes, as well as histological and protein turnover analyses of the resulting regenerative process. The transient upregulation of tissue-specific developmental genes and histological characterization indicated that coleoid arm regeneration is a stepwise process with staged specification of tissues formed de novo, with immune activation potentially affecting the timing but not the result of this process. Together, the data suggest that rather than inducing proliferation of mature cells, developmental pathways are reinstated, and that a pool of naïve progenitors at the blastema site forms the basis for this regeneration.

Corrigendum: Hypoxic Induced Decrease in Oxygen Consumption in Cuttlefish (Sepia officinalis) Is Associated with Minor Increases in Mantle Octopine but No Changes in Markers of Protein Turnover.

[This corrects the article DOI: 10.3389/fphys.2017.00344.].

The benefit of being still: energy savings during winter dormancy in fish come from inactivity and the cold, not from metabolic rate depression.

Winter dormancy is used by many animals to survive the cold and food-poor high-latitude winter. Metabolic rate depression, an active downregulation of resting cellular energy turnover and thus standard (resting) metabolic rate (SMR), is a unifying strategy underlying the persistence of organisms in such energy-limited environments, including hibernating endotherms. However, controversy exists about its involvement in winter-dormant aquatic ectotherms. To address this debate, we conducted simultaneous, multi-day measurements of whole-animal oxygen consumption rate (a proxy of metabolic rate) and spontaneous movement in a model winter-dormant marine fish, the cunner (Tautogolabrus adspersus). Winter dormancy in cunner involved a dampened diel rhythm of metabolic rate, such that a low and stable metabolic rate persisted throughout the 24 h day. Based on the thermal sensitivity (Q10) of SMR as well as correlations of metabolic rate and movement, the reductions in metabolic rate were not attributable to metabolic rate depression, but rather to reduced activity under the cold and darkness typical of the winter refuge among substrate. Previous reports of metabolic rate depression in cunner, and possibly other fish species, during winter dormancy were probably confounded by variation in activity. Unlike hibernating endotherms, and excepting the few fish species that overwinter in anoxic waters, winter dormancy in fishes, as exemplified by cunner, need not involve metabolic rate depression. Rather, energy savings come from inactivity combined with passive physico-chemical effects of the cold on SMR, demonstrating that thermal effects on activity can greatly influence temperature-metabolism relationships, and illustrating the benefit of simply being still in energy-limited environments.

Species-specific low plasma glucose in fish is associated with relatively high tissue glucose content and is inversely correlated with cardiac glycogen content.

The relationship between plasma glucose concentration and intracellular glucose (liver, heart, brain, gill, gonad, intestine, kidney, spleen, white muscle) was determined in fish species with a range in plasma glucose (Atlantic cod, 5.06 mM; cunner, 3.8 mM; rainbow trout, 3.7 mM; lumpfish, 0.9 mM; short-horned sculpin, 0.6 mM; and winter flounder, 0.6 mM). The ratio of intracellular glucose/plasma glucose was always higher than one in liver for all species consistent with a diffusion gradient from the tissue to the plasma. In all other tissues in Atlantic cod, cunner, and rainbow trout the diffusion gradient was from the plasma to the intracellular space. In short-horned sculpin, the mean ratio in heart and white muscle exceeded one and in winter flounder the ratio was significantly greater than one at 5.97 and 2.92 for heart and muscle, respectively. The presence of an active glucose 6-phosphatase in white muscle could account for elevated amounts of free glucose. The white muscle of all species displayed phosphoenolpyruvate carboxykinase and in winter flounder the activity was as high in white muscle as in liver suggesting that gluconeogenesis may be associated with a relatively high-muscle glucose content. The glycogen content was highest in liver followed by heart with lower amounts in all other tissues. There was an inverse correlation between heart glycogen content and plasma glucose concentration when all species were included. It is contended that in species with low plasma glucose, heart glycogen is accumulated at a slow rate under normoxia, to be called upon under hypoxic conditions when the gradient for inward diffusion is unfavourable for high rates of glucose metabolism.

Low plasma glucose limits glucose metabolism by RBCs and heart in some species of teleosts.

