Sex dimorphism of glucosensing parameters and appetite-regulating peptides in the hypothalamus of rainbow trout broodstocks.

Rainbow trout (Oncorhynchus mykiss) is traditionally considered as a poor user of digestible carbohydrates harbouring persistent postprandial hyperglycaemia and decreased growth performances when fed a diet containing more than 20% of digestible carbohydrates. While this glucose-intolerant phenotype is well-described in juveniles, evidence points to a particular regulation of glucose metabolism in rainbow trout broodstrocks. By detecting changes in glucose levels and triggering a specific metabolic response, the hypothalamus plays a key role in the regulation of peripheral glucose metabolism. Therefore, our objective was to assess, for the first time in fish, the short-term consequences in hypothalamus, the glucose sensing and feed intake regulation of feeding mature female and male, and neomale rainbow trout with a diet containing either no or a 33% carbohydrate. The hypothalamic glucosensing capacity was assessed through mRNA levels of glucosensing related-genes and feed intake regulation through appetite-regulating peptides. Our data indicate that a brief period of carbohydrate intake (5 meals at 8 °C) did not induce specific changes in glucosensing capacity and appetite-regulating peptides in the hypothalamus of rainbow trout broodstock. Our results did however demonstrate, for the first time in fish, the existence of sex dimorphism of glucosensing-related genes and appetite-regulating peptides.

Characterization of free fatty acid receptor family in rainbow trout (Oncorhynchus mykiss): towards a better understanding of their involvement in fatty acid signalisation.

Since 20 years of research, free fatty acids receptors (FFARs) have received considerable attention in mammals. To date, four FFARs (FFAR1, FFAR2, FFAR3 and FFAR4) are especially studied owing to their physiological importance in various biological processes. This ubiquitist group of G protein-coupled receptors (GPCRs) are majors reports in the key physiological functions such as the regulation of energy balance, metabolism or fatty acid sensing. However, up till date, even some studies were interested in their potential involvement in fatty acid metabolism, no genome investigation of these FFARs have been carried out in teleost fish. Through genome mining and phylogenetic analysis, we identified and characterised 7 coding sequences for ffar2 in rainbow trout whereas no ffar3 nor ffar4 gene have been found. This larger repertoire of ffar2 genes in rainbow trout results from successive additional whole-genome duplications which occurred in early teleosts and salmonids, respectively. A syntenic analysis was used to assign a new nomenclature to the salmonid ffar2 and showed a clear conservation of genomic organisation, further supporting the identity of these genes as ffar2. RT-qPCR was then used to examine, firstly during ontogenesis and secondly on feeding response the expression pattern of ffar1 and ffar2 genes in proximal gut and brain of all trout ffar genes. Overall, this study presents a comprehensive overview of the ffar family in rainbow trout.

Molecular programming of the hepatic lipid metabolism via a parental high carbohydrate and low protein diet in rainbow trout.

It is now recognised that parental diets could alter their offspring metabolism, concept known as nutritional programming. For agronomic purposes, it has been previously proposed that programming could be employed as a strategy to prepare individual for future nutritional challenges. Concerning cultured fish that belong to high trophic level, plant-derived carbohydrates are a possible substitute for the traditional protein-rich fishmeal in broodstock diet, lowering thus the dietary protein-to-carbohydrate ratio (HC/LP nutrition). However, in mammals, numerous studies have previously demonstrated that parental HC/LP nutrition negatively affects their offspring in the long term. Therefore, the question of possible adaptation to plant-based diets, via parental nutrition, should be explored. First, the maternal HC/LP nutrition induced a global DNA hypomethylation in the liver of their offspring. Interestingly at the gene expression level, the effects brought by the maternal and paternal HC/LP nutrition cumulated in the liver, as indicated by the altered transcriptome. The paternal HC/LP nutrition significantly enhanced cholesterol synthesis at the transcriptomic level. Furthermore, hepatic genes involved in long-chain polyunsaturated fatty acids were significantly increased by the parental HC/LP nutrition, affecting thus both hepatic and muscle fatty acid profiles. Overall, the present study demonstrated that lipid metabolism could be modulated via a parental nutrition in rainbow trout, and that such modulations have consequences on their progeny phenotypes.

Alteration of eggs biochemical composition and progeny survival by maternal high carbohydrate nutrition in a teleost fish.

