Long-term regulation of fat sensing in rainbow trout (Oncorhynchus mykiss) fed a vegetable diet from the first feeding: focus on free fatty acid receptors and their signalling.

Taste plays a fundamental role in an animal's ability to detect nutrients and transmits key dietary information to the brain, which is crucial for its growth and survival. Providing alternative terrestrial ingredients early in feeding influences the growth of rainbow trout (RT, Oncorhynchus mykiss). Thus, the present study aimed to assess the influence, via long-term feeding (from the first feeding to 8 months), of alternative plant ingredients (V diet for vegetable diet v. C diet for a control diet) in RT on the mechanism of fat sensing at the gustatory level. After the feeding trial, we studied the pathways of the fat-sensing mechanism in tongue tissue and the integrated response in the brain. To this end, we analysed the expression pattern of free fatty acid receptors (ffar) 1 and 2, markers of calcium-signalling pathways (phospholipase Cß, Orai, Stim or Serca), the serotonin level (a key neurotransmitter in taste buds) and the expression pattern of appetite-regulating neuropeptides in the hypothalamus (central area of appetite regulation). The results revealed that the V diet modified the expression pattern of ffar1 and paralogs of ffar2 genes in tongue tissue, along with differential regulation of calcium-signalling pathways and a defect in serotonin level and brain turnover, without influencing neuropeptide expression. This study is the first to support that changes in feeding behaviour of RT fed a V diet could be due to the difference in nutrient sensing and a decrease in hedonic sensation. We revealed that RT have similar fat-detection mechanisms as mammals.

Early starvation in European seabass (Dicentrarchus labrax) larvae has no drastic effect on hepatic intermediary metabolism in juveniles.

The present study aims to investigate nutritional programming through early starvation in the European seabass (Dicentrarchus labrax). European seabass larvae were fasted at three different developmental periods for three durations from 60 to 65 dph (F1), 81 to 87 dph (F2), and 123 to 133 dph (F3). Immediate effects were investigated by studying gene expression of npy (neuropeptide Y) and avt (Arginine vasotocin) in the head, while potential long-term effects (i.e., programming) were evaluated on intermediary metabolism later in life (in juveniles). Our findings indicate a direct effect regarding gene expression in the head only for F1, with higher avt mRNA level in fasted larved compared to controls. The early starvation periods had no long-term effect on growth performance (body weight and body length). Regarding intermediary metabolism, we analyzed related key plasma metabolites which reflect the intermediary metabolism: no differences for glucose, triglycerides, and free fatty acids in the plasma were observed in juveniles irrespective of the three early starvation stimuli. As programming is mainly linked to molecular mechanisms, we then studied hepatic mRNA levels for 23 key actors of glucose, lipid, amino acid, and energy metabolism. For many of the metabolic genes, there was no impact of early starvation in juveniles, except for three genes involved in glucose metabolism (glut2-glucose transporter and pk-pyruvate kinase) and lipid metabolism (acly-ATP citrate lyase) which were higher in F2 compared to control. Together, these results highlight that starvation between 81 to 87 dph may have more long-term impact, suggesting the existence of a developmental window for programming by starvation. In conclusion, European seabass appeared to be resilient to early starvation during larvae stages without drastic impacts on intermediary metabolism later in life.

Gut physiology of rainbow trout (Oncorhynchus mykiss) is influenced more by short-term fasting followed by refeeding than by feeding fishmeal-free diets.

Supplementing a fishmeal-free diet with yeast extract improves rainbow trout (Oncorhynchus mykiss) growth performance and modulates the hepatic and intestinal transcriptomic response. These effects are often observed in the long term but are not well documented after short periods of fasting. Fasting for a few days is a common practice in fish farming, especially before handling the fish, such as for short sorting, tank transfers, and vaccinations. In the present study, rainbow trout were subjected to a 4-day fast and then refed, for 8 days, a conventional diet containing fishmeal (control diet) or alternative diets composed of terrestrial animal by-products supplemented or not with a yeast extract. During the refeeding period alone, most of the parameters considered did not differ significantly in response to the different feeds. Only the expression of claudin-15 was upregulated in fish fed the yeast-supplemented diet compared to the control diet. Conversely, fasting followed by refeeding significantly influenced most of the parameters analyzed. In the proximal intestine, the surface area of villi significantly increased, and the density of goblet cell tended to decrease during refeeding. Although no distinct plasma immune response or major signs of gut inflammation were observed, some genes involved in the structure, complement pathway, antiviral functions, coagulation, and endoplasmic reticulum stress response of the liver and intestine were significantly regulated by refeeding after fasting. These results indicate that short-term fasting, as commonly practiced in fish farming, significantly alters the physiology of the liver and intestine regardless of the composition of the diet.

