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For the sustainable development of aquaculture, vegetable ingredients have been used to replace the traditional ingredients fishmeal and fish oil in aquafeeds. On the other hand, selective breeding has also been implemented at research level to obtain strains that are adapted to feeding on the plant-based diet. To better understand the underlying mechanisms prompting the adaptation to plant-based diets in fish, we investigated the hepatic expression of several microRNAs (miRNAs) that are involved in the post-transcriptional regulation of cholesterol and lipid metabolism at 8 h and 16 h after the last meal in two lines of rainbow trout: one selected for better adaptation to plant-based diets and the corresponding control line. Both groups were fed either a fishmeal and fish oil based diet or a 100% plant-based diet. Results showed that the expression of miR-33a in liver was greatly elevated in trout fed the plant-based diet, while the expression of miR-122 and miR-128 was much higher in the selected line at 8 h after the last meal regardless of the diet. Furthermore, our results indicated that some genes involved in immune processes (caspase 6 apoptosis-related cysteine peptidase like 2, casp6l2) and cAMP signal transduction (phosphodiesterase 4B cAMP-specific a, pde4ba) were also potentially regulated by miRNAs. They were newly identified as putative direct targets of miRNAs and affected in trout fed the plant-based diet. Though further investigations are still needed to establish a valid relationship between miRNAs and their target genes, our study found miR-33a, miR-122 and miR-128 as potential candidates for further study and provided new perspectives to understand the role of miRNAs in the selective breeding for adaptation to the plant-based diets.

International audience; 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.

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 ofhk2 (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.

International audience; Cholesterol metabolism is greatly affected in fish fed plant-based diet. The regulation of cholesterol metabolism is mediated by both transcriptional factors such as sterol regulatory element binding proteins (SREBPs) and liver X receptors (LXRs), and posttranscriptional factors including miRNAs. In mammals, SREBP-2 and LXRα are involved in the transcrip-tional regulation of cholesterol synthesis and elimination, respectively. In mammals, miR-33a is reported to directly target genes involved in cholesterol catabolism. The present study aims to investigate the regulation of cholesterol metabolism by SREBP-2 and LXRα and miR-33a in rainbow trout using in vivo and in vitro approaches. In vivo, juvenile rainbow trout of~72 g initial body weight were fed a total plant-based diet (V) or a marine diet (M) containing fishmeal and fish oil. In vitro, primary cell culture hepatocytes were stimulated by graded concentrations of 25-hydroxycholesterol (25-HC). The hepatic expression of cholesterol synthetic genes, srebp-2 and miR-33a as well as miR-33a level in plasma were increased in fish fed the plant-based diet, reversely, their expression in hepatocytes were inhibited with the increasing 25-HC in vitro. However, lxrα was not affected neither in vivo nor in vitro. Our results suggest that SREBP-2 and miR-33a synergistically enhance the expression of cholesterol synthetic genes but do not support the involvement of LXRα in the regulation of cholesterol elimination. As plasma level of miR-33a appears as potential indicator of cholesterol synthetic capacities, this study also highlights circulating miRNAs as promising noninvasive biomarker in aquaculture.

Background:Recently, studies suggest that gut microbiota contributes to the development of obesity in mammals. In rainbow trout, little is known about the role of intestinal microbiota in host physiology.Objective:The aim of this study was to investigate the link between intestinal microbiota and adiposity, by high-throughput 16S RNA gene based illumina Miseq sequencing in two rainbow trout lines divergently selected for muscle lipid content. Fish from these two lines of rainbow trout are known to have a differing lipid metabolism.Methods:Samples from the two lines (L for lean and F for fat) were collected from Midgut (M) and Hindgut (H) in juvenile fish (18 months) to compare intestinal microbiota diversity.Results:Whatever the lines and intestinal localisation,Proteobacteria,FirmicutesandActinobacteriaare the dominant phyla in the bacterial community of rainbow trout (at least 97%). The results indicate that richness and diversity indexes as well as bacterial composition are comparable between all groups even though 6 specific OTUs were identified in the intestinal microbiota of fish from the fat line and 2 OTUs were specific to the microbiota of fish from the lean line. Our work contributes to a better understanding in microbial diversity in intestinal microbiota of rainbow trout.Conclusion:Altogether, our study indicates that no major modification of the intestinal microbiota is induced by selection for muscle lipid content and associated metabolic changes. Finally, we identified members of core microbiota in rainbow trout.

Nutritional programming is a very promising strategy for modifying nutrient metabolism to better adapt animals to new diets. Although it is a well-known approach in fish-farmed species, it has never been applied in marine shrimp. A preliminary requirement to perform nutritional programming is to identify the best developmental window for early stimulus. The objective of the present study was to characterize the ontogenesis of the expression of the main genes involved in digestion and metabolism in white shrimp, Litopenaeus vannamei. Four genes involved in digestion, 11 genes in intermediary metabolism (glucose, amino acid and lipid metabolism) and 5 genes in energy metabolism (mitochondria) were studied at 10 different developmental stages (from eggs – 8 h after fertilization - up to post-larval substage 5) using qRT-PCR for measuring mRNA levels. Our data show that almost all of the studied genes present higher mRNA levels during the protozoea substage Z1, which could be explained by the endogenous-exogenous feeding transition in white shrimp. In conclusion, Z1 substage shows a high molecular plasticity for metabolism which becomes the preferential developmental window for future programming experiments in L. vannamei. Statement of relevance Our study aimed at characterizing for the first time the ontogenesis of expressions of genes involved in intermediary metabolism (nutrient metabolism). This approach determined developmental windows with high molecular plasticity (Z1 and PL stages). These data will support future studies in nutritional programming for the whiteleg shrimp by determining the timing for early stimulus.