Reproductive performances, and the factors affecting them, are of major importance especially for farmed fish in the context of the development of a sustainable aquaculture. Dietary maternal lipids have been identified as a major factor affecting reproductive performances. Nevertheless, the consequences of carbohydrates have been little studied while plant-derived carbohydrates could be increasingly used in broodstock diets. To explore this issue, 2-year-old female trout were fed either a control diet that contains no carbohydrate and a high protein content (65.7%) or a diet formulated with plant-derived carbohydrates containing 32.5% carbohydrate and 42.9% protein ('HC diet') for an entire reproductive cycle. The reproductive performances, the quality of the unfertilized eggs and the development of the progeny were carefully monitored. Although the one year HC nutrition had not impaired female growth nor spawns quality, such nutrition had increased the variability of eggs size within spawns (+ 34.0%). Moreover, the eggs produced had a modified fatty acid profile, including a significant reduction in EPA content (- 22.9%) and a significant increase in the AA/EPA ratio (+ 33.3%). The progeny were impacted by such alterations as their survival rates were significantly reduced. A lower plant-derived carbohydrate inclusion (20%) should be considered in aquafeed for female broodstock in trout.

Tissue origin of circulating microRNAs and their response to nutritional and environmental stress in rainbow trout (Oncorhynchus mykiss).

Stresses associated with changes in diet or environmental disturbances are common situations that fish encounter during their lifetime. The stability and ease of measuring microRNAs (miRNAs) present in biological fluids make these molecules particularly interesting biomarkers for non-lethal assessment of stress in animals. Rainbow trout were exposed for four weeks to abiotic stress (moderate hypoxia) and/or nutritional stress (a high-carbohydrate/low-protein diet). Blood plasma and epidermal mucus were sampled at the end of the experiment, and miRNAs were assessed using small RNA sequencing. We identified four miRNAs (miR-122-5p, miR-184-3p, miR-192-5p and miR-194a-5p) and three miRNAs (miR-210-3p, miR-153a-3p and miR-218c-5p) that accumulated in response to stress in blood plasma and epidermal mucus, respectively. In particular, the abundance of miR-210-3p, a hypoxamiR in mammals, increased strongly in the epidermal mucus of rainbow trout subjected to moderate hypoxia, and can thus be considered a relevant biomarker of hypoxic stress in trout. We explored the contribution of 22 tissues/organs to the abundance of circulating miRNAs (c-miRNAs) in blood plasma and epidermal mucus influenced by the treatments. Some miRNAs were tissue-specific, while others were distributed among several tissues. Some c-miRNAs (e.g., miR-210-3p, miR184-3p) showed similar variations in both tissues and fluids, while others showed an inverse trend (e.g., miR-122-5p) or no apparent relationship (e.g. miR-192-5p, miR-194a-5p. Overall, these results demonstrate that c-miRNAs can be used as non-lethal biomarkers to study stress in fish. In particular, the upregulation of miR-210-3p in epidermal mucus induced by hypoxia demonstrates the potential of using epidermal mucus as a matrix for identifying non-invasive biomarkers of stress. This study provides information about the tissue sources of c-miRNAs and highlights the potential difficulty in relating variations in miRNA abundance in biological fluids to that in tissues.

Hepatic Global DNA Hypomethylation Phenotype in Rainbow Trout Fed Diets Varying in Carbohydrate to Protein Ratio.

BACKGROUND: A high carbohydrate-low protein diet can induce hepatic global DNA hypomethylation in trout. The mechanisms remain unclear. OBJECTIVES: We aimed to investigate whether an increase in dietary carbohydrates (dHCs) or a decrease in dietary proteins (dLPs) can cause hepatic global DNA hypomethylation, as well as explore the underlying mechanisms in trout. METHODS: Two feeding trials were conducted on juvenile males, both of which involved a 4-d fasting and 4-d refeeding protocol. In trial 1, trout were fed either a high protein-no carbohydrate [HP-NC, protein 60% dry matter (DM), carbohydrates 0% DM] or a moderate protein-high carbohydrate (MP-HC, protein 40% DM, carbohydrates 30% DM) diet. In trial 2, fish were fed either a moderate protein-no carbohydrate (MP-NC, protein 40% DM, carbohydrates 0% DM), an MP-HC (protein 40% DM, carbohydrates 30% DM), or a low protein-no carbohydrate (LP-NC, protein 20% DM, carbohydrates 0% DM) diet to separate the effects of dHCs and dLPs on the hepatic methylome. Global CmCGG methylation, DNA demethylation derivative concentrations, and mRNA expression of DNA (de)methylation-related genes were measured. Differences were tested by 1-factor ANOVA when data were normally distributed or by Kruskal-Wallis nonparametric test if not. RESULTS: In both trials, global CmCGG methylation concentrations remained unaffected, but the hepatic 5-mdC content decreased after refeeding (1-3%). The MP-HC group had 3.4-fold higher hepatic 5-hmdC and a similar 5-mdC concentration compared with the HP-NC group in trial 1. Both MP-HC and LP-NC diets lowered the hepatic 5-mdC content (1-2%), but only the LP-NC group had a significantly lower 5-hmdC concentration (P < 0.01) compared with MP-NC group in trial 2. CONCLUSIONS: dHC and dLP independently induced hepatic global DNA demethylation in trout. The alterations in other methylation derivative concentrations indicated the demethylation process was achieved through an active demethylation pathway and probably occurred at non-CmCGG sites.