Circulating miRNA repertoire as a biomarker of metabolic and reproductive states in rainbow trout.

BACKGROUND: Circulating miRNAs (c-miRNAs) are found in most, if not all, biological fluids and are becoming well-established non-invasive biomarkers of many human pathologies. However, their features in non-pathological contexts and whether their expression profiles reflect normal life history events have received little attention, especially in non-mammalian species. The aim of the present study was to investigate the potential of c-miRNAs to serve as biomarkers of reproductive and metabolic states in fish. RESULTS: The blood plasma was sampled throughout the reproductive cycle of female rainbow trout subjected to two different feeding regimes that triggered contrasting metabolic states. In addition, ovarian fluid was sampled at ovulation, and all samples were subjected to small RNA-seq analysis, leading to the establishment of a comprehensive miRNA repertoire (i.e., miRNAome) and enabling subsequent comparative analyses to a panel of RNA-seq libraries from a wide variety of tissues and organs. We showed that biological fluid miRNAomes are complex and encompass a high proportion of the overall rainbow trout miRNAome. While sharing a high proportion of common miRNAs, the blood plasma and ovarian fluid miRNAomes exhibited strong fluid-specific signatures. We further revealed that the blood plasma miRNAome significantly changed depending on metabolic and reproductive states. We subsequently identified three evolutionarily conserved muscle-specific miRNAs or myomiRs (miR-1-1/2-3p, miR-133a-1/2-3p, and miR-206-3p) that accumulated in the blood plasma in response to high feeding rates, making these myomiRs strong candidate biomarkers of active myogenesis. We also identified miR-202-5p as a candidate biomarker for reproductive success that could be used to predict ovulation and/or egg quality. CONCLUSIONS: Together, these promising results reveal the high potential of c-miRNAs, including evolutionarily conserved myomiRs, as physiologically relevant biomarker candidates and pave the way for the use of c-miRNAs for non-invasive phenotyping in various fish species.

Kinetic study of the expression of genes related to hepatic steatosis, glucose and lipid metabolism, and cellular stress during overfeeding in mule ducks.

Induced by overfeeding, hepatic steatosis is a process exploited for the "foie gras" production in mule ducks. To better understand the mechanisms underlying its development, the physiological responses of mule ducks overfed with corn for a duration of 11 days were analyzed. A kinetic analysis of glucose and lipid metabolism and cell protection mechanisms was performed on 96 male mule ducks during overfeeding with three sampling times (after the 4th, the 12th, and the 22nd meal). Gene expression and protein analysis realized on the liver, muscle, and abdominal fat showed an activation of a cholesterol biosynthetic pathway during the complete overfeeding period mainly in livers with significant correlations between its weight and its cholesterolemia (r = 0.88; P < 0.0001) and between the liver weight and the hmgcr and soat1 expression (r = 0.4, P < 0.0001 and r = 0.67; P < 0.0001, respectively). Results also revealed an activation of insulin and amino acid cells signaling a pathway suggesting that ducks boost insulin sensitivity to raise glucose uptake and use via glycolysis and lipogenesis. Cellular stress analysis revealed an upregulation of key autophagy-related gene expression atg8 and sqstm1(P < 0.0001) during the complete overfeeding period, mainly in the liver, in contrast to an induction of cyp2e1(P < 0.0001), suggesting that autophagy could be suppressed during steatosis development. This study has highlighted different mechanisms enabling mule ducks to efficiently handle the starch overload by keeping its liver in a nonpathological state. Moreover, it has revealed potential biomarker candidates of hepatic steatosis as plasma cholesterol for the liver weight.

Proton-NMR Metabolomics of Rainbow Trout Fed a Plant-Based Diet Supplemented with Graded Levels of a Protein-Rich Yeast Fraction Reveal Several Metabolic Processes Involved in Growth.