Background Glucose-6-phosphate (G6pc) is a key enzyme involved in the regulation of the glucose homeostasis. The present study aims at revisiting and clarifying the evolutionary history of g6pc genes in vertebrates. Results g6pc duplications happened by successive rounds of whole genome duplication that occurred during vertebrate evolution. g6pc duplicated before or around Osteichthyes/Chondrichthyes radiation, giving rise to g6pca and g6pcb as a consequence of the second vertebrate whole genome duplication. g6pca was lost after this duplication in Sarcopterygii whereas both g6pca and g6pcb then duplicated as a consequence of the teleost-specific whole genome duplication. One g6pca duplicate was lost after this duplication in teleosts. Similarly one g6pcb2 duplicate was lost at least in the ancestor of percomorpha. The analysis of the evolution of spatial expression patterns of g6pc genes in vertebrates showed that all g6pc were mainly expressed in intestine and liver whereas teleost-specific g6pcb2 genes were mainly and surprisingly expressed in brain and heart. g6pcb2b, one gene previously hypothesised to be involved in the glucose intolerant phenotype in trout, was unexpectedly up-regulated (as it was in liver) by carbohydrates in trout telencephalon without showing significant changes in other brain regions. This up-regulation is in striking contrast with expected glucosensing mechanisms suggesting that its positive response to glucose relates to specific unknown processes in this brain area. Conclusions Our results suggested that the fixation and the divergence of g6pc duplicated genes during vertebrates’ evolution may lead to adaptive novelty and probably to the emergence of novel phenotypes related to glucose homeostasis. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3727-1) contains supplementary material, which is available to authorized users.

International audience; Parental nutrition events may result in permanent changes in the growth potential, health and metabolic status of the next generation. In the present study,we aimed to determine in rainbow trout (Oncorhynchusmykiss), the effect of feeding broodstock with a diet deficient in methionine (one of the main methyl-group donor needed for all biological methylation reactions including DNA and histones methylation) on the response of several metabolism- and growth-related genes and proteins in offspring subjected to different dietary methionine levels (deficiency or adequate). Adult males and females were fed for 6 months with either a methionine deficient diet or a control diet. Males and females of the same feeding group were then crossed and the obtained fry were fed with either a methionine deficient or a control diet for 3 weeks from the first exogenous feeding. The obtained results indicated that, whatever the methionine content of the broodstock diet, a 3weeks dietary methionine deficiency in fry strongly affected the levels of transcripts and/or proteins involved in fatty acid synthesis, cholesterol synthesis and the two main cell quality control mechanisms, namely the ubiquitin-proteasome system and autophagy. In addition, our data also demonstrated that the levels of some of these mRNA and/or proteins are also affected by the dietary methionine deficiency in broodstock. Of interest, some genes (involved in gluconeogenesis and autophagy) were only affected by the parental and not by the fry diet. Together, these results clearly show that the broodstock diet may influence the metabolism of the progeny in rainbow trout and highlight the potential to apply new feeding strategies adapted to plant feedstuffs

International audience; Diadromy in eel is facultative and the diverse propensities in glass eel estuarine migration may lead to a large spatial dispersion, which may has profound influences on the species sex determination. In this study, we sought to clarify the relationship between European glass eel energetics and their pattern of migration behavior, in the framework of the conditional strategy. Marine glass eels were sampled in autumn and spring, stratifying high and low energetic status, respectively. Their migration behavior was determined in experimental installations that mimic tides using a change in water current direction every 6.2 h. When synchronized to the current reversal, glass eels were called active while individuals hiding in the substratum were considered as non-active. Then, for each active fish, a level of swimming activity was determined and both migration behavior and level of swimming activity were correlated to the individual wet weight (used as a proxy of energy stores), standard metabolic rate (relative SMR) and transcriptomic profile of metabolism related genes. Results showed that spring glass eels presented a lower probability to migrate, a lower wet weight and a higher expression of genes involved in energy stress resistance than autumn ones, supporting a conditional strategy based on individual's energy status. However, within each season, no wet weight difference was observed between active and non-active fish. In autumn glass eels, migration behavior was weakly related to relative SMR while in spring, none of the parameters analyzed allows discriminating active and non-active glass eels. The level of swimming activity in active fish was related to their relative SMR in autumn and to their wet weight in spring. Altogether, our results could not validate a conditional strategy but spring glass eels displayed some signs of energy distress and a lower level of swimming activity than autumn ones

