Glucose metabolism and gene expression in juvenile zebrafish (Danio rerio) challenged with a high carbohydrate diet: effects of an acute glucose stimulus during late embryonic life

Knowledge on the role of early nutritional stimuli as triggers of metabolic pathways in fish is extremely scarce. The objective of the present study was to assess the long-term effects of glucose injection in the yolk (early stimulus) on carbohydrate metabolism and gene regulation in zebrafish juveniles challenged with a high-carbohydrate low-protein (HC) diet. Eggs were microinjected at 1 d post-fertilisation (dpf) with either glucose (2 M) or saline solutions. Up to 25 dpf, fish were fed a low-carbohydrate high-protein (LC) control diet, which was followed by a challenge with the HC diet. Survival and growth of 35 dpf juveniles were not affected by injection or the HC diet. Glucose stimulus induced some long-term metabolic changes in the juveniles, as shown by the altered expression of genes involved in glucose metabolism. On glycolysis, the expression levels of hexokinase 1 (HK1) and phosphofructokinase-6 (6PFK) were up-regulated in the visceral and muscle tissues, respectively, of juveniles exposed to the glucose stimulus, indicating a possible improvement in glucose oxidation. On gluconeogenesis, the inhibition of the expression levels of PEPCK in fish injected with glucose suggested lower production of hepatic glucose. Unexpectedly, fructose-1,6-bisphosphatase (FBP) expression was induced and 6PFK expression reduced by glucose stimulus, leaving the possibility of a specific regulation of the FBP-6PFK metabolic cycle. Glucose metabolism in juveniles was estimated using a [C-14]glucose tracer; fish previously exposed to the stimulus showed lower retention of [C-14]glucose in visceral tissue (but not in muscle tissue) and, accordingly, higher glucose catabolism, in comparison with the saline group. Globally, our data suggest that glucose stimulus at embryo stage has the potential to alter particular steps of glucose metabolism in zebrafish juveniles.Fundacao para a Ciencia e Tecnologia (FCT, Portugal) [PTDC/CVT/102481/2008]; FCT (Portugal) [SFRH/BD/74921/2010, SFRH/BPD/49051/2008]info:eu-repo/semantics/publishedVersio

High-glucose feeding of gilthead seabream (Sparus aurata) larvae: effects on molecular and metabolic pathways

Nutritional programming has begun to arouse interest as a novel tool to alter specific metabolic pathways or functions in farmed animals. The aim of the present study was to explore the potential of early glucose stimuli to induce changes in nutrient metabolism of gilthead seabream. Nutritional conditioning was performed by delivering glucose-rich feed at three distinct recurrent periods of larval feeding regime: during first-feeding with rotifers (3 days after hatching, DAH) and mid-feeding with Artemia metanauplii (20DAH) and the beginning of inert diet feeding (30DAH), called the Recurrent treatment (REC). As opposed, the control treatment (CTRL) did not experience any glucose stimuli. At post-larval stage (from 50 to 60DAH), both treatments were challenged with a high-carbohydrate diet (50%). The immediate response to the early stimuli was assessed through gene expression of metabolic markers and by nutrient metabolism using [C-14] tracers. Each dietary stimulus induced metabolic changes on REC larvae, shown by altered expression of some genes, including those involved in glycolysis, and by a different pattern of glucose utilization. However, none of the molecular adaptations (except G6PDH gene) were persistent in the viscera and muscle of challenged post-larvae from REC group. In contrast, the glucose metabolism of challenged REC post-larvae revealed a shift towards a higher catabolism and lower glucose retention in tissues, compared to the CTRL group, suggesting an improvement of glucose oxidation pathways. In addition, the REC group showed a higher bio-conversion of glucose into lipids, indicating enhanced hepatic lipogenesis. The early stimuli did not affect the relative retention or use of amino acids or the growth and survival of challenged fish, up to 60DAH. In summary, although not substantiated at a molecular level, our data reveal that a recurrent high-glucose stimulus during larval stages affects the short-term modulation of pathways for glucose utilization in gilthead seabream. (C) 2015 Elsevier B.V. All rights reserved

Glucose overload in yolk has little effect on the long-term modulation of carbohydrate metabolic genes in zebrafish (Danio rerio)