BACKGROUND: Plant raw materials are commonly used in aquafeeds, as marine resources are unsustainable. However, full plant-based diets lead to poorer fish growth performance. OBJECTIVE: We aimed to understand the metabolic effects of a yeast fraction as a protein supplement in a plant-based diet and to integrate such effects with phenotypic traits as a new approach to assess the interest of this raw material. METHODS: Juvenile (49 g) rainbow trout (Oncorhynchus mykiss) were fed graded levels of a yeast protein-rich fraction (5% YST05, 10% YST10, 15% YST15) in a plant-based diet (PB) for 84 d. Final body weight, feed conversion ratio, and hepatosomatic and viscerosomatic indexes were measured. Plasma, liver, and muscle 1H-NMR fingerprints were analyzed with principal component analyses, and their metabolite patterns were clustered according to the yeast level to identify concomitant metabolic effects. A regression modeling approach was used to predict tissue metabolite changes from plasma fingerprints. RESULTS: In tissues, the patterns of metabolite changes followed either linear trends with the gradual inclusion of a yeast fraction (2 patterns out of 6 in muscle, 1 in liver) or quadratic trends (4 patterns in muscle, 5 in liver). Muscle aspartate and glucose (395 and 138% maximum increase in relative content compared with PB, respectively) revealing modification in energy metabolism, as well as modification of liver betaine (163% maximum increase) and muscle histidine (57% maximum decrease) related functions, indicates that the yeast fraction could improve growth in several ways. The highest correlation between measured and predicted metabolite intensities in a tissue based on plasma fingerprints was observed for betaine in liver (r = 0.80). CONCLUSIONS: These findings herald a new approach to assess the plurality of metabolic effects induced by diets and establish the optimal level of raw materials. They open the way for using plasma as a noninvasive matrix in trout nutrition studies.

Impact on cerebral function in rainbow trout fed with plant based omega-3 long chain polyunsaturated fatty acids enriched with DHA and EPA.

Characterization and modulation of cerebral function by ω-3 long chain polyunsaturated fatty acids (ω-3 LC-PUFAs), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) enrichment in plant based-diet were studied in rainbow trout (Oncorhynchus mikyss). We hypothesized that ω-3 LC-PUFAs are involved in the regulation of cerebral function in fish. During nine weeks, we examined the growth performance of rainbow trout for three experimental plant based-diets containing distinct levels of EPA and DHA. Using RT-qPCR, we assessed mRNA genes related to feeding behavior regulated by the central nervous system of humans, rodents and fish. These include markers of neuropeptides, indicators of cellular specification, animal stress, oxidant status, cytokines and genes regulating animal behaviour. ω-3 LC-PUFAs enrichment decreased daily food intake and induced a simultaneous mRNA expression increase in orexigenic transcript npy peptide and a decrease in anorexigen transcript pomcA peptide in the hypothalamus. Overall transcript genes related to proinflammatory cytokines, inflammation, antioxidant status, cortisol pathway, serotoninergic pathways and dopaminergic pathways were down-regulated in the juveniles fed the high ω-3 LC-PUFAs diet. However, the mRNA expression of transcripts related to cell specification were down regulated, namely tmem119 markers of microglial cell in forebrain and midbrain, gfap markers of astrocyte in the midbrain, and rbfox3 markers of neurons in the midbrain and hindbrain in juveniles fed high ω-3 experimental diet. In conclusion, this study revealed that a diet rich in ω-3 LC-PUFAs affected a relatively high proportion of the brain function in juvenile rainbow trout through mechanisms comparable to those characterized previously in mammals.

Regulation of genes related to cholesterol metabolism in rainbow trout (Oncorhynchus mykiss) fed a plant-based diet.

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.

Characterizing alternative feeds for rainbow trout (O. mykiss) by 1H NMR metabolomics.