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International audience; MicroRNAs (miRNAs) are a class of small non-coding RNAs which are known to posttranscriptionally regulate the expression of most genes in both animals and plants. Meanwhile, studies have shown that numbers of miRNAs are present in body fluids including the plasma. Despite the mode of action of these circulating miRNAs still remains unknown, they have been found to be promising biomarkers for disease diagnosis, prognosis and response to treatment. In order to evaluate the potential of miRNAs as non-invasive biomarkers in aquaculture, a time-course experiment was implemented to investigate the postprandial regulation of miRNAs levels in liver and plasma as well as the hepatic expression of genes involved in cholesterol metabolism. We showed that miR-1, miR-33a, miR-122, miR-128 and miR-223 were expressed in the liver of rainbow trout and present at detectable level in the plasma. We also demonstrated that hepatic expression of miR-1, miR-122 and miR-128 were regulated by feed intake and reached their highest levels 12 hours after the meal. Interestingly, we observed that circulating levels of miR-128 and miR-223 are subjected to postprandial regulations similar to that observed in their hepatic counterparts. Statistical correlations were observed between liver and plasma for miR-128 and miR-223 and between hepatic and circulating miR-122, miR-128 and miR-223 and expression of genes related to cholesterol synthesis and efflux or glucose phosphorylation. These results demonstrated that circulating miR-122, miR-128 and miR-223 are potential biomarkers of cholesterol metabolism in rainbow trout.

When compared with fish meal and fish oil, plant ingredients differ not only in their protein content and amino acid and fatty acid profiles but are also devoid of cholesterol, the major component of cell membrane and precursor of several bioactive compounds. Based on these nutritional characteristics, plant-based diets can affect fish physiology and cholesterol metabolism. To investigate the mechanisms underlying cholesterol homeostasis, rainbow trout were fed from 1 g body wt for 6 mo with a totally plant-based diet (V), a marine diet (M), and a marine-restricted diet (MR), with feed intake adjusted to that of the V group. The expression of genes involved in cholesterol synthesis, esterification, excretion, bile acid synthesis, and cholesterol efflux was measured in liver. Results showed that genes involved in cholesterol synthesis were upregulated in trout fed the V diet, whereas expression of genes related to bile acid synthesis ( cyp7a1) and cholesterol elimination ( abcg8) were reduced. Feeding trout the V diet also enhanced the expression of srebp-2 while reducing that of lxrα and miR-223. Overall, these data suggested that rainbow trout coped with the altered nutritional characteristics and absence of dietary cholesterol supply by increasing cholesterol synthesis and limiting cholesterol efflux through molecular mechanisms involving at least srebp-2, lxrα, and miR-223. However, plasma and body cholesterol levels in trout fed the V diet were lower than in fish fed the M diet, raising the question of the role of cholesterol in the negative effect of plant-based diet on growth.

International audience; The objective of this study was to examine the long-term effects of different levels of dietary carbohydrates on glucose metabolism in the omnivorous Nile Tilapia (Oreochromis niloticus). Fish were fed from the first feeding stage until the adult stage, over a period of 40 weeks, using three different levels of carbohydrates (dextrin): 0% (CHO-L), 30% (CHO-M) and 50% (CHO-H). Growth performance, blood metabolite parameters, the proximate composition of the whole body, muscle and liver tissue, and the mRNA levels for genes involved in glycolysis, gluconeogenesis, lipogenesis and glucose transport in liver and muscle tissue were analyzed at week 26 and week 40. Fish fed the CHO-M diet exhibited the best growth performance, while the fish fed with the CHO-H diet showed the lowest growth performance. These data suggest that the incorporation of a certain level dietary carbohydrates can benefit Nile Tilapia. However, if the proportion of carbohydrates is very high, and concomitantly, the level of dietary proteins too low, this will not promote growth. Fish fed with carbohydrates exhibited a significant increase in lipid deposition in the whole body, muscle and liver tissues in. This seems to be due to increased lipogenic capacity, as reflected by higher hepatic fatty acid synthase mRNA levels and activity, as well as higher levels of plasma triglyceride. Nile Tilapia appear to be able to effectively adapt to the intake of carbohydrates, as reflected by: (i) the weak postprandial hyperglycemia (5.3–6.1 mM), (ii) the higher level of hepatic glycogen, (iii) the higher glycolytic pyruvate kinase enzyme in muscle (mRNA level and activity) and (iv) the inhibition of the gluconeogenic pathway for glucose-6-phosphatase and phosphoenolpyruvate carboxykinase enzymes (at molecular and enzymatic levels). Overall, these data show that the well-known differences in the capacity of carnivorous and omnivorous fish species to use dietary carbohydrates could be linked to differences in the regulation of the gluconeogenic and lipogenic pathways.