Within teleosts there is a species range in plasma glucose levels from undetectable to 20mM. At low plasma glucose levels the gradient from the extracellular to the intracellular space is decreased. The impact of this on glucose metabolism by RBCs and heart from species with different steady state levels of plasma glucose (Atlantic cod ~5mM; Atlantic salmon ~5mM, cunner ~1mM, lumpfish <1mM; short-horned sculpin <1mM) is the subject of this review. Under normoxia, at physiological levels of extracellular glucose, RBCs and heart produce lactate although the contribution of anaerobic metabolism to ATP production is small. Sustained lactate production from extracellular glucose appears to be the primary fate of extracellular glucose. In many cases, glycogen is not mobilized and the rate of glucose metabolism=two times the rate of lactate production. As such, alternative metabolic sources are required to fuel oxidative metabolism. Under hypoxia, hearts from Atlantic cod and rainbow trout increase rates of both glucose metabolism and lactate production, partially supported by glycogen reserves. But in lumpfish and short-horned sculpin hearts there is no change in rates of glucose metabolism. The most likely explanation is that glucose uptake is compromised in lumpfish and short-horned sculpin hearts due to a low diffusion gradient. Under these conditions rates of lactate production are well below that of Atlantic cod or rainbow trout. Energy demand must be reduced under hypoxia in lumpfish and short-horned sculpin hearts in order to maintain ATP balance.

Hypoxic Induced Decrease in Oxygen Consumption in Cuttlefish (Sepia officinalis) Is Associated with Minor Increases in Mantle Octopine but No Changes in Markers of Protein Turnover.

The common cuttlefish (Sepia officinalis), a dominant species in the north-east Atlantic ocean and Mediterranean Sea, is potentially subject to hypoxic conditions due to eutrophication of coastal waters and intensive aquaculture. Here we initiate studies on the biochemical response to an anticipated level of hypoxia. Cuttlefish challenged for 1 h at an oxygen level of 50% dissolved oxygen saturation showed a decrease in oxygen consumption of 37% associated with an 85% increase in ventilation rate. Octopine levels were increased to a small but significant level in mantle, whereas there was no change in gill or heart. There were no changes in mantle free glucose or glycogen levels. Similarly, the hypoxic period did not result in changes in HSP70 or polyubiquinated protein levels in mantle, gill, or heart. As such, it appears that although there was a decrease in metabolic rate there was only a minor increase in anaerobic metabolism as evidenced by octopine accumulation and no biochemical changes that are hallmarks of alterations in protein trafficking. Experiments with isolated preparations of mantle, gill, and heart revealed that pharmacological inhibition of protein synthesis could decrease oxygen consumption by 32 to 42% or Na+/K+ ATPase activity by 24 to 54% dependent upon tissue type. We propose that the decrease in whole animal oxygen consumption was potentially the result of controlled decreases in the energy demanding processes of both protein synthesis and Na+/K+ ATPase activity.

Low levels of extracellular glucose limit cardiac anaerobic metabolism in some species of fish.

There is a wide interspecific range in plasma glucose levels in teleosts from less than 0.5 to greater than 10 mmol l-1 Here we assessed how glucose availability influences glucose metabolism in hearts of Atlantic cod (Gadus morhua), rainbow trout (Oncorhynchus mykiss), lumpfish (Cyclopterus lumpus) and short-horned sculpin (Myoxocephalus scorpius) under normoxic and hypoxic conditions. These species had plasma glucose levels of 5.1, 4.8, 0.9 and 0.5 mmol l-1, respectively. Rates of glucose metabolism and lactate production were determined in isolated hearts perfused with medium containing physiological levels of glucose. Under normoxic conditions there was no significant difference in rates of either glucose metabolism (average 15 nmol g-1 min-1) or lactate production (average 30 nmol g-1 min-1) across species. Under hypoxia (12% of air saturation) there were significant increases in rates of glucose metabolism and lactate production in hearts from Atlantic cod (glucose-130; lactate-663 nmol g-1 min-1) and rainbow trout (glucose-103; lactate-774 nmol g-1 min-1); however, there was no change in rate of glucose metabolism in hearts from either lumpfish or short-horned sculpin and only increases in lactate production to rates much lower than the other species. Furthermore, Atlantic cod hearts perfused with medium containing low non-physiological levels of glucose (0.5 mmol l-1) had the same rates of glucose metabolism under normoxic and hypoxic treatments. Anaerobic metabolism supported by extracellular glucose is compromised in fish with low levels of plasma glucose, which in turn may decrease performance under oxygen-limiting conditions at the whole-animal level.