INTRODUCTION: Fish feed formulations are constantly evolving to improve the quality of diets for farmed fish and to ensure the sustainability of the aquaculture sector. Nowadays, insect, microalgae and yeast are feedstuff candidates for new feeds. However, the characterization of aquafeed is still based on proximate and targeted analyses which may not be sufficient to assess feed quality. OBJECTIVES: Our aim was to highlight the soluble compounds that specifically differ between selected plant-based feeds complemented with alternative feedstuffs and discuss their origin and potential for fish nutrition. METHODS: A growth trial was carried out to evaluate growth performances and feed conversion ratios of fish fed plant-based, commercial, insect, spirulina and yeast feeds. 1H NMR metabolomics profiling of each feed was performed using a CPMG sequence on polar extracts. Spectra were processed, and data were analyzed using multivariate and univariate analyses to compare alternative feeds to a plant-based feed. RESULTS: Fish fed insect or yeast feed showed the best growth performances associated with the lowest feed conversion ratios compared to plant-based feed. Soluble compound 1H NMR profiles of insect and spirulina alternative feeds differed significantly from the plant-based one that clustered with yeast feed. In insect and spirulina feeds, specific differences compared to plant-based feed concerned glycerol and 3-hydroxybutyrate, respectively. CONCLUSION: This strategy based on compositional differences between plant-based and alternative feeds can be useful for detecting compounds unsuspected until now that could impact fish metabolism.

Hepatic fatty acid biosynthesis is more responsive to protein than carbohydrate in rainbow trout during acute stimulations.

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.

New insights into the nutritional regulation of gluconeogenesis in carnivorous rainbow trout (Oncorhynchus mykiss): a gene duplication trail.

The rainbow trout (Oncorhynchus mykiss) is considered to be a strictly carnivorous fish species that is metabolically adapted for high catabolism of proteins and low utilization of dietary carbohydrates. This species consequently has a "glucose-intolerant" phenotype manifested by persistent hyperglycemia when fed a high-carbohydrate diet. Gluconeogenesis in adult fish is also poorly, if ever, regulated by carbohydrates, suggesting that this metabolic pathway is involved in this specific phenotype. In this study, we hypothesized that the fate of duplicated genes after the salmonid-specific 4th whole genome duplication (Ss4R) may have led to adaptive innovation and that their study might provide new elements to enhance our understanding of gluconeogenesis and poor dietary carbohydrate use in this species. Our evolutionary analysis of gluconeogenic genes revealed that pck1, pck2, fbp1a, and g6pca were retained as singletons after Ss4r, while g6pcb1, g6pcb2, and fbp1b ohnolog pairs were maintained. For all genes, duplication may have led to sub- or neofunctionalization. Expression profiles suggest that the gluconeogenesis pathway remained active in trout fed a no-carbohydrate diet. When trout were fed a high-carbohydrate diet (30%), most of the gluconeogenic genes were non- or downregulated, except for g6pbc2 ohnologs, whose RNA levels were surprisingly increased. This study demonstrates that Ss4R in trout involved adaptive innovation via gene duplication and via the outcome of the resulting ohnologs. Indeed, maintenance of ohnologous g6pcb2 pair may contribute in a significant way to the glucose-intolerant phenotype of trout and may partially explain its poor use of dietary carbohydrates.

Amino Acids Attenuate Insulin Action on Gluconeogenesis and Promote Fatty Acid Biosynthesis via mTORC1 Signaling Pathway in trout Hepatocytes.

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 Ser(302) 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 Ser(302) 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 Ser(302) 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.

Metabolic consequences of microRNA-122 inhibition in rainbow trout, Oncorhynchus mykiss.

BACKGROUND: MicroRNAs (miRNAs) are small regulatory molecules which post-transcriptionally regulate mRNA stability and translation. Several microRNAs have received attention due to their role as key metabolic regulators. In spite of the high evolutionary conservation of several miRNAs, the role of miRNAs in lower taxa of vertebrates has not been studied with regard to metabolism. The liver-specific and highly abundant miRNA-122 is one of the most widely studied miRNA in mammals, where it has been implicated in the control of hepatic lipid metabolism. Following our identification of acute postprandial, nutritional and endocrine regulation of hepatic miRNA-122 isomiRNA expression in rainbow trout, we used complementary in silico and in vivo approaches to study the role of miRNA-122 in rainbow trout metabolism. We hypothesized that the role of miRNA-122 in regulating lipid metabolism in rainbow trout is conserved to that in mammals and that modulation of miRNA-122 function would result in altered lipid homeostasis and secondarily altered glucose homeostasis, since lipogenesis has been suggested to act as glucose sink in trout. RESULTS: Our results show that miRNA-122 was functionally inhibited in vivo in the liver. Postprandial glucose concentrations increased significantly in rainbow trout injected with a miRNA-122 inhibitor, and this effect correlated with decreases in hepatic FAS protein abundance, indicative of altered lipogenic potential. Additionally, miRNA-122 inhibition resulted in a 20% decrease in plasma cholesterol concentration, an effect associated with increased expression of genes involved in cholesterol degradation and excretion. CONCLUSIONS: Overall evidence suggests that miRNA-122 may have evolved in early vertebrates to support liver-specific metabolic functions. Nevertheless, our data also indicate that metabolic consequences of miRNA-122 inhibition may differ quantitatively between vertebrate species and that distinct direct molecular targets of miRNA-122 may mediate metabolic effects between vertebrate species, indicating that miRNA-122 - mRNA target relationships may have undergone species-specific evolutionary changes.