The objective of the study was to test for the first time the molecular adaptation of the glucose metabolism to this fish species know to be a good user of carbohydrates. In this way, Nile tilapia were fed with 3 different levels of carbohydrates 0% (CHO-L) 30% (CHO-M) and 50% (CHO-H) dextrin. After 45 days and 90 days of feeding we analyzed the plasma parameters, zootechnical performance and the mRNA levels for genes involved in glycolysis, gluconeogenesis, lipogenesis and glucose transport in liver and muscle. The best growth performance was found for fish fed M-CHO diet, the fish fed with the H-CHO diets showing the worst growth performance. Increased of hepatic and muscle glycogen, hepatic somatic index and plasma metabolites (glucose, triglycerides and cholesterol) was linked to the increased of dietary carbohydrates. However, no hyperglycemia, no change of body compositions was found showing that dietary carbohydrates are efficiently used as an energy source in tilapia. Moreover, in contrats to the dietary protein-linked decreased of amino acid catabolic mRNA levels, no clear molecular adaptation for glycolysis, gluconeogenesis, lipogenesis in liver and glycolysis in muscle was detected except higher mRNA levels for glucose transporter in muscle. Our data suggest that these metabolic pathways at a molecular level are not the main actors explaining the efficient use of glucose in tilapia. Statement of relevance Our study aimed at characterizing for the first time the molecular effects of increased dietary carbohydrates (dextrin) from 0% up to 50% in Nile tilapia during 45 and 90 days. Our study confirmed that Nile tilapia can use high level of carbohydrates without any deregulation of glucose homeostasis. However, no strong regulations of expression for genes involved in glucose metabolism in liver and muscle - which could explain the reasons for an efficient use of carbohydrate by this fish species - were detected.

International audience; It is well known that change in environmental parameters experienced during early life can have profound effect on the metabolism of individuals later in life. The purpose of the present study was to evaluate the impact of a chronic hypoxic stimulus (15 days, 50% dissolved O2, 5.0 mg·L-1) applied at hatching (oxygen history) alone or combined with a 60% high carbohydrate (HC) dietary stimulus (8 days) applied at first-feeding (dietary history) on growth performance and glucose metabolism of juvenile trout. The effectiveness of the hypoxic and the HC dietary stimuli were verified by monitoring the modulation in mRNA levels of oxygen-sensitive and dietary carbohydrate-sensitive genes, respectively. At juvenile stage (28 weeks after first-feeding), neither dietary history nor oxygen history had long-term effect on growth performance. These juveniles were then subjected to a 5 days challenge test with a 30% carbohydrate diet. Our data showed that mRNA levels of some glucose transport-related genes in liver and muscle, and plasma triglycerides were down-regulated in fish which were exposed to hypoxia during early life. Regarding to the effect of dietary history, the mRNA level of muscular pfkmaa was observably increased in fish previously subjected to the HC dietary stimulus. In summary, the present study indicated that a chronic hypoxic stimulus applied at hatching alone or combined with a high carbohydrate stimulus at first-feeding can affect plasma triglycerides level and glucose metabolism-related genes in juvenile trout.

International audience; Salmonids belong to a high trophic level and are thus considered as strictly carnivorous species, metabolically adapted for high catabolism of proteins and low utilisation of dietary carbohydrates. However they conserved a “mammalian-type” nutritional regulation of glucokinase encoding gene and its enzymatic activity by dietary carbohydrates which remains puzzling regarding their dietary regime. The present study investigates the hypothesis that this conservation could be linked to a real consumption by trout of this nutrient in their natural habitat. To do so, brown trout were sampled in the sub-Antarctic Kerguelen Islands, a site presenting oligotrophic hydrosystems and no local freshwater fish fauna prior the introduction of salmonids fifty years ago. Qualitative and quantitative analysis of carbohydrate content within Kerguelen trout stomachs demonstrate that these animals are fed on food resources containing digestible carbohydrates. Additionally, glycaemia and more particularly gck mRNA level and gck enzymatic activity prove that Kerguelen trout digest and metabolise dietary carbohydrates. Physiological and molecular analyses performed in the present study thus strongly evidence for consumption of dietary carbohydrates by wild trout in natural environments. Investigating differences between Kerguelen individuals, we found that smaller individuals presented higher glycaemia, as well as higher carbohydrates contents in stomach. However no relationship between scaled mass index and any physiological indicator was found. Thus it appears that Kerguelen trout do not turn to carbohydrate diet because of a different condition index, or that the consumption of carbohydrates does not lead to a generally degraded physiological status. As a conclusion, our findings may explain the evolutionary conservation of a “mammalian-type” nutritional regulation of gck by dietary carbohydrates in these carnivorous fish

Background/Aims: Carnivores exhibit poor utilization of dietary carbohydrates and glucose intolerant phenotypes, yet it remains unclear what are the causal factors and underlying mechanisms. We aimed to evaluate excessive amino acids (AAs)-induced effects on insulin signaling, fatty acid biosynthesis and glucose metabolism in rainbow trout and determine the potential involvement of mTORC1 and p38 MAPK pathway. Methods: We stimulated trout primary hepatocytes with different AA levels and employed acute administration of rapamycin to inhibit mTORC1 activation. Results: Increased AA levels enhanced the phosphorylation of ribosomal protein S6 kinase (S6K1), S6, and insulin receptor substrate 1 (IRS-1) on Ser302 but suppressed Akt and p38 phosphorylation; up-regulated the expression of genes related to gluconeogenesis and fatty acid biosynthesis. mTORC1 inhibition not only inhibited the phosphorylation of mTORC1 downstream targets, but also blunted IRS-1 Ser302 phosphorylation and restored excessive AAs-suppressed Akt phosphorylation. Rapamycin also inhibited fatty acid biosynthetic and gluconeogenic gene expression. Conclusion: High levels of AAs up-regulate hepatic fatty acid biosynthetic gene expression through an mTORC1-dependent manner, while attenuate insulin-mediated repression of gluconeogenesis through elevating IRS-1 Ser302 phosphorylation, which in turn impairs Akt activation and thereby weakening insulin action. We propose that p38 MAPK probably also involves in these AAs-induced metabolic changes.