High rates of glucose utilization in the gas gland of Atlantic cod (Gadus morhua) are supported by GLUT1 and HK1b.

The gas gland of physoclistous fish utilizes glucose to generate lactic acid that leads to the off-loading of oxygen from haemoglobin. This study addresses characteristics of the first two steps in glucose utilization in the gas gland of Atlantic cod (Gadus morhua). Glucose metabolism by isolated gas gland cells was 12- and 170-fold higher, respectively, than that in heart and red blood cells (RBCs) as determined by the production of (3)H2O from [2-(3)H]glucose. In the gas gland, essentially all of the glucose consumed was converted to lactate. Glucose uptake in the gas gland shows a very high dependence upon facilitated transport as evidenced by saturation of uptake of 2-deoxyglucose at a low extracellular concentration and a requirement for high levels of cytochalasin B for uptake inhibition despite the high efficacy of this treatment in heart and RBCs. Glucose transport is via glucose transporter 1 (GLUT1), which is localized to the glandular cells. GLUT1 western blot analysis from whole-tissue lysates displayed a band with a relative molecular mass of 52 kDa, consistent with the deduced amino acid sequence. Levels of 52 kDa GLUT1 in the gas gland were 2.3- and 33-fold higher, respectively, than those in heart and RBCs, respectively. Glucose phosphorylation is catalysed by hexokinase Ib (HKIb), a paralogue that cannot bind to the outer mitochondrial membrane. Transcript levels of HKIb in the gas gland were 52- and 57-fold more abundant, respectively, than those in heart and RBCs. It appears that high levels of GLUT1 protein and an unusual isoform of HKI are both critical for the high rates of glycolysis in gas gland cells.

Enzymatic capacities of metabolic fuel use in cuttlefish (Sepia officinalis) and responses to food deprivation: insight into the metabolic organization and starvation survival strategy of cephalopods.

Food limitation is a common challenge for animals. Cephalopods are sensitive to starvation because of high metabolic rates and growth rates related to their "live fast, die young" life history. We investigated how enzymatic capacities of key metabolic pathways are modulated during starvation in the common cuttlefish (Sepia officinalis) to gain insight into the metabolic organization of cephalopods and their strategies for coping with food limitation. In particular, lipids have traditionally been considered unimportant fuels in cephalopods, yet, puzzlingly, many species (including cuttlefish) mobilize the lipid stores in their digestive gland during starvation. Using a comprehensive multi-tissue assay of enzymatic capacities for energy metabolism, we show that, during long-term starvation (12 days), glycolytic capacity for glucose use is decreased in cuttlefish tissues, while capacities for use of lipid-based fuels (fatty acids and ketone bodies) and amino acid fuels are retained or increased. Specifically, the capacity to use the ketone body acetoacetate as fuel is widespread across tissues and gill has a previously unrecognized capacity for fatty acid catabolism, albeit at low rates. The capacity for de novo glucose synthesis (gluconeogenesis), important for glucose homeostasis, likely is restricted to the digestive gland, contrary to previous reports of widespread gluconeogenesis among cephalopod tissues. Short-term starvation (3-5 days) had few effects on enzymatic capacities. Similar to vertebrates, lipid-based fuels, putatively mobilized from fat stores in the digestive gland, appear to be important energy sources for cephalopods, especially during starvation when glycolytic capacity is decreased perhaps to conserve available glucose.

Metabolic rate and rates of protein turnover in food-deprived cuttlefish, Sepia officinalis (Linnaeus 1758).

To determine the metabolic response to food deprivation, cuttlefish (Sepia officinalis) juveniles were either fed, fasted (3 to 5 days food deprivation), or starved (12 days food deprivation). Fasting resulted in a decrease in triglyceride levels in the digestive gland, and after 12 days, these lipid reserves were essentially depleted. Oxygen consumption was decreased to 53% and NH4 excretion to 36% of the fed group following 3-5 days of food deprivation. Oxygen consumption remained low in the starved group, but NH4 excretion returned to the level recorded for fed animals during starvation. The fractional rate of protein synthesis of fasting animals decreased to 25% in both mantle and gill compared with fed animals and remained low in the mantle with the onset of starvation. In gill, however, protein synthesis rate increased to a level that was 45% of the fed group during starvation. In mantle, starvation led to an increase in cathepsin A-, B-, H-, and L-like enzyme activity and a 2.3-fold increase in polyubiquitin mRNA that suggested an increase in ubiquitin-proteasome activity. In gill, there was a transient increase in the polyubiquitin transcript levels in the transition from fed through fasted to the starved state and cathepsin A-, B-, H-, and L-like activity was lower in starved compared with fed animals. The response in gill appears more complex, as they better maintain rates of protein synthesis and show no evidence of enhanced protein breakdown through recognized catabolic processes.

