Transcriptional analysis of LPS-stimulated activation of trout (Oncorhynchus mykiss) monocyte/macrophage cells in primary culture treated with cortisol.

Primary immune responses to pathogen invasion are mediated by the innate immune system in which tissue macrophages play a key role. During infectious processes glucocorticoids generally may function to dampen inflammatory responses. In this study, the ability of cortisol to directly modulate the transcriptional response of rainbow trout macrophages to the cellular activator lipopolysaccharide (LPS) was investigated. The results indicate that cortisol significantly inhibits the well-described LPS-dependent induction of the expression of TNF-alpha2, a pro-inflammatory cytokine. In order to further characterize the molecular effects of LPS and the immunomodulatory role of cortisol, the in vitro macrophage response to LPS in the absence or presence of 12-h cortisol exposure was analyzed utilizing a salmonid-specific microarray platform. Genes that were stimulated or inhibited with LPS plus cortisol fell into several major functional groups. The first, a general "response" group comprising genes within ontology classes including the response to external stimuli, stress, humoral immunity and apoptosis, exhibited a significant increase after LPS stimulation, whereas suppression of this response was observed in the presence of cortisol. LPS stimulated other genes in a second group involved in cell signalling and also genes in a third group involved in the activation of transcription. Categories activated with cortisol were mainly related to various aspects of metabolism (including protein biosynthesis, binding and transport of ions) and structural proteins (mainly cytoskeleton and microtubules). The immunomodulatory action of cortisol on LPS-stimulated macrophages therefore appears more complex than simply the antagonism of LPS-induced transcriptional responses.

Rainbow trout glucose transporter (OnmyGLUT1): functional assessment in Xenopus laevis oocytes and expression in fish embryos.

Recently, we reported the cloning of a putative glucose transporter (OnmyGLUT1) from rainbow trout embryos. In this paper, we describe the functional characteristics of OnmyGLUT1 and its expression during embryonic development of rainbow trout. Transport of D-glucose was analysed in Xenopus laevis oocytes following microinjection of mRNA transcribed in vitro. These experiments confirmed that OnmyGLUT1 is a facilitative Na(+)-independent transporter. Assessment of substrate selectivity, sensitivity to cytochalasin B and phloretin and kinetic parameters showed that the rainbow trout glucose transporter was similar to a carp transporter and to mammalian GLUT1. Embryonic expression of OnmyGLUT1 was studied using whole-mount in situ hybridization. Ubiquitous distribution of transcripts was observed until the early phase of somitogenesis. During the course of organogenesis, somitic expression decreased along the rostro-caudal axis, finally ceasing in the mature somites. The OnmyGLUT1 transcripts were detected in the neural crest during the whole study period. Transcripts were also found in structures that are likely to originate from the neural crest cells (gill arches, pectoral fins, upper jaw, olfactory organs and primordia of mouth lips). Hexose transport activity was detected at all developmental stages after blastulation. Cytochalasin B blocked the accumulation of phosphorylated 2-deoxy-D-glucose by dissociated embryonic cells, suggesting an important role for transport in glucose metabolism.

Rainbow trout (Onchorhynchus mykiss) hepatic glucose transporter.

We report identification of a rainbow trout hepatic glucose transporter sharing 58% and 52% amino acid identity with avian and mammalian GLUT2 sequences, respectively. The functionality of OnmyGLUT2 was assessed by expression in rainbow trout embryos. We also measured the transport of hexose in isolated rainbow trout hepatocytes. Inhibition of 3-O-methylglucose uptake by cytochalasin B, phloretin and 2-deoxy-D-glucose suggested the existence of a functional facilitative transporter in these cells. Expression of OnmyGLUT2 was found in the liver, kidney and intestine.

Monosaccharide uptake in common carp (Cyprinus carpio) EPC cells is mediated by a facilitative glucose carrier.