Based on the concept of nutritional programming in mammals, we tested whether a short term hyper or hypo vitamin stimulus during first-feeding could induce long-lasting changes in nutrient metabolism in rainbow trout. Trout alevins received during the 4 first weeks of exogenous feeding a diet either without supplemental vitamins (NOSUP), a diet supplemented with a vitamin premix to satisfy the minimal requirement in all the vitamins (NRC) or a diet with a vitamin premix corresponding to an optimal vitamin nutrition (OVN). Following a common rearing period on the control diet, all three groups were then evaluated in terms of metabolic marker gene expressions at the end of the feeding period (day 119). Whereas no gene modifications for proteins involved in energy and lipid metabolism were observed in whole alevins (short-term effect), some of these genes showed a long-term molecular adaptation in the muscle of juveniles (long-term effect). Indeed, muscle of juveniles subjected at an early feeding of the OVN diet displayed up-regulated expression of markers of lipid catabolism (3-hydroxyacyl-CoA dehydrogenase – HOAD - enzyme) and mitochondrial energy metabolism (Citrate synthase - cs, Ubiquitinol cytochrome c reductase core protein 2 - QCR2, cytochrome oxidase 4 - COX4, ATP synthase form 5 - ATP5A) compared to fish fed the NOSUP diet. Moreover, some key enzymes involved in glucose catabolism (Muscle Pyruvate kinase - PKM) and amino acid catabolism (Glutamate dehydrogenase - GDH3) were also up regulated in muscle of juvenile fish fed with the OVN diet at first-feeding compared to fish fed the NOSUP diet. We researched if these permanently modified gene expressions could be related to global modifications of epigenetic marks (global DNA methylation and global histone acetylation and methylation). There was no variation of the epigenetic marks in muscle. However, we found changes in hepatic DNA methylation, global H3 acetylation and H3K4 methylation, dependent on the vitamin intake at early life. In summary, our data show, for the first time in fish, that a short-term vitamin-stimulus during early life may durably influence muscle energy and lipid metabolism as well as some hepatic epigenetic marks in rainbow trout. Statement of relevance Programming at first feeding with different levels of vitamins was successfully tested in fish for the first time. These data proove one more time the importance of the early life on the fish metabolism in adults.

Environmental conditions experienced during early life play an important role in the long-term metabolic status of individuals. The present study investigated whether early hypoxia exposure (24 h, 2.5 mg·l−1, 20% dissolved O2) during embryo stage alone (hypoxic history) or combined with a 5-day high carbohydrate diet (60%) stimulus at first-feeding (high carbohydrate diet history) can affect the glucose metabolism later in life, i.e. in fish juveniles. After 19 weeks of growth, we observed a decrease in final body weight in fish with a high carbohydrate diet history. Feed efficiency was significantly affected by both hypoxic and dietary histories. After a short challenge test (5 days) performed with a 30% carbohydrate diet in trout juveniles, our results also showed that in trout which experienced hypoxic history, mRNA levels of gluconeogenic genes in liver and glucose transport genes in both liver and muscle of fish juveniles were significantly increased. Besides, mRNA levels of glycolytic genes were decreased in fish with a high carbohydrate dietary history. Both hypoxic and dietary histories barely affected plasma metabolites and global epigenetic modifications in fish juveniles after the challenge test. In conclusion, our results demonstrated that an acute hypoxic stimulus during early development alone or combined with a hyperglucidic stimulus at first feeding can modify growth performance and glucose metabolism at the molecular level in trout juveniles.

Rainbow trout (Oncorhynchus mykiss) is considered a “glucose-intolerant” species. With the aim of programming trout to improve their metabolic use of dietary carbohydrates, we hypothesised that a hypoxic stimulus applied during embryogenesis could later affect glucose metabolism at the first-feeding stage. An acute hypoxic stimulus (2.5 or 5.0 mg·L−1 O2) was applied for 24 h to non-hatched embryos or early hatched alevins followed by a challenge test with a high carbohydrate diet at first-feeding. The effectiveness of the early hypoxic stimulus was confirmed by the induction of oxygen-sensitive markers such as egln3. At first-feeding, trout previously subjected to the 2.5 mg·L−1 O2 hypoxia displayed a strong induction of glycolytic and glucose transport genes, whereas these glucose metabolism-related genes were affected much less in trout subjected to the less severe (5.0 mg·L−1 O2) hypoxia. Our results demonstrate that an acute hypoxic stimulus during early development can affect glucose metabolism in trout at first-feeding.