The use of early nutritional stimuli to program metabolic pathways in fish is ill defined. Therefore, studies were undertaken with zebrafish to assess the effect of high glucose levels during the embryonic stage as a lifelong modulator of genes involved in carbohydrate metabolism. Genes related to carbohydrate metabolism were expressed at low levels at 0.2 and 1 day post-fertilization (dpf). However, from 4 dpf onwards there was a significant increase on expression of all genes, suggesting that all analysed pathways were active. By microinjection, we successfully enriched zebrafish egg yolk with glucose (a 43-fold increase of basal levels). Acute effects of glucose injection on gene expression were assessed in larvae up to 10 dpf, and the programming concept was evaluated in juveniles (41 dpf) challenged with a hyperglucidic diet. At 4 dpf, larvae from glucose-enriched eggs showed a downregulation of several genes related to glycolysis, glycogenolysis, lipogenesis and carbohydrate digestion in comparison with control (saline-injected) embryos. This inhibitory regulation was suppressed after 10 dpf. At the juvenile stage, and upon switching from a low to a high digestible carbohydrate diet, early glucose enrichment had no significant effect on most analysed genes. However, these same fish showed altered expression of the genes for cytosolic phosphoenolpyruvate carboxykinase, sodium-dependent glucose cotransporter 1 and glycogen synthase, suggesting changes to the glucose storage capacity in muscle and glucose production and transport in viscera. Overall, supplementation of egg yolk with high glucose levels had little effect on the long-term modulation of carbohydrate metabolic genes in zebrafish

Metabolic utilization of dietary carbohydrates in lean and fat lines of rainbow trout (Oncorhynchus mykiss)

L objectif de la thèse était d'identifier s il existait des différences d utilisation digestive et métabolique des glucides alimentaires entre deux lignes de truite sélectionnées pour leur teneur divergente en lipides intramusculaires: la lignée maigre (L) à faible taux de lipides dans le muscle et la lignée grasse (F) à teneur élevée de lipides musculaires. Dans ce but les deux lignées ont été nourries avec deux aliments, un contenant des glucides alimentaires (20% d amidon), l autre ne contenant pas. Chez les deux lignées, la présence d amidon dans les régimes n a pas eu d effets sur la croissance, a été associée à une épargne protéique, et a provoqué une hyperglycémie postprandiale modérée, en induisant les acteurs impliqués dans la signalisation cellulaire (TOR/AMPK). Aucune différence entre les deux lignées n a été observée pour la digestion de l'amidon, le niveau d expression des gènes codant les transporteurs de glucose dans l intestin, la glycémie postprandiale et l'utilisation périphérique du glucose (muscle et tissu adipeux). Par contre, la lignée F se caractérise par une croissance et une efficacité alimentaire plus faibles, des capacités plus élevées de stockage du glucose sous forme de glycogène hépatiques et de lipogenèse (sans impact sur l utilisation des glucides) et un métabolisme lipidique spécifique (plus faible catabolisme des acides gras, plus fortes capacités à synthétiser des chylomicrons et à bio-convertir des acides gras). Dans une seconde étude, le remplacement de l'huile de poisson de l aliment par un mélange d'huiles végétales a supprimé la plupart des différences moléculaires observées précédemment entre les deux génotypes. Toutefois, l apport de glucides alimentaires a alors entrainé des modifications métaboliques au niveau intestinal et hépatique avec, par exemple, l induction des capacités moléculaires de bioconversions des acides gras. Globalement, nos données ont donc démontré qu il existait des différences métaboliques importantes entre les deux lignées mais que cela n aboutissait pas à une meilleure utilisation des glucides alimentaires. En outre, nos travaux soulignent l'importance de la composition des aliments dans les réponses' des différents génotypes de poissons.The aim of the thesis was to characterise the differences in digestive and metabolic utilisation of dietary carbohydrates between two lines of rainbow trout divergently selected for muscle fat content (Fat-F and Lean-L), when fed diets with (20%) or without gelatinised starch. In both lines, starch intake did not adversely affect growth, resulted in a moderate postprandial hyperglycemia, enhanced protein/lipid retention and a distinct intracellular signalling pattern in the liver involving the energy sensor AMPK and nutrient sensor TOR-S6. No difference between the two lines was observed for the apparent digestibility of starch, the mRNA levels of genes encoding glucose transporters in the intestine, regulation of postprandial glycemia and utilisation of glucose in the peripheral tissues (muscle and adipose tissue). However when compared to the L line, the F line was characterised by lower growth and feed efficiency; better capacity to store excess glucose as indicated by higher glycogen levels and mRNA levels of lipogenic markers in the liver (with no impact on glucose homeostasis); and a specific lipid metabolism (a greater potential to synthesise chylomicrons and to bioconvert fatty acids, coupled with a lower potential to oxidise fatty acids). In a separate study, replacement of fish oil in the diet with a blend of vegetable oils was found to suppress most of the molecular differences previously observed between the two genotypes. Starch intake under the vegetable oil diet regime led to different metabolic changes in the intestine and liver, for example, elicited a higher transcriptional response of key desaturase and elongase enzymes in both lines. Overall, our data demonstrated the existence of significant metabolic differences between the two trout lines, but it did not lead to better utilisation of dietary carbohydrates. In addition, our work highlights the importance of diet composition in the growth and metabolic response of different genotypes of fish.PAU-BU Sciences (644452103) / SudocSudocFranceF