Effects of Low Stocking Densities on Zootechnical Parameters and Physiological Responses of Rainbow Trout (Oncorhynchus mykiss) Juveniles.

The present study investigated the effect of low stocking density on growth, survival, feed parameters and physiological responses (blood metabolites, welfare indicators, immune biomarkers, and transcriptomic responses of stress and immune-related genes) on juvenile rainbow trout (Oncorhynchus mykiss) reared under a recirculating aquaculture system during 12 weeks. Fish (average weight 29.64 g) were reared in triplicate under four initial densities: nine fish per tank (D9, 3.76 ± 0.06 kg/m3), 18 fish per tank (D18, 7.66 ± 0.18 kg/m3), 27 fish per tank (D27, 9.67 ± 0.01 kg/m3) and 36 fish per tank (D36, 12.94 ± 0.14 kg/m3). Results showed that lower stocking density D9 significantly altered survival with several fish dying during the experiment and an alteration of growth and feed efficiency for the remaining fish. In parallel, the study revealed that low stocking density induced a chronic stress altering the physiological responses of trout by dysregulation of the inflammatory, immune system, and indolamine/catecholamine brain levels. In conclusion, regarding all the variables observed, low stocking density (D9) alters survival, growth and feed efficiency of rainbow trout with alteration of their physiological responses. Selecting appropriate fish density relating to rearing conditions proved to be an essential concern to improve welfare in an aquaculture context.

No adverse effect of a maternal high carbohydrate diet on their offspring, in rainbow trout (Oncorhynchus mykiss).

In order to develop a sustainable salmonid aquaculture, it is essential to continue to reduce the use of the protein-rich fishmeal. One promising solution to do so is the use of plant-derived carbohydrates in diet destined to broodstock. However, in mammals, the reduction of protein content (replaced by carbohydrates) in parental diet is known to have strong adverse effects on offspring phenotypes and metabolism. For the first time, the effect of a paternal and a maternal high carbohydrate-low protein diet was assessed on progeny at long term in the rainbow trout. A 30% protein diminution in both males and females broodstock diet during 10 month and 5 months, respectively, did not trigger adverse consequences on their offspring. At the molecular level, offspring transcriptomes were not significantly altered, emphasizing no effect on metabolism. Tenuous differences in the biochemical composition of the liver and the viscera were observed. The recorded effects remained in the normal range of value and accordingly offspring growth were not negatively affected over the long term. Overall, we demonstrated here that a 30% protein diminution during gametogenesis is feasible, confirming the possibility to increase the proportion of plant-derived carbohydrates in female broodstock diets to replace fishmeal proteins.

Modulation of Energy Metabolism and Epigenetic Landscape in Rainbow Trout Fry by a Parental Low Protein/High Carbohydrate Diet.

It is now recognized that parental diets could highly affect offspring metabolism and growth. Studies in fish are, however, lacking. In particular, the effect of a parental diet high in carbohydrate (HC) and low in protein (LP) on progeny has never been examined in higher trophic level teleost fish. Thus, two-year old male and female rainbow trout (Oncorhynchus mykiss) were fed either a control diet (0% carbohydrate and 63.89% protein) or a diet containing 35% carbohydrate and 42.96% protein (HC/LP) for a complete reproductive cycle for females and over a 5-month period for males. Cross-fertilizations were then carried out. To evaluate the effect of the parental diet on their offspring, different phenotypic and metabolic traits were recorded for offspring before their first feeding and again three weeks later. When considering the paternal and maternal HC/LP nutrition independently, fry phenotypes and transcriptomes were only slightly affected. The combination of the maternal and paternal HC/LP diets altered the energy metabolism and mitochondrial dynamics of their progeny, demonstrating the existence of a synergistic effect. The global DNA methylation of whole fry was also highly affected by the HC/LP parental diet, indicating that it could be one of the fundamental mechanisms responsible for the effects of nutritional programming.