Environmental conditions experienced during early life play an important role in the long-term metabolic status of individuals. The present study investigated whether hypoxia exposure [for 24 h: 2.5 mg O

International audience; The impact of increased incorporation of plant ingredients on diets for rainbow trout was evaluated in terms of gene expression of gastric (gastric lipase, pepsinogen) and intestinal (prolidase, maltase, phospholipase A2) digestive enzymes and nutrient transporters (peptide and glucose transporters), as well as of postprandial levels of plasma glucose, triglycerides and total free amino acids. For that purpose, trout alevins were fed from the start of exogenous feeding one of three different experimental diets: a diet rich in fish meal and fish oil (FM–FO), a plant-based diet (noFM–noFO) totally free from fish meal and fish oil, but containing plant ingredients and a Mixed diet (Mixed) intermediate between the FM–FO and noFM–noFO diets. After 16 months of rearing, all fish were left unfed for 72 h and then given a single meal to satiation. Blood, stomach and anterior intestine were sampled before the meal and at 2, 6 and 12 h after this meal. The postprandial kinetics of gene expression of gastric and intestinal digestive enzymes and nutrient transporters were then followed in trout fed the FM–FO diet. The postprandial profiles showed that the expression of almost all genes studied was stimulated by the presence of nutrients in the digestive tract of trout, but the timing (appearance of peaks) varied between genes. Based on these data, we have focused on the molecular response to dietary factors in the stomach and the intestine at 6 and 12 h after feeding, respectively. The reduction in FM and FO levels of dietary incorporation induced a significant decrease in the gene expression of gastric lipase, GLUT2 and PEPT1. The plasma glucose and triglycerides levels were also reduced in trout fed the noFM–noFO diet. Consequently, the present study suggests a decrease in digestive capacities in trout fed a diet rich in plant ingredients.

The link between dietary carbohydrate/protein and de novo lipogenesis (DNL) remains debatable in carnivorous fish. We aimed to evaluate and compare the response of hepatic lipogenic gene expression to dietary carbohydrate intake/glucose and dietary protein intake/amino acids (AAs) during acute stimulations using both in vivo and in vitro approaches. For the in vivo trial, three different diets and a controlled-feeding method were employed to supply fixed amount of dietary protein or carbohydrate in a single meal; for the in vitro trial, primary hepatocytes were stimulated with a low or high level of glucose (3 mM or 20 mM) and a low or high level of AAs (one-fold or four-fold concentrated AAs). In vitro data showed that a high level of AAs upregulated the expression of enzymes involved in DNL [fatty acid synthase (FAS) and ATP citrate lyase (ACLY)], lipid bioconversion [elongation of very long chain fatty acids like-5 (Elovl5), Elovl2, Δ6 fatty acyl desaturase (D6D) and stearoyl-CoA desaturase-1 (SCD1)], NADPH production [glucose-6-phosphate dehydrogenase (G6PDH) and malic enzyme (ME)], and transcriptional factor sterol regulatory element binding protein 1-like, while a high level of glucose only elevated the expression of ME. Data in trout liver also showed that high dietary protein intake induced higher lipogenic gene expression (FAS, ACLY, and Elovl2) regardless of dietary carbohydrate intake, while high carbohydrate intake markedly suppressed the expression of acetyl-CoA carboxylase (ACC) and Elovl5. Overall, we conclude that, unlike rodents or humans, hepatic fatty acid biosynthetic gene expression in rainbow trout is more responsive to dietary protein intake/AAs than dietary carbohydrate intake/glucose during acute stimulations. This discrepancy probably represents one important physiological and metabolic difference between carnivores and omnivores.

The rainbow trout (Oncorhynchus mykiss) exhibits high dietary amino acid requirements and an apparent inefficiency to use dietary carbohydrates. Using this species, we investigated the metabolic consequences of long-term high carbohydrates/low protein feeding. Fish were fed two experimental diets containing either 20 % carbohydrates/50 % proteins (C20P50), or high levels of carbohydrates at the expense of proteins (35 % carbohydrates/35 % proteins – C35P35). The expression of genes related to hepatic and muscle glycolysis (glucokinase (GK), pyruvate kinase and hexokinase) illustrates the poor utilisation of carbohydrates irrespective of their dietary levels. The increased postprandial GK activity and the absence of inhibition of the gluconeogenic enzyme glucose-6-phosphatase activity support the hypothesis of the existence of a futile cycle around glucose phosphorylation extending postprandial hyperglycaemia. After 9 weeks of feeding, the C35P35-fed trout displayed lower body weight and feed efficiency and reduced protein and fat gains than those fed C20P50. The reduced activation of eukaryotic translation initiation factor 4-E binding protein 1 (4E-BP1) in the muscle in this C35P35 group suggests a reduction in protein synthesis, possibly contributing to the reduction in N gain. An increase in the dietary carbohydrate:protein ratio decreased the expression of genes involved in amino acid catabolism (serine dehydratase and branched-chain α-keto acid dehydrogenase E1α and E1β), and increased that of carnitine palmitoyltransferase 1, suggesting a higher reliance on lipids as energy source in fish fed high-carbohydrate and low-protein diets. This probably also contributes to the lower fat gain. Together, these results show that different metabolic pathways are affected by a high-carbohydrate/low-protein diet in rainbow trout.

