NADPH production, a growth marker, is stimulated by maslinic acid in gilthead sea bream by increased NADP-IDH and ME expression.

NADPH plays a central role in reductive biosynthesis of membrane lipids, maintenance of cell integrity, protein synthesis and redox balance maintenance. Hence, NADPH is involved in the growth and proliferation processes. In addition, it has been shown that changes in nutritional conditions produced changes in NADPH levels and growth rate. Maslinic acid (MA), a pentacyclic triterpene of natural origin, is able to stimulate NADPH production, through regulation of the two oxidative phase dehydrogenases of the pentose phosphate pathway. Our main objective was to study the effects of MA on the kinetic behaviour and on the molecular expression of two NADPH-generating systems, NADP-dependent isocitrate dehydrogenase (NADP-IDH) and malic enzyme (ME), in the liver and white muscle of gilthead sea bream (Sparus aurata). Four groups of 12g of a mean body mass were fed for 210days in a fish farm, with diets containing 0 (control), and 0.1g of MA per kg of diet. Two groups were fed ad libitum (C-AL and MA-AL) and another's two, with restricted diet of 1% of fish weight (C-R and MA-R). Results showed that MA significantly increased the main kinetic parameter of the NADPH-forming enzymes (NADP-IDH and ME). In this sense, specific activity, maximum velocity, catalytic efficiency and activity ratio values were higher in MA conditions than control groups. Moreover, these changes were observed in both feeding regimen, AL and R. Meanwhile, the Michaelis constant changed mainly in groups fed with the MA and restricted diet, these changes are related to the best substrate affinity by enzyme. Moreover, in the Western-blot result, we found that MA increased both protein levels studied, this behaviour being consistent with the regulation of the number of enzyme molecules. All results, indicate that MA, independently of the fed regimen, could potentially be a nutritional additive for fish as it improved the metabolic state of fish, as consequence of increased activity and expression of NADP-IDH and ME enzymes.

Proteomics in the liver of gilthead sea bream (Sparus aurata) to elucidate the cellular response induced by the intake of maslinic acid.

Maslinic acid (MA) is a pentacyclic triterpene used as a feed additive to stimulate growth, protein-turnover rates, and hyperplasia in fish. To further our understanding of cellular mechanisms underlying the action of MA, we have used 2-DE coupled with MS to identify proteins differentially expressed in the livers of juvenile gilthead sea bream (Sparus aurata) grown under fish-farm conditions and fed with a 100 mg/kg MA-enriched diet (MA(100)). After the comparison of the protein profiles from MA(100) fed fish and from control, 49 protein spots were found to be altered in abundance (≥2-fold). Analysis by MALDI-TOF/TOF allowed the unambiguous identification of 29 spots, corresponding to 19 different proteins. These proteins were: phosphoglucomutase, phosphoglucose isomerase, S-adenosyl methionine-dependent methyltransferase class I, aldehyde dehydrogenase, catalase, 6-phosphogluconate dehydrogenase, fumarylacetoacetate hydrolase, 4-hydroxyphenylpyruvic dioxygenase, methylmalonate-semialdehyde dehydrogenase, lysozyme, urate oxidase, elongation factor 2, 60 kDa heat-shock protein, 58 kDa glucose-regulated protein, cytokeratin E7, type-II keratin, intermediate filament proteins, 17-ß-hydroxysteroid dehydrogenase type 4, and kinase suppressor of Ras1. Western blot analysis of kinase suppressor of Ras1, glucose 6-phosphate dehydrogenase, elongation factor 2, 60 kDa heat-shock protein, and catalase supported the proteome evidence. Based on the changes found in the protein-expression levels of these proteins, we proposed a cellular-signalling pathway to explain the hepatic-cell response to the intake of a diet containing MA.

Dietary protein differentially regulates the kinetic behaviour of serine dehydratase and tyrosine aminotransferase of liver and white muscle of rainbow trout (Oncorhynchus mykiss).

We have determined the kinetic behaviour of serine dehydratase (SerDH) and tyrosine aminotransferase (TyrAT) in the liver and white muscle of juvenile rainbow trout (Oncorhynchus mykiss) fed on a low-protein/high-fat (LP/HF) and control diets. The relationship between the kinetic parameters and tissue-DNA concentration has also been determined. SerDH and TyrAT showed hyperbolic kinetics in all cases. The partial replacement of protein with fat significantly increased hepatic SerDH-specific activity, maximum velocity (V(max)) and Michaelis constant (K(m)) while no significant changes were detected in the values of these parameters in white muscle. Nevertheless, hepatic and white-muscle TyrAT specific activity and V(max) decreased in trout fed on LP/HF diet with respect to control. The K(m) of hepatic TyrAT was also lower in trout fed on LP/HF diet than in control. The TyrAT kinetic parameters expressed per cell unit also changed in the same sense as previously described. These results show that, in trout, SerDH and TyrAT are two enzymes regulated by the partial replacement of dietary protein by fat.

Blood antioxidant defenses and hematological adjustments in crowded/uncrowded rainbow trout (Oncorhynchus mykiss) fed on diets with different levels of antioxidant vitamins and HUFA.