In most animal cells, transport of monosaccharides across the plasma membrane is mediated by glucose transporters (GLUT). Mammals express at least five distinct transporters (GLUTs 1--5), which are well characterised both functionally and genetically. In contrast, the glucose transport system of fish remains poorly studied. Here we report studies of hexose uptake in carp EPC cells and cloning of a glucose transporter cDNA from these cells. Transport of radio-labelled methylglucose (3-OMG) followed Michaelis--Menten kinetics with a K(m) value (8.5 mM) similar to that of mammalian cells. The inhibition of transport by cytochalasin B and phloretin, but not by phloridzin or cyanide, strongly suggested the existence of a facilitative carrier. D-Glucose, 2-deoxyglucose, 3-OMG, D-mannose and D-xylose were competitive inhibitors of 3-OMG uptake, while L-glucose, mannitol, D-fructose, D-ribose and sucrose did not compete with 3-OMG. We cloned a carp glucose transporter (CyiGLUT1), using RT-PCR and RACE strategies. CyiGLUT1 was different from known carp and zebrafish EST sequences. The complete cDNA (3060 bp) contained one open reading frame encoding a predicted protein of 478 amino acids. The deduced amino acid sequence shared 78% identity with mammalian and avian GLUT1 proteins. Key amino acids involved in substrate selection and catalysis of mammalian GLUTs were conserved in the carp transporter.

Changes in tissue cellularity are associated with growth enhancement in genetically modified arctic char (Salvelinus alpinus L.) carrying recombinant growth hormone gene.

Biochemical and histological analyses were used to study the number and size of cells (cellularity) in tissues of fast-growing, genetically modified Arctic char (Salvelinus alpinus L.), overexpressing sockeye salmon (Oncorhynchus nerka) growth hormone gene (OnGH1). DNA contents of muscle, heart, and liver were compared in transformed, sibling (age control) and 1 year older (size control) char. Total white muscle cross-sectional area, white muscle fiber number, and total nuclei number within the muscle tissue were determined from one complete half-section of each fish. The analyzed tissues responded differently to growth hormone overproduction. In muscle tissue of OnGH1-transformed char, the enhanced growth was clearly associated with proliferation of muscle cells (hyperplasia), whereas in heart tissue both cell proliferation and increase in cell size (hypertrophy) were enhanced. The relative DNA concentration in the liver of transformed char was significantly greater than that of control fish, suggesting reduction in size of hepatic cells.

Cloning and characterization of glucose transporter in teleost fish rainbow trout (Oncorhynchus mykiss).

The facilitated diffusion of monosaccharides across the plasma membrane is mediated by glucose transporters (GLUTs). In contrast to mammals, the glucose transport system of lower vertebrates remains unexplored. We detected glucose transport activity in rainbow trout embryos. Two GLUTs sharing 83% amino acid identity were cloned from juvenile fish, these have been denoted OnmyGLUT1A and OnmyGLUT1B. In adult trout OnmyGLUT1A is predominantly expressed in the heart with low expression in other tissues. An inverse terminal repeat of a Tc1-like transposable element was found in the 3'-untranslated region of OnmyGLUT1B. Phylogenetic analysis suggested that rainbow trout genes share a common ancestor with higher vertebrate GLUT1. We also found GLUT genes in several salmonid species.

Transfer of growth hormone (GH) transgenes into Arctic charr. (Salvelinus alpinus L.) II. Nutrient partitioning in rapidly growing fish.

To examine whether the utilization of protein and lipids is altered in the genetically modified, rapidly growing charr, we compared CMVOnGH1 transgenic and sibling fish. Muscle composition and rates of gas exchange were analyzed. Plasma metabolites were determined in the recently fed and post-absorptive state. No difference was found in muscle composition. At equal rates of protein accretion, the rate of NH4 excretion was 43% greater in sibling charr. The lower molar ratio of NH4 to O2 exchange implied the reduced expenditure of metabolized protein in transgenic charr. Plasma NH4 concentration in transgenic fish did not differ from that in sibling charr whereas the greater level of total CO2 indicated enhanced oxidation of non-protein nutrients. Decreased plasma triglycerides concentration and lower triglyceride to cholesterol ratio showed faster utilization of ingested lipids in transgenic charr, especially of energy-containing fraction. However, this was not accompanied with a reduced lipid content or altered fatty acid composition of muscle triglycerides or phospholipids. Comparative studies suggested that the transgenic charr had acquired features of domesticated salmonid fish. Their increased metabolic rate and enhanced utilization of dietary lipids, especially triglycerides, resembled the characteristics of domestic rainbow trout rather than wild counterparts.