Short-Term Effect of a Low-Protein High-Carbohydrate Diet on Mature Female and Male, and Neomale Rainbow Trout.

Rainbow trout are considered as a poor user of dietary carbohydrates, displaying persistent postprandial hyperglycaemia when fed a diet containing high amounts of carbohydrates. While this phenotype is well-described in juveniles, less attention was given to broodstock. Our objective was to assess for the first time the short-term consequences of feeding mature female and male, and neomale trout with a low-protein high-carbohydrate diet on glucose and lipid metabolism. Fish were fed for two days with a diet containing either no or 32% of carbohydrates. We analysed plasma metabolites, mRNA levels and enzymatic activities of glycolysis, gluconeogenesis, de novo lipogenesis and ß-oxidation in the liver. Results demonstrated that the glucose and lipid metabolism were regulated by the nutritional status in all sexes, irrespective of the carbohydrate intake. These data point out that carbohydrate intake during a short period (5 meals) at 8 °C did not induce specific metabolic changes in broodstock. Finally, we demonstrated, for the first time, sex differences regarding the consequences of two days of feeding on glucose and lipid metabolism.

Early feeding with hyperglucidic diet during fry stage exerts long-term positive effects on nutrient metabolism and growth performance in adult tilapia (Oreochromis niloticus).

The present study aimed to investigate nutritional programming of carbohydrate metabolism in Nile tilapia. Early nutritional intervention stimulus was achieved by feeding fry with high-protein/low-carbohydrate (HP/LC) or low-protein/high-carbohydrate (LP/HC) diet since first feeding for 4 weeks, and the effect of nutritional stimulus on carbohydrate and its related metabolism was evaluated through the adult stage. Our findings indicated that at week 1, LP/HC diet-fed fry had lower levels of mRNA for genes coding gluconeogenesis and amino acid catabolism and higher levels of hk2 (P < 0⋅05). As expected, in adult tilapia, although LP/HC diet-fed fish had poorer growth (end of stimulus), the fish showed compensatory growth. There were permanent effects of early high-carbohydrate (HC) intake on several parameters, including (1) modulating hepatic composition, (2) increased muscle glycogen, (3) lower levels of enzymes involved in amino acid catabolism and (4) higher levels of glycolytic enzymes in glycolysis. Finally, HP/LC diet- and LP/HC diet-fed fish were challenged with different dietary carbohydrate levels. Irrespective of challenging diets, the early HC stimulus had significant effects on adult tilapia by (1) promoting utilisation of glucose, which had protein-sparing effects for better growth, (2) inducting lipogenesis and (3) decreasing amino acid catabolism. Taken together, for the first time, we demonstrated that early HC feeding was effective for positive nutritional programming of metabolism in Nile tilapia (an omnivorous fish). It led to the improvement of growth performance in adult fish associated with early feeding, which is linked to a better ability to use glucose, to induce lipogenesis, and to suppress amino acid catabolism.

Hepatic Glycerol Metabolism-Related Genes in Carnivorous Rainbow Trout (Oncorhynchus mykiss): Insights Into Molecular Characteristics, Ontogenesis, and Nutritional Regulation.

Glycerol metabolism in rainbow trout is poorly studied even though it is at the interface between lipid and glucose metabolism. Moreover, glycerol can be an important ingredient in new aquafeed formulation to decrease the catabolism of dietary amino acids. Thus, the present study aimed to characterize for the first time the different genes coding for key enzymes and proteins involved in hepatic glycerol metabolism. From the trout genomes, all the paralogous genes coding for glycerol transport (aqp9b), glycerol kinase (gk2a and gk5), glycerol-3-phosphate phosphatase (pgp), and glycerol-3-phosphate dehydrogenase (gpd1a, gpd1b, and gpd1c) were identified. The ontogenesis determined that the capacity to metabolize glycerol begins with the apparition of the liver during the development (stage 22) and are more expressed at the endogenous-exogenous feeding period (stage 35). The postprandial regulation of the expression of these genes in juvenile trout showed that the postprandial peak of expression is between 4 and 24 h after the last meal for many of the genes, demonstrating that glycerol metabolism could be nutritionally regulated at a molecular level. However, surprisingly, no regulation of the mRNA abundance for the glycerol metabolism-related genes by different levels of dietary glycerol (0, 2.5, and 5%) have been detected, showing that hepatic glycerol metabolism is poorly regulated at a molecular level by dietary glycerol in rainbow trout juveniles.