Extracellular glucose supports lactate production but not aerobic metabolism in cardiomyocytes from both normoglycemic Atlantic cod and low glycemic short-horned sculpin.

Fish exhibit a wide range of species-specific blood glucose levels. How this relates to glucose utilization is yet to be fully realized. Here, we assessed glucose transport and metabolism in myocytes isolated from Atlantic cod (Gadus morhua) and short-horned sculpin (Myoxocephalus scorpius), species with blood glucose levels of 3.7 and 0.57 mmol l(-1), respectively. Glucose metabolism was assessed by the production of (3)H2O from [2-(3)H]glucose. Glucose metabolism was 3.5- to 6-fold higher by myocytes from Atlantic cod than by those from short-horned sculpin at the same level of extracellular glucose. In Atlantic cod myocytes, glucose metabolism displayed what appears to be a saturable component with respect to extracellular glucose, and cytochalasin B inhibited glucose metabolism. These features revealed a facilitated glucose diffusion mechanism that accounts for between 30% and 55% of glucose entry at physiological levels of extracellular glucose. Facilitated glucose diffusion appears to be minimal in myocytes for short-horned sculpin. Glucose entry by simple diffusion occurs in both cell types with the same linear relationship between glucose metabolism and extracellular glucose concentration, presumably due to similarities in membrane composition. Oxygen consumption by myocytes incubated in medium containing physiological levels of extracellular glucose (Atlantic cod 5 mmol l(-1), short-horned sculpin 0.5 mmol l(-1)) was similar in the two species and was not decreased by cytochalasin B, suggesting that these cells have the capability of oxidizing alternative on-board metabolic fuels. Cells produced lactate at low rates but glycogen levels did not change during the incubation period. In cells from both species, glucose utilization assessed by both simple chemical analysis of glucose disappearance from the medium and (3)H2O production was half the rate of lactate production and as such extracellular glucose was not available for oxidative metabolism. Overall, extracellular glucose makes only a minor contribution to ATP production but a sustained glycolysis may be necessary to support Ca(2+) transport mechanisms at either the sarcoplasmic reticulum or the sarcolemmal membrane.

Physiological responses to digestion in low salinity in the crabs Carcinus maenas and Cancer irroratus.

Osmoregulation and digestion are energetically demanding, and crabs that move into low salinity environments to feed must be able to balance the demands of both processes. Achieving this balance may pose greater challenges for weak than for efficient osmoregulators. This study examined the rate of oxygen consumption (MO2) of Carcinus maenas (efficient osmoregulator) and Cancer irroratus (weak osmoregulator) as a function of feeding and hyposaline stress. The MO2 increased 2-fold in both species following feeding. The MO2 increased and remained elevated in fasted crabs during acute hyposaline exposure. When hyposaline stress occurred after feeding, C. maenas responded with an immediate summation of the MO2 associated with feeding and hyposaline stress, whereas C. irroratus reacted with a partial summation of responses in a salinity of 24‰, but were unable to sum responses in 16‰. C. irroratus exhibited longer gut transit times. This may be due to an inability to regulate osmotic water onload as efficiently as C. maenas. Mechanical digestion in crabs can account for a significant portion of SDA, and a short term interruption led to the delay in summation of metabolic demands. Although protein synthesis is reported to account for the majority of SDA, this did not appear to be the case here. Protein synthesis rates were higher in C. irroratus but neither feeding or salinity affected protein synthesis rates of either species which suggests that protein synthesis can continue in low salinity as long as substrates are available.

Cloning and characterization of aquaglyceroporin genes from rainbow smelt (Osmerus mordax) and transcript expression in response to cold temperature.