Transfer of growth hormone (GH) transgenes into Arctic charr. (Salvelinus alpinus L.) I. Growth response to various GH constructs.

Four constructs containing salmonid growth hormone (GH) genes were transferred to Arctic charr (Salvelinus alpinus L.). Cytomegalovirus (CMV) and piscine metallothionein B (OnMT) and histone 3 (OnH3) promoters connected to sockeye salmon growth hormone 1 gene (OnGH1) were used for ectopic expression, and Atlantic salmon growth hormone 2 gene with 5'flanking region (SsGH2) was tested for pituitary-specific expression. Charr carrying the OnGH1 constructs showed a dramatic increase in growth rate. The 10-month old transformed fish were 14-fold heavier than control siblings. The ability of the CMVGH1 construct to promote growth was greater than that obtained in fish with piscine promoters. Analysis of individual growth curves of charr carrying the OnH3GH1 transgene indicated a stable ratio of specific growth rates in transformed and control fish regardless of fish size. No alteration in growth performance was found in fish carrying the SsGH2 transgene. There was evidence that the transformed rainbow trout (Oncorhynchus mykiss) were unable to produce SsGH2 mRNA in their pituitary glands. The presence of the transgene in various tissues was examined in trout to evaluate the reliability of one-tissue sampling.

Gene transfer for targeted modification of salmonid fish metabolism.

The reviewed studies addressed the possibility of using gene transfer for correction of L-ascorbic acid biosynthesis and carbohydrate utilization in rainbow trout. Analyses of enzymatic activities in the L-AAB pathway indicated that reasons for the lack of L-AA production can be common in fish and scurvy-prone animals. Rat gulonolactone oxidase cDNA was transferred into trout. Regardless of the fact that rGLO transcription occurred in embryos, neither GLO protein, nor enzyme activity were detected. There was no production of L-AA in transgenic fish raised on vitamin C-free diets or injected with L-gulonolactone. These results indicated that the conditions required for translation or stability of rGLO were not present in trout tissues. To augment carbohydrates utilization, human glucose transporter 1 and rat hexokinase II cDNAs were tested. In the transfected embryos. HK activity, rates of hexose uptake and glucose oxidation were increased. The effect of hGLUT1 on glucose metabolism was greater than that of rHKII. Trout carrying hGLUT1 and rHKII with viral or piscine promoters were created. Though interpretation of the metabolic effects of the transgenes was complicated with mosaicism, a tendency to improved carbohydrate utilization was revealed in some of the transgenic individuals.

Rainbow trout (Oncorhynchus mykiss) exposed to oxygen supersaturation and handling stress: plasma cortisol and hepatic glutathione status.

Three groups of one summer old rainbow trout were exposed for 22 days either to normoxia (100%) or moderate oxygen supersaturation; 120% and 140%. After the exposure, all groups were transported for three hours in hyperoxic conditions (123% O2) thus simultaneously experiencing density and handling stress. The recovery of rainbow trout to multiple stressors was measured in normoxic conditions. Moderate oxygen supersaturation did not have any negative effects on growth, feed conversion and blood hematology measured over 22 days. On the other hand, the combined effects of the stressful environment in the fish farm and oxygen supersaturation resulted in a 3-fold increase in plasma cortisol levels in those with 100% and 120% O2 supersaturation and a 2-fold increase in the 140% supersaturation group. Furthermore, the stress response after transportation was lowest in the 140% group 24 hours after recovery but highest after 70 hours. Moderate hyperoxia or transportation stress did not change glutathione concentrations in liver indicating that routine sampling does not affect hepatic glutathione status. Our results indicate that moderate O2 supersaturation (<140%) could be considered as feasible in cultivation of rainbow trout since no harmful effects were found.