Acute rapamycin treatment improved glucose tolerance through inhibition of hepatic gluconeogenesis in rainbow trout (Oncorhynchus mykiss).

Our aim was to investigate the potential role of TOR (target of rapamycin) signaling pathway in the regulation of hepatic glucose metabolism in rainbow trout. Fasted fish were first treated with a single intraperitoneal injection of rapamycin or vehicle and then submitted to a second intraperitoneal administration of glucose 4 h later. Our results revealed that intraperitoneal administration of glucose induced hyperglycemia for both vehicle and rapamycin treatments, which peaked at 2 h. Plasma glucose level in vehicle-treated fish was significantly higher than in rapamycin-treated fish at 8 and 17 h, whereas it remained at the basal level in rapamycin-treated fish. Glucose administration significantly enhanced the phosphorylation of Akt and ribosomal protein S6 kinase (S6K1) in vehicle-treated fish, while rapamycin completely abolished the activation of S6K1 in rapamycin-treated fish, without inhibiting the phosphorylation of Akt on Thr-308 or Ser-473. Despite the lack of significant variation in phosphoenolpyruvate carboxykinase mRNA abundance, mRNA abundance for glucokinase (GK), glucose 6-phosphatase (G6Pase) I and II, and fructose 1,6-bisphosphatase (FBPase) was reduced by rapamycin 17 h after glucose administration. The inhibition effect of rapamycin on GK and FBPase was further substantiated at the activity level. The suppression of GK gene expression and activity by rapamycin provided the first in vivo evidence in fish that glucose regulates hepatic GK gene expression and activity through a TORC1-dependent manner. Unlike in mammals, we observed that acute rapamycin treatment improved glucose tolerance through the inhibition of hepatic gluconeogenesis in rainbow trout.

Dietary methionine availability affects the main factors involved in muscle protein turnover in rainbow trout (Oncorhynchus mykiss).

Methionine is a limiting essential amino acid in most plant-based ingredients of fish feed. In the present study, we aimed to determine the effect of dietary methionine concentrations on several main factors involved in the regulation of mRNA translation and the two major proteolytic pathways (ubiquitin-proteasome and autophagy-lysosomal) in the white muscle of rainbow trout (Oncorhynchus mykiss). The fish were fed for 6 weeks one of the three isonitrogenous diets providing three different methionine concentrations (deficient (DEF), adequate (ADQ) and excess (EXC)). At the end of the experiment, the fish fed the DEF diet had a significantly lower body weight and feed efficiency compared with those fed the EXC and ADQ diets. This reduction in the growth of fish fed the DEF diet was accompanied by a decrease in the activation of the translation initiation factors ribosomal protein S6 and eIF2α. The levels of the main autophagy-related markers (LC3-II and beclin 1) as well as the expression of several autophagy genes (atg4b, atg12 l, Uvrag, SQSTM1, Mul1 and Bnip3) were higher in the white muscle of fish fed the DEF diet. Similarly, the mRNA levels of several proteasome-related genes (Fbx32, MuRF2, MuRF3, ZNF216 and Trim32) were significantly up-regulated by methionine limitation. Together, these results extend our understanding of mechanisms regulating the reduction of muscle growth induced by dietary methionine deficiency, providing valuable information on the biomarkers of the effects of low-fishmeal diets.