Rainbow trout maintained at crowding or noncrowding conditions were fed on five experimental diets that were formulated considering two levels of vitamin E (25.6 and 275.6 mg/kg diet), vitamin C (0 and 1000 mg/kg diet) and HUFA (12.5 and 30.5 g/kg diet): -E-HUFA, -E+HUFA, +E-HUFA, +E+HUFA, -C+E+HUFA. Hematological parameters, the activity of some antioxidant enzymes and lipid peroxidation from RBC were evaluated. The SOD isoenzyme pattern was analyzed by nondenaturing PAGE. Hematological response to crowding stress was manifested by increased hemoglobin and RBC count in most of the crowded groups. Antioxidant enzyme activity was clearly affected by dietary HUFA levels, with uncrowded fish fed on +HUFA diets showing a higher SOD activity compared to those fed on -HUFA diets. In uncrowded groups, only one CuZn-SOD isozyme was detected, whereas in the crowded fish a great variability was revealed with up to five isozymes. G6PDH activity was increased in uncrowded -E+HUFA fish compared to the remaining groups. Lipid peroxidation was significantly increased in -E+HUFA fish regardless of fish density. Data supported the negative correlation of lipid peroxidation and hematocrit or hemoglobin explained by decreased erythrocyte stability. Dietary imbalances in vitamin E and HUFA supplementation may promote oxidative stress which triggers hematological deterioration, which in turn would affect the whole hematological status and ultimately fish welfare.

Maslinic acid added to the diet increases growth and protein-turnover rates in the white muscle of rainbow trout (Oncorhynchus mykiss).

Maslinic acid (2-alpha, 3-beta-dihydroxiolean-12-en-28-oic acid) is a triterpenoid compound present in fruit and leaves of Olea europaea that can be used as an additive in the diet of trout. The present work investigates the effects of maslinic acid on growth, protein-turnover rates and nucleic acid concentration in trout white muscle. Five groups of 180 trout of a mean body mass of 20 g were fed for 225 days with diets containing 0, 1, 5, 25 and 250 mg of maslinic acid per kg of diet. At the end of the experiment, white-muscle weight and protein-accumulation rate of trout fed with maslinic acid were higher than in control. The total content of DNA, RNA, and protein in trout fed with 25 and 250 mg of maslinic acid kg(-1) were significantly higher than in control. The protein:DNA ratio was also slightly higher than control. In the same groups of trout, fractional (K(S)) and absolute (A(S)) protein-synthesis rates increased to more than 80% over the control values while no differences were found in the fractional protein-degradation rate (K(D)). These results, similar to previous findings in liver, show that maslinic acid can act as a growth factor when added to a standard trout diet.

Maslinic acid as a feed additive to stimulate growth and hepatic protein-turnover rates in rainbow trout (Onchorhynchus mykiss).

Maslinic acid is a triterpene present in a considerable proportion in solid residues from olive-oil production. In the present work the effects of maslinic acid on growth, protein-turnover rates and nucleic-acid concentration on liver were investigated in the rainbow trout. Five groups of 120 fish of a mean body mass of 20 g were fed for 225 days with diets containing 0, 1, 5, 25 and 250 mg of maslinic acid per kg diet. At the end of the experiment, whole-body and liver weight and growth rate of trout fed with maslinic acid were higher than controls. The highest weight increase was registered for the group fed 250 mg kg(-1), representing a 29% increase over controls. The total hepatic DNA or liver cell hyperplasia levels in trout fed with 25 and 250 mg of maslinic acid kg(-1) were 37% and 68% higher than controls. Also in these same groups of trout, fractional and absolute hepatic protein-synthesis rates were significantly higher than in control, and significant increments in hepatic protein-synthesis efficiency and protein-synthesis capacity were reported. In close agreement with these results, microscopy studies showed that trout fed on 25 and 250 mg kg(-1) hepatocytes appeared to be more compact, with a larger rough-endoplasmic reticulum and larger glycogen stores than controls. These results suggest that maslinic acid can act as a growth factor when added to trout diet.

Oxidative stress generated by dietary Zn-deficiency: studies in rainbow trout (Oncorhynchus mykiss).

To date, there is scarce information on the metabolic and biochemical repercussions of Zn-deficiency in fish. In this work, the effect of dietary Zn-deficiency on the diet utilization and the metabolism of activated oxygen species in rainbow trout (Oncorhynchus mykiss) has been studied. Fish were randomly separated in different lots according to their Zn-starvation and diet intake. In crude extracts of liver, gut and muscle, total and isoenzymatic superoxide dismutase and catalase activities were analysed. Lipid peroxidation was also determined in the same tissues. Western blotting was performed using antibodies against manganese- and copperzinc-containing superoxide dismutase. Lots fed on the Zn-deficient diet and with low intake showed significantly lower weight gain and feed-conversion efficiency indexes than control trouts. However, these parameters returned to control values when trouts were recovered by feeding them a control diet ad libitum. In control trouts, three independent copperzinc superoxide dismutase isozymes were detected in liver, whereas only one isozyme was present in the other lots. However, by Western blotting analysis the presence of a manganese superoxide dismutase was found in liver from all lots except in control trouts. Catalase activity and lipid peroxidation values were mainly detected in liver and gut, respectively, and both parameters increased in all lots with respect to the control group. Our results thus suggest that in rainbow trout an oxidative stress appears to occur as a consequence of Zn-deficiency.