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- Glucose overload in yolk has little effect on the long-term modulation of carbohydrate metabolic genes in zebrafish (Danio rerio)Publication . Rocha, Filipa; Dias, J.; Engrola, S.; Gavaia, Paulo J.; Geurden, Inge; Dinis, Maria Teresa; Panserat, S.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.
- Requisitos de ácido araquidónico em microdietas para larvas de dourada (Sparus aurata, L., 1758)Publication . Rocha, Filipa; Conceição, L. E. C.; Martins, Dulce AlvesOs ácidos gordos (AG) essenciais presentes nas dietas são tidos como factores críticos no sucesso do cultivo larvar. Tem sido dado um maior destaque aos ácidos docosahexaenóico (DHA) e eicosapentaenóico (EPA) devido às suas funções na integridade celular membranar e predominância nos tecidos. A importância do ácido araquidónico (ARA) no crescimento, sobrevivência e resistência ao stress é também cada vez mais reconhecida. Realizou-se um estudo para determinar os efeitos que diferentes concentrações de ARA em microdietas causam no crescimento, sobrevivência e perfil de ácidos gordos das larvas de dourada. As larvas foram alimentadas durante 25 dias com microdietas, incluindo um período inicial de coalimentação com rotíferos enriquecidos. Foram testadas, em triplicado, quatro microdietas experimentais que diferiam nos níveis de ARA, variando entre 0,4% e 3,0% da matéria seca (ARA0,4/ ARA0,8/ ARA1,5/ ARA3,0). As microdietas apresentavam níveis de HUFA n-3 de 4,6% e um rácio DHA/EPA de 1,4. Mediu-se o crescimento com base no peso seco e taxa de crescimento relativo, e obteve-se a sobrevivência por contagem directa. O perfil em AG dos lípidos totais das larvas foi determinado por cromatografia em fase gasosa. Após 34 dias, não se registaram diferenças significativas no crescimento e sobrevivência, apesar de existir uma tendência nos tratamentos intermédios para um menor crescimento. A composição lipídica dos tecidos reflectiu o perfil de AG das dietas, particularmente quanto ao ARA. O DHA manteve-se constante, enquanto o EPA diminuiu com o aumento de ARA, mesmo com níveis semelhantes de EPA nas dietas, sugerindo uma competição entre estes AG para a incorporação nos tecidos. Até 1,5% nas dietas, o ARA mostrou ser preferencialmente depositado nos tecidos, sendo metabolizado em concentrações superiores (3,0%). O DHA e alguns AG saturados foram preferencialmente retidos enquanto que o EPA e os ácidos linoleico (LA), α- linolénico (ALA) e oleico (OA) foram metabolizados, provavelmente para fins energéticos. Nas condições testadas, as larvas de dourada mostraram uma boa tolerância à variação de ARA nas dietas, de 0,4% a 3,0%. No entanto, o crescimento não foi afectado nem se verificou uma melhoria da sobrevivência.
- Early nutritional programming in fish: tailoring the metabolic use of dietary carbohydratesPublication . Rocha, Filipa; Dinis, Maria Teresa; Dias, Jorge; Panresat, StéphaneSustainable feeding practices in aquaculture require a higher use of vegetable feedstuffs, which are naturally rich in carbohydrates. This can raise some constrains to fish species with carnivorous feeding habits, due to their poor ability to use dietary carbohydrates. This thesis aimed to explore the potential of nutritional programming as a new strategy to better understand the mechanisms underlying the impaired utilization of dietary carbohydrates in fish. The experimental work relied on multiple approaches: a) rearing trials with larvae and juvenile fish for assessment of zootechnical criteria; b) analysis with radiolabeled tracers to follow the metabolic flux of nutrients; c) and genomic expression of key metabolic-genes. In Chapters 2 and 3, we evaluated how the supplementation of egg-yolk with glucose, through microinjection, could act as a stimulus and permanently alter some metabolic pathways in zebrafish. Microinjection was proven an efficient technique to alter the nutritional composition of embryo’s yolk. We saw that the embryonic window for stimulus delivery is crucial in determining future outcomes. The late embryo stage was found more suitable to exert a glucidic stimulus compared with incipient stages of embryogenesis, based on molecular and metabolic analyses that suggested an improved capacity for glucose utilization. Chapters 4 and 5 aimed the nutritional programming of the carbohydrate-related metabolic pathways in gilthead seabream. Recurrent hyperglucidic stimuli were delivered at early larval development using live preys and a glucose-rich diet, demonstrating that nutritional stimuli can be performed in a marine fish species during sensitive stages of development, without compromising survival and growth. We found some short-term effects after stimulus delivery, on gene expression pattern and metabolic utilization of glucose of post-larvae. In contrast, juvenile fish exposed to the early stimuli showed only few changes on glucose utilization, namely a higher absorption of dietary starch. This thesis has generated new knowledge on the triggering effect of early glucidic events upon the regulation of key metabolic processes, contributing to a better comprehension over the concept of nutritional programming in fish.