Exploring the Impact of a Low-Protein High-Carbohydrate Diet in Mature Broodstock of a Glucose-Intolerant Teleost, the Rainbow Trout.

Sustainable aquaculture production requires a greater reduction in the use of marine-derived ingredients, and one of the most promising solutions today is the augmentation in the proportion of digestible carbohydrates in aquafeed. This challenge is particularly difficult for high trophic level teleost fish as they are considered to be glucose-intolerant (growth delay and persistent postprandial hyperglycemia observed in juveniles fed a diet containing more than 20% of carbohydrates). It was previously suggested that broodstock could potentially use carbohydrates more efficiently than juveniles, probably due to important metabolic changes that occur during gametogenesis. To investigate this hypothesis, 2-year old male and female rainbow trout (Oncorhynchus mykiss) were either fed a diet containing no carbohydrates (NC) or a 35%-carbohydrate diet (HC) for an entire reproductive cycle. Zootechnical parameters as well as the activities of enzymes involved in carbohydrate metabolism were measured in livers and gonads. Fish were then reproduced to investigate the effects of such a diet on reproductive performance. Broodstock consumed the HC diet, and in contrast to what is commonly observed in juveniles, they were able to grow normally and they did not display postprandial hyperglycemia. The modulation of their hepatic metabolism, with an augmentation of the glycogenesis, the pentose phosphate pathway and a possible better regulation of gluconeogenesis, may explain their improved ability to use dietary carbohydrates. Although the HC diet did induce precocious maturation, the reproductive performance of fish was not affected, confirming that broodstock are able to reproduce when fed a low-protein high-carbohydrate diet. In conclusion, this exploratory work has shown that broodstock are able to use a diet containing digestible carbohydrates as high as 35% and can then grow and reproduce normally over an entire reproductive cycle for females and at least at the beginning of the cycle for males. These results are highly promising and suggest that dietary carbohydrates can at least partially replace proteins in broodstock aquafeed.

Glucose Injection Into Yolk Positively Modulates Intermediary Metabolism and Growth Performance in Juvenile Nile Tilapia (Oreochromis niloticus).

The aim of this study was to explore for the first time in omnivorous fish the concept of nutritional programming. A nutritional stimulus was accomplished by microinjecting 2 M glucose into yolk reserves during the alevin stage in Nile tilapia (Oreochromis niloticus). At the molecular level in fry, at 1 week post-injection, glucose stimuli were associated with the up-regulation of genes involved in glycolysis (pklr, hk1, hk2, and pkma), glucose transport (glut4) pathways and down-regulation of genes related to gluconeogenesis (g6pca1, g6pca2, and pck1) and amino acid catabolism (asat, alat) (P < 0.05), demonstrating that the larvae well received the glucose stimulus at a molecular level. Moreover, 20 weeks after glucose injection, early glucose stimuli were always linked to permanent effects in juvenile fish, as reflected by a higher level of glycolytic enzymes [gck, hk1 and hk2 at both mRNA and enzymatic levels and pyruvate kinase (PK) activity]. Finally, the effects of the glucose stimulus history were also examined in fish fed with two different dietary carbohydrate/protein levels (medium-carbohydrate diet, CHO-M; high-carbohydrate diet, CHO-H) in juvenile fish (during weeks 20-24). As expected, the CHO-H diet induced the expression of glycolytic and lipogenic genes (gck, pklr, hk1, hk2, fpkma, fasn, and g6pd) and suppressed the expression of gluconeogenic and amino acid catabolism genes (g6pca1, pck1, pck2, asat, alat, and gdh). Nevertheless, the early glucose stimulus led to persistent up-regulation of glycolytic enzymes (gck, pklr, hk1, and hk2) at both the mRNA and enzyme activity levels and glucose transporter glut4 as well as lower gluconeogenic pck1 gene expression (P < 0.05). More interestingly, the early glucose stimulus was associated with a better growth performance of juvenile fish irrespective of the diets. These permanent changes were associated with DNA hypomethylation in the liver and muscles, suggesting the existence of epigenetic mechanisms at the origin of programming. In conclusion, for the first time in tilapia, early glucose stimuli were found to be clearly associated with a positive metabolic programming effect later in life, improving the growth performance of the fish.