Aquaglyceroporins (GLPs) are integral membrane proteins that facilitate passive movement of water, glycerol and urea across cellular membranes. In this study, GLP-encoding genes were characterized in rainbow smelt (Osmerus mordax mordax), an anadromous teleost that accumulates high glycerol and modest urea levels in plasma and tissues as an adaptive cryoprotectant mechanism in sub-zero temperatures. We report the gene and promoter sequences for two aqp10b paralogs (aqp10ba, aqp10bb) that are 82% identical at the predicted amino acid level, and aqp9b. Aqp10bb and aqp9b have the 6 exon structure common to vertebrate GLPs. Aqp10ba has 8 exons; there are two additional exons at the 5' end, and the promoter sequence is different from aqp10bb. Molecular phylogenetic analysis suggests that the aqp10b paralogs arose from a gene duplication event specific to the smelt lineage. Smelt GLP transcripts are ubiquitously expressed; however, aqp10ba transcripts were highest in kidney, aqp10bb transcripts were highest in kidney, intestine, pyloric caeca and brain, and aqp9b transcripts were highest in spleen, liver, red blood cells and kidney. In cold-temperature challenge experiments, plasma glycerol and urea levels were significantly higher in cold- compared to warm-acclimated smelt; however, GLP transcript levels were generally either significantly lower or remained constant. The exception was significantly higher aqp10ba transcript levels in kidney. High aqp10ba transcripts in smelt kidney that increase significantly in response to cold temperature in congruence with plasma urea suggest that this gene duplicate may have evolved to allow the re-absorption of urea to concomitantly conserve nitrogen and prevent freezing.

Rainbow smelt: the unusual case of cryoprotection by sustained glycerol production in an aquatic animal.

Rainbow smelt flourish at -1.8 °C, the freezing point of sea water. An antifreeze protein contributes to freeze point depression but, more importantly, cryoprotection is due to an elevation in osmotic pressure, by the accumulation of glycerol. The lower the water temperature, the higher the plasma glycerol with levels recorded as high as 400 mmol l(-1). Glycerol freely diffuses out in direct relation to the glycerol concentration and fish may lose as much as 15% of their glycerol reserve per day. Glycerol levels decrease from a maximum in February/March while water temperature is still sub-zero. The decrease in glycerol may respond to a photoperiod signal as opposed to initiation which is triggered by low temperature. The initial increase in glycerol level is supported by liver glycogen but high sustained glycerol level is dependent upon dietary carbohydrate and protein. The metabolic pathways leading to glycerol involve flux from glycogen/glucose to the level of dihydroxyacetone phosphate (DHAP) via the initial part of glycolysis and from amino acids via a truncated gluconeogenesis again to the level of DHAP. DHAP in turn is converted to glycerol 3-phosphate (G3P) and then directly to glycerol. The key to directing DHAP to G3P is a highly active glycerol 3-P dehydrogenase. G3P is converted directly to glycerol via G3P phosphatase, the rate-limiting step in the process. The transition to glycerol production is associated with increased activities of enzymes at key loci in the top part of glycogenolysis/glycolysis. Curtailment of the final section of glycolysis may reside at the level of pyruvate oxidation with an inactivation of pyruvate dehydrogenase (PDH) driven by increased levels of PDH kinase. Enzymes associated with amino acid trafficking are elevated as is the pivotal enzyme phosphoenolpyruvate carboxykinase.

Gut transport characteristics in herbivorous and carnivorous serrasalmid fish from ion-poor Rio Negro water.