International audience; The objective of the present study was to identify postprandial molecular events associated with dietary fish oil level in rainbow trout using trout cDNA microarray tools (9 K). Our genomics data showed that relatively few hepatic genes (n=41) are differentially expressed between fish fed a high fat diet compared to those fed a diet without added fish oil. We then focus our analysis of specific events involved in intermediary metabolism by analysing them using qRT-PCR. Removal of fish oil was associated with higher lipid biosynthesis (reflected by the fatty acid synthase gene) and lower lipid catabolism (reflected by the acyl-CoA oxidase gene). More interestingly, some genes involved in proteasomal-dependant proteolysis (proteasome and ubiquitin ligase genes) were down regulated in fish fed without fish oil, suggesting a relationship between dietary lipid intake and regulation of hepatic protein metabolism in rainbow trout. (c) 2008 Elsevier B.V. All rights reserved.

The rainbow trout (Oncorhynchus mykiss), a carnivorous fish species, displays a “glucose-intolerant” phenotype when fed a high carbohydrate diet. The importance of carbohydrate metabolism during embryogenesis and the timing of establishing this latter phenotype are currently unclear. In addition, the mechanisms underlying the poor ability of carnivorous fish to use dietary carbohydrates as a major energy substrate are poorly understood. It has recently been shown in trout that duplicated genes involved in glucose metabolism may be involved in establishing the glucose-intolerant phenotype. The aim of this study was therefore to provide new understanding of glucose metabolism during ontogenesis and nutritional transition, taking into consideration the complexity of the trout genome. Trout were sampled at several stages of development from fertilization to hatching, and alevins were then fed a non-, or a high carbohydrate diet during first-feeding. mRNA levels of all glucose metabolism-related genes increased in embryos during the setting up of the primitive liver. After the first meal, genes rapidly displayed expression patterns equivalent to those observed in the livers of juveniles. g6pcb2.a (one glucose-6-phosphatase encoding gene) was up-regulated in alevins fed a high carbohydrate diet, mimicking the expression pattern of gck genes. The g6pcb2.a gene may contribute to the non-inhibition of the last step of gluconeogenesis and thus to establishing the glucose-intolerant phenotype in trout fed a high carbohydrate diet as early as first-feeding. This information is crucial for nutritional programming investigations as it suggests that first feeding would be too late to programme glucose metabolism in the long term.

International audience; Hepatic lipogenesis represents a crucial part of intermediary metabolism and is acutely regulated by endocrine factors and nutrients. The liver-specific and highly abundant microRNA-122 has emerged as a powerful regulator of lipogenesis in higher vertebrates, but little is known about its endocrine and nutritional regulation. In this study, we investigated the hypothesis that insulin regulates hepatic expression of omy-miRNA-122 isomiRNAs (omy-miRNA-122a and omy-miRNA-122b) by using in vivo and in vitro approaches. Since the hepatic insulin pathway and lipogenesis are acutely regulated by dietary macronutrient ratios in rainbow trout, we further investigated the effect of single meals with altered carbohydrate/protein ratio and lipid/protein ratio on the postprandial expression of omy-miRNA-122 isomiRNAs. Insulin acutely induced omy-miRNA-122b expression in vivo and in vitro. Conversely, a single meal with increased lipid to protein ratio acutely decreased expression of both omy-miRNA-122 isomiRNAs. As a direct proof of lipogenic effects of miRNA-122 is currently still lacking in fish, we investigated the correlated expression between omy-miRNA-122 isomiRNAs and the rate-limiting lipogenic gene fas, an indirect target gene of miRNA-122 in mammals. Our results show a significant positive correlation of omy-miRNA-122b and fas, consistent with a potential evolutionary conserved role for miRNA-122 in the regulation of postprandial lipogenesis in trout

International audience; Salmonids are generally known to be glucose intolerant.However,previous studies have shown that growth hor- mone (GH) transgenic coho salmon display modified nutritional regulation of glycolysis and lipogenesis com- pared to non-transgenic fish, suggesting the potential for better use of glucose in GH transgenic fish. To examine this in detail, GH transgenic and non-transgenic coho salmon were subjected to glucose tolerance test and subsequent metabolic assessments. After intra-peritoneal injection of 250 mg/kg glucose, we analysed post-injectionkineticsofglycaemiaandexpressionofseveralkeytargetgeneshighlyinvolvedinglucosehomeo- stasis in muscle and liver tissues. Our data show no significant differences in plasma glucose levels during peak hyperglycaemia (3–6 h after injection), demonstrating a similar glucose tolerance between transgenic and non transgenic. However, and unrelated to the hyperglycaemic episode, GH transgenic fish return to a slightly lower basal glycaemia values 24 h after injection. Correspondingly, GH transgenic fish exhibited higher mRNA levels of glucokinase (GK) and glucose-6-phosphate dehydrogenase (G6PDH) in liver, and glucose transporter (GLUT4) in muscle. These data suggest that these metabolic actors may be involved in different glucose use in GHtransgenic fish,which would be expected to influence the glucose challenge response.Overall,our data dem- onstrate that GH transgenic coho salmon may be a pertinent animal model for further study of glucose metabo- lism in carnivorous fish

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.