Nutritional regulation of glucose metabolism-related genes in the emerging teleost model Mexican tetra surface fish: a first exploration.

Astyanax mexicanus has gained importance as a laboratory model organism for evolutionary biology. However, little is known about its intermediary metabolism, and feeding regimes remain variable between laboratories holding this species. We thus aimed to evaluate the intermediary metabolism response to nutritional status and to low (NC) or high (HC) carbohydrate diets in various organs of the surface-dwelling form of the species. As expected, glycaemia increased after feeding. Fish fed the HC diet had higher glycaemia than fish fed the NC diet, but without displaying hyperglycaemia, suggesting that carbohydrates are efficiently used as an energy source. At molecular level, only fasn (Fatty Acid Synthase) transcripts increased in tissues after refeeding, suggesting an activation of lipogenesis. On the other hand, we monitored only moderate changes in glucose-related transcripts. Most changes observed were related to the nutritional status, but not to the NC versus HC diet. Such a metabolic pattern is suggestive of an omnivorous-related metabolism, and this species, at least at adult stage, may adapt to a fish meal-substituted diet with high carbohydrate content and low protein supply. Investigation to identify molecular actors explaining the efficient use of such a diet should be pursued to deepen our knowledge on this species.

Evolutionary history of DNA methylation related genes in chordates: new insights from multiple whole genome duplications.

DNA methylation is an important epigenetic mechanism involved in many biological processes, i.e. gametogenesis and embryonic development. However, increased copy numbers of DNA methylation related genes (dnmt, tet and tdg) have been found during chordate evolution due to successive whole genome duplication (WGD) events. Their evolutionary history and phylogenetic relationships remain unclear. The present study is the first to clarify the evolutionary history of DNA methylation genes in chordates. In particular, our results highlight the fixation of several dnmt3-related genes following successive WGD throughout evolution. The rainbow trout genome offered a unique opportunity to study the early evolutionary fates of duplicated genes due to a recent round of WGD at the radiation of salmonids. Differences highlighted in transcriptional patterns of these genes during gametogenesis and ontogenesis in trout indicated that they might be subjected to sub- or neo-functionalisation after WDG. The fixation of multiple dnmt3 genes in genomes after WGD could contribute to the diversification and plastic adaptation of the teleost.

Higher glycolytic capacities in muscle of carnivorous rainbow trout juveniles after high dietary carbohydrate stimulus at first feeding.

BACKGROUND: Rainbow trout is a "glucose-intolerant" carnivorous species. Using the metabolic programming strategy, we used early nutritional stimuli in order to modify carbohydrate utilization in trout juveniles. METHOD: Fish were fed two diets during the first feeding, namely HP (no carbohydrate / high protein) diet and LP (high carbohydrate / low protein) diet. HP diet was used as the control diet and LP diet as an early stimulus diet. We also used another early stimulus with fish fed HP diet every other day during the first feeding (HP restriction feeding - HPR). After the first-feeding stage (4 weeks), all fish were subsequently subjected to a growth trial with a commercial diet followed by a challenge test with the LP diet (11 weeks). At the end of the first feeding stimulus and of the challenge test, we investigated growth performance, glucose metabolism-related parameters and global DNA CmCGG methylation in trout. RESULTS: LP and HPR dietary stimuli have been a success as shown by the direct modifications of growth performance and mRNA levels for glucose metabolism-related genes at the end of first feeding compared to alevins fed the HP diet. At the end of the challenge trial, no variation in growth performance and hepatic metabolism of LP-history and HPR-history in trout juveniles were observed. However, in muscle of trout juvenile subjected to LP diet at the first feeding, we found an up-regulation of mRNA levels of some glucose metabolism (glucose transport and glycolysis)-related genes and an increase of activities of important glycolysis-related enzymes (hexokinase, phosphofructokinase and pyruvate kinase). These observations are associated with a decrease in the content of glycogen compared to fish fed the HP diet. Moreover, global CmCGG DNA methylation in the muscle of fish with LP history was significantly lower than those fed the HP diet. CONCLUSION: Dietary LP stimulus at first feeding could permanently modify glucose metabolism and global CmCGG DNA methylation level in muscle of trout juveniles, showing that the first feeding stage is efficient for programming the glucose metabolism in fish.