Postprandial molecular responses in the liver of the barramundi, Lates calcarifer.

The regulation of gene expression by nutrients is an important mechanism governing energy storage and growth in most animals, including fish. At present, very few genes that regulate intermediary metabolism have been identified in barramundi, nor is there any understanding of their nutritional regulation. In this study, a partial barramundi liver transcriptome was assembled from next-generation sequencing data and published barramundi EST sequences. A large number of putative metabolism genes were identified in barramundi, and the changes in the expression of 24 key metabolic regulators of nutritional pathways were investigated in barramundi liver over a time series immediately after a meal of a nutritionally optimised diet for this species. Plasma glucose and free amino acid levels showed a mild postprandial elevation which peaked 2 h after feeding, and had returned to basal levels within 4 or 8 h, respectively. Significant activation or repression of metabolic nuclear receptor regulator genes were observed, in combination with activation of glycolytic and lipogenic pathways, repression of the final step of gluconeogenesis and activation of the Akt-mTOR pathway. Strong correlations were identified between a number of different metabolic genes, and the coordinated co-regulation of these genes may underlie the ability of this fish to utilise dietary nutrients. Overall, these data clearly demonstrate a number of unique postprandial responses in barramundi compared with other fish species and provide a critical step in defining the response to different dietary nutrient sources.

Exploring the effects of dietary inulin in rainbow trout fed a high-starch, 100% plant-based diet.

BACKGROUND: High dietary carbohydrates can spare protein in rainbow trout (Oncorhynchus mykiss) but may affect growth and health. Inulin, a prebiotic, could have nutritional and metabolic effects, along with anti-inflammatory properties in teleosts, improving growth and welfare. We tested this hypothesis in rainbow trout by feeding them a 100% plant-based diet, which is a viable alternative to fishmeal and fish oil in aquaculture feeds. In a two-factor design, we examined the impact of inulin (2%) as well as the variation in the carbohydrates (CHO)/plant protein ratio on rainbow trout. We assessed the influence of these factors on zootechnical parameters, plasma metabolites, gut microbiota, production of short-chain fatty acids and lactic acid, as well as the expression of free-fatty acid receptor genes in the mid-intestine, intermediary liver metabolism, and immune markers in a 12-week feeding trial. RESULTS: The use of 2% inulin did not significantly change the fish intestinal microbiota, but interestingly, the high CHO/protein ratio group showed a change in intestinal microbiota and in particular the beta diversity, with 21 bacterial genera affected, including Ralstonia, Bacillus, and 11 lactic-acid producing bacteria. There were higher levels of butyric, and valeric acid in groups fed with high CHO/protein diet but not with inulin. The high CHO/protein group showed a decrease in the expression of pro-inflammatory cytokines (il1b, il8, and tnfa) in liver and a lower expression of the genes coding for tight-junction proteins in mid-intestine (tjp1a and tjp3). However, the 2% inulin did not modify the expression of plasma immune markers. Finally, inulin induced a negative effect on rainbow trout growth performance irrespective of the dietary carbohydrates. CONCLUSIONS: With a 100% plant-based diet, inclusion of high levels of carbohydrates could be a promising way for fish nutrition in aquaculture through a protein sparing effect whereas the supplementation of 2% inulin does not appear to improve the use of CHO when combined with a 100% plant-based diet.

Adverse effects of excessive dietary arachidonic acid on survival, PUFA-derived enzymatic and non-enzymatic oxylipins, stress response in rainbow trout fry.