Characterization and comparison of fatty acyl Delta 6 desaturase cDNAs from freshwater and marine teleost fish species

Fish are the most important dietary source of the n-3 highly unsaturated fatty acids (HUFA), eicosapentaenoic (EPA) and docosahexaenoic acid (DHA), that have particularly important roles in human nutrition reflecting their roles in critical physiological processes. The objective of the study described here was to clone, functionally characterise and compare expressed fatty acid desaturase genes involved in the production of EPA and DHA in freshwater and marine teleost fish species. Putative fatty acid desaturase cDNAs were isolated and cloned from common carp (Cyprinus carpio) and turbot (Psetta maximus). The enzymic activities of the products of these cDNAs, together with those of cDNAs previously cloned from rainbow trout (Oncorhynchus mykiss) and gilthead seabream (Sparus aurata), were determined by heterologous expression in the yeast Saccharomyces cerevisiae. The carp and turbot desaturase cDNAs included open reading frames of 1335 and 1338 base pairs, respectively, specifying proteins of 444 and 445 amino acids. The protein sequences possessed all the characteristic features of microsomal fatty acid desaturases, including three histidine boxes, two transmembrane regions, and N-terminal cytochrome b5 domains containing the haem-binding motif, HPGG. Functional expression showed all four fish cDNAs encode basically unifunctional Δ6 fatty acid desaturase enzymes responsible for the first and rate-limiting step in the biosynthesis of HUFA from 18:3n-3 and 18:2n-6. All the fish desaturases were more active towards the n-3 substrate with 59.5%, 31.5%, 23.1% and 7.0% of 18:3n-3 being converted to 18:4n-3 in the case of turbot, trout, seabream and carp, respectively. The enzymes also showed very low, probably physiologically insignificant, levels of Δ5 desaturase activity, but none of the products showed Δ4 desaturase activity. The cloning and characterisation of desaturases from these fish is an important advance, as they are species in which there is a relative wealth of data on the nutritional regulation of fatty acid desaturation and HUFA synthesis, and between which substantive differences occur

Dietary carbohydrate in land-locked salmon

A common-garden experiment was carried out to compare two genetically distinct strains of Atlantic salmon (Salmo salar) fed diets with either high (CHO) or low (NoCHO) digestible carbohydrate (starch). Twenty salmon from either a commercial farmed strain (F) or a land-locked population (G) were placed in two tanks (10 fish of each population in each tank) and fed either CHO or NoCHO feeds. At the end of the experiment fish were fasted for 8 h, euthanized and blood and liver collected. Both diet and population had an effect on circulating glucose levels with G showing hypoglycaemia and dietary starch increasing this parameter. In contrast, G showed increased plasma triacylglycerol levels regardless of dietary treatment suggesting faster conversion of glucose to triacylglycerol. This different ability to metabolise dietary starch among strains was also reflected at a molecular (gene) level as most of the metabolic pathways evaluated were mainly affected by the factor population rather than by diet. The data are promising and suggest different regulatory capacities towards starch utilization between land-locked salmon and the farmed stock. Further analyses are necessary in order to fully characterise the capacity of land-locked salmon to utilise dietary carbohydrate

The Autophagic Flux Inhibitor Bafilomycine A1 Affects the Expression of Intermediary Metabolism-Related Genes in Trout Hepatocytes

Autophagy is an evolutionarily conserved process of cellular self-eating which emerged these last years as a major adaptive metabolic response to various stresses such as fasting, hypoxia, or environmental pollutants. However, surprisingly very few data is currently available on its role in fish species which are directly exposed to frequent environmental perturbations. Here, we report that the treatment of fasted trout hepatocytes with the autophagy inhibitor Bafilomycine A1 lowered the mRNA levels of many of the gluconeogenesis-related genes and increased those of genes involved in intracellular lipid stores. Concurrently, intracellular free amino acid levels dropped and the expression of the main genes involved in the endoplasmic reticulum (ER) stress exhibited a sharp increase in autophagy inhibited cells. Together these results highlight the strong complexity of the crosstalk between ER, autophagy and metabolism and support the importance of considering this function in future studies on metabolic adaptation of fish to environmental stresses

The Positive Impact of the Early-Feeding of a Plant-Based Diet on Its Future Acceptance and Utilisation in Rainbow Trout