Early feeding of rainbow trout (Oncorhynchus mykiss) with methionine-deficient diet over a 2 week period: consequences for liver mitochondria in juveniles.

Methionine is a key factor in modulating the cellular availability of the main biological methyl donor S-adenosylmethionine (SAM), which is required for all biological methylation reactions including DNA and histone methylation. As such, it represents a potential critical factor in nutritional programming. Here, we investigated whether early methionine restriction at first feeding could have long-term programmed metabolic consequences in rainbow trout. For this purpose, trout fry were fed with either a control diet (C) or a methionine-deficient diet (MD) for 2 weeks from the first exogenous feeding. Next, fish were subjected to a 5 month growth trial with a standard diet followed by a 2 week challenge (with the MD or C diet) to test the programming effect of the early methionine restriction. The results showed that, whatever the dietary treatment of fry, the 2 week challenge with the MD diet led to a general mitochondrial defect associated with an increase in endoplasmic reticulum stress, mitophagy and apoptosis, highlighting the existence of complex cross-talk between these different functions. Moreover, for the first time, we also observed that fish fed the MD diet at the first meal later exhibited an increase in several critical factors of mitophagy, hinting that the early nutritional stimulus with methionine deficiency resulted in long-term programming of this cell function. Together, these data extend our understanding of the role of dietary methionine and emphasize the potential for this amino acid in the application of new feeding strategies, such as nutritional programming, to optimize the nutrition and health of farmed fish.

Profiling the rainbow trout hepatic miRNAome under diet-induced hyperglycemia.

Carnivorous rainbow trout exhibit prolonged postprandial hyperglycemia when fed a diet exceeding 20% carbohydrate content. This poor capacity to utilize carbohydrates has led to rainbow trout being classified as "glucose-intolerant" (GI). The metabolic phenotype has spurred research to identify the underlying cellular and molecular mechanisms of glucose intolerance, largely because carbohydrate-rich diets provide economic and ecological advantages over traditionally used fish meal, considered unsustainable for rainbow trout aquaculture operations. Evidence points to a contribution of hepatic intermediary carbohydrate and lipid metabolism, as well as upstream insulin signaling. Recently, microRNAs (miRNAs), small noncoding RNAs acting as negative posttranscriptional regulators affecting target mRNA stability and translation, have emerged as critical regulators of hepatic control of glucose-homeostasis in mammals, revealing that dysregulated hepatic miRNAs might play a role in organismal hyperglycemia in metabolic disease. To determine whether hepatic regulatory miRNA networks may contribute to GI in rainbow trout, we induced prolonged postprandial hyperglycemia in rainbow trout by using a carbohydrate-rich diet and profiled genome-wide hepatic miRNAs in hyperglycemic rainbow trout compared with fasted trout and trout fed a diet devoid of carbohydrates. Using small RNA next-generation sequencing and real-time RT-PCR validation, we identified differentially regulated hepatic miRNAs between these groups and used an in silico approach to predict bona fide mRNA targets and enriched pathways. Diet-induced hyperglycemia resulted in differential regulation of hepatic miRNAs compared with fasted fish. Some of the identified miRNAs, such as miRNA-27b-3p and miRNA-200a-3p, are known to be responsive to hyperglycemia in the liver of hyperglycemic glucose-tolerant fish and mammals, suggesting an evolutionary conserved regulation. Using Gene Ontology term-based enrichment analysis, we identify intermediate carbohydrate and lipid metabolism and insulin signaling as potential targets of posttranscriptional regulation by hyperglycemia-regulated miRNAs and provide correlative expression analysis of specific predicted miRNA-target pairs. This study identifies hepatic miRNAs in rainbow trout that exhibit differential postprandial expression in response to diets with different carbohydrate content and predicts posttranscriptionally regulated target mRNAs enriched for pathways involved in glucoregulation. Together, these results provide a framework for testable hypotheses of functional involvement of specific hepatic miRNAs in GI in rainbow trout.

Impact of Dietary Carbohydrate/Protein Ratio on Hepatic Metabolism in Land-Locked Atlantic Salmon (Salmo salar L.).

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 metabolize 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 toward starch utilization between land-locked salmon and the farmed stock. Further analyses are necessary in order to fully characterize the capacity of land-locked salmon to utilize dietary carbohydrate.