Arachidonic acid (C20: 4n-6, AA) plays a fundamental role in fish physiology, influencing growth, survival and stress resistance. However, imbalances in dietary AA can have detrimental effects on fish health and performance. Optimal AA requirements for rainbow trout have not been established. This study aimed to elucidate the effects of varying dietary AA levels on survival, growth, long-chain polyunsaturated fatty acid (LC-PUFA) biosynthetic capacity, oxylipin profiles, lipid peroxidation, and stress resistance of rainbow trout fry. Over a period of eight weeks, 4000 female rainbow trout fry at the resorptive stage (0.12 g) from their first feeding were fed diets with varying levels of AA (0.6%, 1.1% or 2.5% of total fatty acids) while survival and growth metrics were closely monitored. The dietary trial was followed by an acute confinement stress test. Notably, while the fatty acid profiles of the fish reflected dietary intake, those fed an AA-0.6% diet showed increased expression of elongase5, highlighting their inherent ability to produce LC-PUFAs from C18 PUFAs and suggesting potential AA or docosapentaenoic acidn-6 (DPAn-6) biosynthesis. However, even with this biosynthetic capacity, the trout fed reduced dietary AA had higher mortality rates. The diet had no effect on final weight (3.38 g on average for the three diets). Conversely, increased dietary AA enhanced eicosanoid production from AA, suggesting potential inflammatory and oxidative consequences. This was further evidenced by an increase in non-enzymatic lipid oxidation metabolites, particularly in the AA-2.5% diet group, which had higher levels of phytoprostanes and isoprostanes, markers of cellular oxidative damage. Importantly, the AA-1.1% diet proved to be particularly beneficial for stress resilience. This was evidenced by higher post-stress turnover rates of serotonin and dopamine, neurotransmitters central to the fish's stress response. In conclusion, a dietary AA intake of 1.1% of total fatty acids appears to promote overall resilience in rainbow trout fry.

Ontogenetic expression of metabolic genes and microRNAs in rainbow trout alevins during the transition from the endogenous to the exogenous feeding period.

As oviparous fish, rainbow trout change their nutritional strategy during ontogenesis. This change is divided into the exclusive utilization of yolk-sac reserves (endogenous feeding), the concurrent utilization of yolk reserves and exogenous feeds (mixed feeding) and the complete dependence on external feeds (exogenous feeding). The change in food source is accompanied by well-characterized morphological changes, including the development of adipose tissue as an energy storage site, and continuous muscle development to improve foraging. The aim of this study was to investigate underlying molecular mechanisms that contribute to these ontogenetic changes between the nutritional phenotypes in rainbow trout alevins. We therefore analyzed the expression of marker genes of metabolic pathways and microRNAs (miRNAs) important in the differentiation and/or maintenance of metabolic tissues. In exogenously feeding alevins, the last enzyme involved in glucose production (g6pca and g6pcb) and lipolytic gene expression (cpt1a and cpt1b) decreased, while that of gk, involved in hepatic glucose use, was induced. This pattern is consistent with a progressive switch from the utilization of stored (gluconeogenic) amino acids and lipids in endogenously feeding alevins to a utilization of exogenous feeds via the glycolytic pathway. A shift towards the utilization of external feeds is further evidenced by the increased expression of omy-miRNA-143, a homologue of the mammalian marker of adipogenesis. The expression of its predicted target gene abhd5, a factor in triglyceride hydrolysis, decreased concurrently, suggesting a potential mechanism in the onset of lipid deposition. Muscle-specific omy-miRNA-1/133 and myod1 expression decreased in exogenously feeding alevins, a molecular signature consistent with muscle hypertrophy, which may be linked to nutritional cues or increased foraging.

Post-prandial regulation of hepatic glucokinase and lipogenesis requires the activation of TORC1 signalling in rainbow trout (Oncorhynchus mykiss).

To assess the potential involvement of TORC1 (target of rapamycin complex 1) signalling in the regulation of post-prandial hepatic lipid and glucose metabolism-related gene expression in trout, we employed intraperitoneal administration of rapamycin to achieve an acute inhibition of the TOR pathway. Our results reveal that rapamycin inhibits the phosphorylation of TORC1 and its downstream effectors (S6K1, S6 and 4E-BP1), without affecting Akt and the Akt substrates Forkhead-box Class O1 (FoxO1) and glycogen synthase kinase 3α/ß (GSK 3α/ß). These results indicate that acute administration of rapamycin in trout leads to the inhibition of TORC1 activation. No effect is observed on the expression of genes involved in gluconeogenesis, glycolysis and fatty acid oxidation, but hepatic TORC1 inhibition results in decreased sterol regulatory element binding protein 1c (SREBP1c) gene expression and suppressed fatty acid synthase (FAS) and glucokinase (GK) at gene expression and activity levels, indicating that FAS and GK activity is controlled at a transcriptional level in a TORC1-dependent manner. This study demonstrates for the first time in fish that post-prandial regulation of hepatic lipogenesis and glucokinase in rainbow trout requires the activation of TORC1 signalling.