Expression of Human Glucose Transporter Type 1 and Rat Hexokinase Type II Complementary DNAs in Rainbow Trout Embryos: Effects on Glucose Metabolism.

: Sugars are utilized poorly in fish mainly because of low rates of transport across plasma membrane and phosphorylation. To evaluate whether it is possible to augment carbohydrate metabolism in fish using heterologous genes, expression of human glucose transporter type 1 (hGLUT1) and rat hexokinase type II (rHKII) complementary DNAs cloned with cytomegalovirus promoter was followed in rainbow trout embryos. Both genes were transcribed. Hexokinase activity, undetectable in control, was found in transformed blastulas. Increased rates of 14C-methylglucose uptake and sensitivity to cytochalasin B indicated the presence of facilitative hexose transport due to hGLUT1 expression. Effect of hGLUT1 on production of 14CO2 from glucose was greater than that of rHKII. Coexpression of the genes did not increase the rate of glucose oxidation compared with expression of hGLUT1 alone.

The effect of photoperiod on diel rhythms in serum melatonin, cortisol, glucose, and electrolytes in the common dentex, Dentex dentex.

Diel rhythms in serum concentrations of melatonin, cortisol, glucose, sodium, chloride, and potassium were studied in the common dentex, Dentex dentex, under different photoperiods (DD, 8L:16D, 12L:12D, 16L:8D). Photoperiod affected both the diel rhythms and the absolute values of the estimated blood components. Regardless of the photoperiod, melatonin titers were elevated during the scotophase (384.3 +/- 13.9 pg/ml) compared with a mean baseline level of 54.4 +/- 2.7 pg/ml during the photophase. Serum melatonin concentrations reflected the prevailing photoperiod and constantly elevated melatonin levels with no diel rhythmicity were evident in fish held in the DD protocol. A circadian-like pattern in serum cortisol was observed in fish that were kept at the DD and 8L:16D protocols with cortisol peak at 18:00 h in the night. Fish exposed to the 16L:8D regime showed highest cortisol levels at 10:00 h, while no rhythmicity was evident under the 12L:12D protocol. A phase shift of 4 h between the peaks of cortisol and glucose was evident in fish exposed to the DD, 8L:16D, and 12L:12D regimes. Diel patterns of changes in serum Na+ and Cl- were observed only in the fish held in the DD protocol. Serum K+ values were lowest during the first part of the scotophase under all regimes, except the 16L:8D where no diel rhythmicity was detected. During the photophase, cortisol was positively correlated with glucose, Na+, and Cl- and negatively with K+. During the scotophase, melatonin was positively correlated with glucose and electrolytes. Results indicated that cortisol may be responsible for the observed rhythmicity of glucose and that melatonin may play a role in glucose and ion regulation in common dentex.

Expression of rat gene for L-gulono-gamma-lactone oxidase, the key enzyme of L-ascorbic acid biosynthesis, in guinea pig cells and in teleost fish rainbow trout (Oncorhynchus mykiss).

The ability of rainbow trout liver and kidney preparations to produce L-ascorbic acid with an added source of L-gulono-gamma-lactone oxidase (GLO) and the absence of their own GLO activity suggested that the reason for the absence of L-ascorbic acid biosynthesis in fish and in guinea pig, a scurvy-prone mammal, can be similar. Nevertheless, results of rat GLO cDNA expression in guinea pig cells and in rainbow trout proved different. In guinea pig cells, rat GLO was expressed in a functional form. Regardless of recombinant GLO transcripts detected in rainbow trout embryos, alevins and in juvenile fish, neither GLO protein nor GLO activity were found. Furthermore, production of L-ascorbic acid in transgenic rainbow trout was not revealed in feeding tests with vitamin C-free diets or after direct administration of L-gulono-gamma-lactone. These results indicate that conditions required for translation or stability of rat GLO are absent in rainbow trout tissues.