International audience; Using rainbow trout hepatocytes stimulated with L-leucine, L-methionine, or L-lysine in the presence or absence of bovine insulin, we investigated the ability of these amino acids to mimic the effects of a pool of amino acids on protein kinase B (Akt)/target of rapamycin (TOR) signaling pathways and expression of 6 genes known to be subjected to insulin and/or amino acid regulation [glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase (PEPCK), glucokinase (GK), pyruvate kinase (PK), fatty acid synthase (FAS), and serine dehydratase (SDH)]. Emphasis was placed on leucine, known to be a signaling molecule in mammals, and methionine and lysine that are essential amino acids limiting in plant-based diets for fish. In the presence of insulin, leucine (but not methionine or lysine) phosphorylated Akt and ribosomal protein 56 as previously observed with a pool of amino acids, suggesting that leucine might participate in the activation of the TOR pathway by amino acids in f sh, as in mammals. G6Pase, PEPCK, GK, and SDH gene expression were higher in leucine-treated cells compared with control cells. Leucine combined with insulin reduced G6Pase gene expression by 90% and increased FAS gene expression > 4-fold compared with the control treatment. Methionine weakly decreased G6Pase, GK, and SDH gene expression and lysine weakly but significantly decreased the mRNA level of PEPCK. Thus, leucine regulated gluconeogenesis and lipogenesis, but not glycolysis, in the same way as a pool of amino acids. Methionine appeared to be involved in the regulation of specific genes, whereas lysine only had limited effects. These findings are particularly relevant regarding the involvement of amino acids in the regulation of metabolism-related gene expression. J. Nutr. 141: 75-80, 2011.

International audience; Most teleost fish are known to require high levels of dietary proteins. Such high-protein intake could have significant effects, particularly on insulin-regulated gene expression. We therefore analyzed the effects of an increase in the ratio of dietary carbohydrates/proteins on the refeeding activation of the Akt-target of rapamycin (TOR) signaling pathways in rainbow trout and the effects on the expression of several genes related to hepatic and muscle metabolism and known to be regulated by insulin, amino acids, and/or glucose. Fish were fed once one of three experimental diets containing high (H), medium (M), or low (L) protein (P) or carbohydrate (C) levels after 48 h of feed deprivation. Activation of the Akt/TOR signaling pathway by refeeding was severely impaired by decreasing the proteins-to-carbohydrates ratio. Similarly, postprandial regulation of several genes related to glucose (Glut4, glucose-6-phosphatase isoform 1), lipid (fatty acid synthase, ATP-citrate lyase, sterol responsive element binding protein, carnitine palmitoyltransferase 1, and 3-hydroxyacyl-CoA dehydrogenase), and amino acid metabolism (serine dehydratase and branched-chain alpha-keto acid dehydrogenase E2 subunit) only occurred when fish were fed the high-protein diet. On the other hand, diet composition had a low impact on the expression of genes related to muscle protein degradation. Interestingly, glucokinase was the only gene of those monitored whose expression was significantly upregulated by increased carbohydrate intake. In conclusion, this study demonstrated that macro-nutrient composition of the diet strongly affected the insulin/amino acids signaling pathway and expression pattern of genes related to metabolism.

International audience; Amino acids are considered to be regulators of metabolism in several species, and increasing importance has been accorded to the role of amino acids as signalling molecules regulating protein synthesis through the activation of the TOR transduction pathway. Using rainbow trout hepatocytes, we examined the ability of amino acids to regulate hepatic metabolism-related gene expression either alone or together with insulin, and the possible involvement of TOR. We demonstrated that amino acids alone regulate expression of several genes, including glucose-6-phosphatase, phosphoenolpyruvate carboxykinase, pyruvate kinase, 6-phospho-fructo-1-kinase and serine dehydratase, through an unknown molecular pathway that is independent of TOR activation. When insulin and amino acids were added together, a different pattern of regulation was observed that depended upon activation of the TOR pathway. This pattern included a dramatic up-regulation of lipogenic (fatty acid synthase, ATP-citrate lyase and sterol responsive element binding protein 1) and glycolytic (glucokinase, 6-phospho-fructo-1-kinase and pyruvate kinase) genes in a TOR-dependent manner. Regarding gluconeogenesis genes, only glucose-6-phosphatase was inhibited in a TOR-dependent manner by combination of insulin and amino acids and not by amino acids alone. This study is the first to demonstrate an important role of amino acids in combination with insulin in the molecular regulation of hepatic metabolism.

Energy intake and genetic background are major determinants of muscle fat content in most animals, including man. We combined genetic selection and dietary energy supply to study the metabolic pathways involved in genetic and nutritional control of fat deposition in the muscle of rainbow trout (Oncorhynchus mykiss). Two experimental lines of rainbow trout, selected for lean (L) or fat (F) muscle, were fed with diets containing either 10 or 23 % lipids from the first feeding, up to 6 months. At the end of the trial, trout exhibited very different values of muscle fat content (from 4·2 to 10·1 % wet weight). Using microarrays made from a rainbow trout multi-tissue cDNA library, we analysed the molecular changes occurring in the muscle of the two lines when fed the low-energy or high-energy diet. The results from microarray analysis revealed that eleven metabolism-related genes were differentially expressed according to the diet while selection resulted in expression change for twenty-six genes. The most striking observation was the increased level of transcripts encoding the VLDL receptor and fatty acid translocase/CD36 following both the high-fat diet and upward selection for muscle fat content, suggesting that these two genes are relevant molecular markers of fat deposition in the white muscle of rainbow trout.