Three closely related characids, Tambaqui (omnivore), black Piranha (carnivore), and Pacu (herbivore), all Serrasalmidae, inhabit the ion-poor, acidic Rio Negro. We compared O2-consumption and N excretion rates in vivo, and sodium, chloride, glucose, and ammonia transport characteristics of gut sac preparations in vitro. The Pacu had a significantly higher weight-specific oxygen consumption, and a lower N/Q ratio than the omnivorous Tambaqui, and a significantly lower urea-N excretion rate than the carnivorous black Piranha, suggesting N-limitation in the herbivorous Pacu. With a value of 2.62 ± 0.15, gut to fork length ratio in the Pacu was about 2.5 times higher than in the black Piranha, and 2.0 times higher than in the Tambaqui. Anterior intestinal activities of three enzymes involved in N-fixation for amino acid synthesis (glutamate dehydrogenase, glutamate-oxaloacetate transferase, and glutamate-pyruvate transferase) were generally greatest in the carnivore and lowest in the herbivore species. In all three species, sodium, chloride, glucose, and ammonia were taken up at high rates from the intestine, resulting in an isosmotic fluid flux. Comparing the area-specific fluid flux of the anterior, mid, and posterior gut sections, no difference was detected between the three sections of the Pacu, while in the Tambaqui, it was highest in the anterior section, and in the black Piranha highest in the middle section. Overall, the area-specific uptake rates for sodium, chloride, glucose, and ammonia of anterior, mid, and posterior sections were similar in all three species, indicating that there is no difference in the area-specific transport rates associated with trophic position. The net ammonia uptake flux from gut interior was not significantly different from the net ammonia efflux to the serosal fluid, so that the ammonia removed from the intestine by the mucosal epithelium was quantitatively transferred through the tissue to the serosal side in all three species. Thus, metabolic activity of gut tissue did not significantly influence the net ammonia transfer. Due to the much higher gut to fork length ratio, the overall transport capacity of the gut of the herbivorous Pacu by far exceeded the transport capacity of their carnivorous and omnivorous relatives, thus compensating for the lower digestibility and the low Na(+), Cl(-), and N-content of the plant diet. Accordingly, in order to cope with the more difficult digestible plant material and the very low nitrogen content of plants, herbivorous fish have not evolved more effective area-specific transport capacities, but rather have increased the length of the gut.

Extracellular glucose can fuel metabolism in red blood cells from high glycemic Atlantic cod (Gadus morhua) but not low glycemic short-horned sculpin (Myoxocephalus scorpius).

Energy metabolism was assessed in red blood cells (RBCs) from Atlantic cod and short-horned sculpin, two species that have markedly different levels of blood glucose. The objective was to determine whether the level of extracellular glucose has an impact on rates of glucose metabolism. The blood glucose level was 2.5 mmol l(-1) in Atlantic cod and 0.2 mmol l(-1) in short-horned sculpin, respectively. Oxygen consumption, lactate production and glucose utilization were measured in whole blood and related to grams of RBCs. Glucose utilization was assessed by measuring both glucose disappearance and the production of (3)H2O from [2-(3)H]-glucose. RBCs from both species have an aerobic-based metabolism. In Atlantic cod, extracellular glucose is sufficient to provide the sum of glucosyl equivalents to support both oxidative metabolism and lactate production. In contrast, extracellular glucose can account for only 10% of the metabolic rate in short-horned sculpin RBCs. In both species, about 70% of glucose enters the RBCs via facilitated transport. The difference in rates of extracellular glucose utilization is related to the extremely low levels of blood glucose in short-horned sculpin. In this species energy metabolism by RBCs must be supported by alternative fuels.

Transcript levels of class I GLUTs within individual tissues and the direct relationship between GLUT1 expression and glucose metabolism in Atlantic cod (Gadus morhua).

GLUTs 1-4 are sodium-independent facilitated glucose transporters and are considered to play a major role in glucose trafficking. The relative transcript levels of GLUTs 1-4 were determined in tissues of Atlantic cod (Gadus morhua). The distribution profile of GLUTs normalized to RNA is similar to mammals and with a few exceptions other fish. GLUT1 is ubiquitous, GLUT2 is relatively abundant in tissues that release glucose, GLUT3 expression is relatively strong in brain, and GLUT4 is relatively high in heart and muscle. The functionally significant level of transcript is presumably the level in the cell. Normalization of relative GLUT levels to tissue mass reveals there are extremely high levels of GLUT1 transcript in gas gland consistent with the high lactate production rates, GLUT3 is dominant in gill and head kidney as well as brain, and GLUT4 expression in gill is elevated relative to other tissues. Consideration of GLUTs within tissues reveals that GLUT1 is the dominant transcript in a group of tissues including gas gland, heart, white muscle, and RBCs. Brain, gill, and spleen display a co-dominance of GLUTs 1 and 3. There are relatively low levels of GLUT4 in most tissues, the highest being found in white muscle where GLUT4 accounts for only 12 % of the total transcript level. The apparent low level of GLUT4 transcript may reflect two tissues that were not included in the current study, red muscle and adipose tissue, due to their low abundance in Atlantic cod. The rate of glucose metabolism in isolated cells prepared from gas gland, heart, and RBCs was determined by tracking the rate of (3)H2O production from [2-(3)H]-glucose. The steady-state rate of basal glycolysis in these three tissues correlates with relative transcript levels of GLUT1.