- Dietary glucose stimulus at larval stage modifies the carbohydrate metabolic pathway in gilthead seabream (Sparus aurata) juveniles: An in vivo approach using C-14-starchPublication . Rocha, Filipa; Dias, Jorge; Geurden, Inge; Dinis, Maria Teresa; Panserat, Stephane; Engrola, S.The concept of nutritional programming was investigated in order to enhance the use of dietary carbohydrates in gilthead seabream juveniles. We assessed the long-term effects of high-glucose stimuli, exerted at the larval stage, on the growth performance, nutrient digestibility and metabolic utilization and gene expression of seabream juveniles, challenged with a high-carbohydrate intake. During early development, a group of larvae (control, CTRL) were kept under a rich-protein-lipid feeding regime whereas another group (GLU) was subjected to high-glucose stimuli, delivered intermittently over time. At juvenile stage, triplicate groups (IBW: 2.5 g) from each fish nutritional background were fed a high-protein (59.4%) low-carbohydrate (2.0%) diet before being subjected to a low-protein (43.0%) high-carbohydrate (33.0%) dietary challenge for 36-days. Fish from both treatments increased by 8-fold their initial body weight, but neither growth rate, feed intake, feed and protein efficiency, nutrient retention (except lipids) nor whole-body composition were affected (P (>) 0.05) by fish early nutritional history. Nutrient digestibility was also similar among both groups. The metabolic fate of C-14-starch and C-14-amino acids tracers was estimated; GLU juveniles showed higher absorption of starch-derived glucose in the gut, suggesting an enhanced digestion of carbohydrates, while amino acid use was not affected. Moreover, glucose was less used for de novo synthesis of hepatic proteins and muscle glycogen from GLU fish (P < 0.05). Our metabolic data suggests that the early glucose stimuli may alter carbohydrate utilization in seabream juveniles. (C) 2016 Elsevier Inc. All rights reserved.
- Does a ghrelin stimulus during zebrafish embryonic stage modulate its performance on the long-term?Publication . Navarro-Guillén, Carmen; Dias, Jorge; Rocha, Filipa; Castanheira, M. F.; Martins, Catarina I. M.; Laizé, Vincent; Gavaia, Paulo; Engrola, SofiaMetabolic programming refers to the induction, deletion, or impaired development of a somatic structure or "setting" of a physiological system by an early life stimulus operated at a critical period during development. Ghrelin is the only known orexigenic gut hormone, is an acylated peptide that acts as an endogenous ligand specific for growth-hormone secretagogue-receptor. The aim of the present work was to evaluate if an in ovo ghrelin administration could positively influence the zebrafish performance in the long-term and to gain insight on the mechanisms associated to ghrelin regulation of food intake during the larval phase. Food intake, growth potential, protein metabolism, expression of target genes involved in ghrelin, feeding behaviour regulation and locomotor activity were assessed in zebrafish (Danio rerio) larvae at 25 days post-fertilization. Elevated levels of acylated ghrelin in zebrafish eggs did not result in increased growth or food intake. Differences in mRNA expression between larvae fasted for 16 h before and 1 h after feeding were found for igf1ra, gh1 and pomca. Moreover, ghrelin treated larvae showed higher swimming activity, indicating that the peptide may have an important role on foraging activity. The present study addressed for the first time the effects of an early stimulus of ghrelin during the embryonic stage of zebrafish, however, further studies are needed to clarify the metabolic pathways affected by the early stimulus as well as focus on the effects on metabolic regulation of energy balance through lipid and carbohydrate metabolism.
- 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 lifePublication . Rocha, Filipa; Dias, Jorge; Engrola, S.; Gavaia, Paulo; Geurden, Inge; Dinis, Maria Teresa; Panserat, StephaneKnowledge 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.
- Teleost fish larvae adapt to dietary arachidonic acid supply through modulation of the expression of lipid metabolism and stress response genesPublication . Alves Martins, Dulce; Rocha, Filipa; Martinez-Rodriguez, Gonzalo; Bell, Gordon; Morais, Sofia; Castanheira, Maria Filipa; Bandarra, Narcisa; Coutinho, Joana; Yufera, Manuel; Conceicao, LuisDietary fatty acid supply can affect stress response in fish during early development. Although knowledge on the mechanisms involved in fatty acid regulation of stress tolerance is scarce, it has often been hypothesised that eicosanoid profiles can influence cortisol production. Genomic cortisol actions are mediated by cytosolic receptors which may respond to cellular fatty acid signalling. An experiment was designed to test the effects of feeding gilthead sea-bream larvae with four microdiets, containing graded arachidonic acid (ARA) levels (0.4, 0.8, 1.5 and 3.0 %), on the expression of genes involved in stress response (steroidogenic acute regulatory protein, glucocorticoid receptor and phosphoenolpyruvate carboxykinase), lipid and, particularly, eicosanoid metabolism (hormone-sensitive lipase, PPAR alpha, phospholipase A(2), cyclo-oxygenase-2 and 5-lipoxygenase), as determined by real-time quantitative PCR. Fish fatty acid phenotypes reflected dietary fatty acid profiles. Growth performance, survival after acute stress and similar whole-body basal cortisol levels suggested that sea-bream larvae could tolerate a wide range of dietary ARA levels. Transcription of all genes analysed was significantly reduced at dietary ARA levels above 0.4%. Nonetheless, despite practical suppression of phospholipase A(2) transcription, higher leukotriene B-4 levels were detected in larvae fed 3.0% ARA, whereas a similar trend was observed regarding PGE(2) production. The present study demonstrates that adaptation to a wide range of dietary ARA levels in gilthead sea-bream larvae involves the modulation of the expression of genes related to eicosanoid synthesis, lipid metabolism and stress response. The roles of ARA, other polyunsaturates and eicosanoids as signals in this process are discussed.
- High-glucose feeding of gilthead seabream (Sparus aurata) larvae: effects on molecular and metabolic pathwaysPublication . Rocha, Filipa; Dias, Jorge; Geurden, Inge; Dinis, Maria Teresa; Panserat, Stephane; Engrola, S.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.