Sustainable aquaculture, which entails proportional replacement of fish-based feed sources by plant-based ingredients, is impeded by the poor growth response frequently seen in fish fed high levels of plant ingredients. This study explores the potential to improve, by means of early nutritional exposure, the growth of fish fed plant-based feed. Rainbow trout swimup fry were fed for 3 weeks either a plant-based diet (diet V, V-fish) or a diet containing fishmeal and fish oil as protein and fat source (diet M, M-fish). After this 3-wk nutritional history period, all V- or M-fish received diet M for a 7-month intermediate growth phase. Both groups were then challenged by feeding diet V for 25 days during which voluntary feed intake, growth, and nutrient utilisation were monitored (V-challenge). Three isogenic rainbow trout lines were used for evaluating possible family effects. The results of the V-challenge showed a 42% higher growth rate (P = 0.002) and 30% higher feed intake (P = 0.005) in fish of nutritional history V compared to M (averaged over the three families). Besides the effects on feed intake, V-fish utilized diet V more efficiently than M-fish, as reflected by the on average 18% higher feed efficiency (P = 0.003). We noted a significant family effect for the above parameters (P,0.001), but the nutritional history effect was consistent for all three families (no interaction effect, P.0.05). In summary, our study shows that an early shortterm exposure of rainbow trout fry to a plant-based diet improves acceptance and utilization of the same diet when given at later life stages. This positive response is encouraging as a potential strategy to improve the use of plant-based feed in fish, of interest in the field of fish farming and animal nutrition in general. Future work needs to determine the persistency of this positive early feeding effect and the underlying mechanisms

The Metabolic Consequences of Hepatic AMP-Kinase Phosphorylation in Rainbow Trout

AMP-activated protein kinase (AMPK), a phylogenetically conserved serine/threonine protein kinase, is proposed to function as a “fuel gauge” to monitor cellular energy status in response to nutritional environmental variations. However, in fish, few studies have addressed the metabolic consequences related to the activation of this kinase. This study demonstrates that the rainbow trout (Oncorhynchus mykiss) possesses paralogs of the three known AMPK subunits that co-diversified, that the AMPK protein is present in the liver and in isolated hepatocytes, and it does change in response to physiological (fasting-re-feeding cycle) and pharmacological (AICAR and metformin administration and incubations) manipulations. Moreover, the phosphorylation of AMPK results in the phosphorylation of acetyl-CoA carboxylase, a main downstream target of AMPK in mammals. Other findings include changes in hepatic glycogen levels and several molecular actors involved in hepatic glucose and lipid metabolism, including mRNA transcript levels for glucokinase, glucose-6-phosphatase and fatty acid synthase both in vivo and in vitro. The fact that most results presented in this study are consistent with the recognized role of AMPK as a master regulator of energy homeostasis in living organisms supports the idea that these functions are conserved in this piscine model

Effects of the total replacement of fish-based diet with plant-based diet on the hepatic transcriptome of two European sea bass (Dicentrarchus labrax) half-sibfamilies showing different growth rates with the plant-based diet

Background: Efforts towards utilisation of diets without fish meal (FM) or fish oil (FO) in finfish aquaculture have been being made for more than two decades. Metabolic responses to substitution of fishery products have been shown to impact growth performance and immune system of fish as well as their subsequent nutritional value, particularly in marine fish species, which exhibit low capacity for biosynthesis of long-chain poly-unsaturated fatty acids (LC-PUFA). The main objective of the present study was to analyse the effects of a plant-based diet on the hepatic transcriptome of European sea bass (Dicentrarchus labrax). Results: We report the first results obtained using a transcriptomic approach on the liver of two half-sibfamilies of the European sea bass that exhibit similar growth rates when fed a fish-based diet (FD), but significantly different growth rates when fed an all-plant diet (VD). Overall gene expression was analysed using oligo DNA microarrays (GPL9663). Statistical analysis identified 582 unique annotated genes differentially expressed between groups of fish fed the two diets, 199 genes regulated by genetic factors, and 72 genes that exhibited diet-family interactions. The expression of several genes involved in the LC-PUFA and cholesterol biosynthetic pathways was found to be up-regulated in fish fed VD, suggesting a stimulation of the lipogenic pathways. No significant diet-family interaction for the regulation of LC-PUFA biosynthesis pathways could be detected by microarray analysis. This result was in agreement with LC-PUFA profiles, which were found to be similar in the flesh of the two half-sibfamilies. In addition, the combination of our transcriptomic data with an analysis of plasmatic immune parameters revealed a stimulation of complement activity associated with an immunodeficiency in the fish fed VD, and different inflammatory status between the two half-sibfamilies. Biological processes related to protein catabolism, amino acid transaminations, RNA splicing and blood coagulation were also found to be regulated by diet, while the expression of genes involved in protein and ATP synthesis differed between the half-sibfamilies. Conclusions: Overall, the combined gene expression, compositional and biochemical studies demonstrated a large panel of metabolic and physiological effects induced by total substitution of both FM and FO in the diets of European sea bass and revealed physiological characteristics associated with the two half-sibfamilies