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Polakof S, Skiba-Cassy S, Panserat S. Glucose homeostasis is impaired by a paradoxical interaction between metformin and insulin in carnivorous rainbow trout. Am J Physiol Regul Integr Comp Physiol 297: R1769-R1776, 2009. First published September 30, 2009; doi: 10.1152/ajpregu.00369.2009.--Utilizing rainbow trout (Oncorhynchus mykiss) as a known model of a 'glucose-intolerant' and poor dietary glucose user, we assessed glucose utilization in fish chronically receiving two molecules able to improve glucose homeostasis: insulin and metformin. Our objectives were to assess the ability of rainbow trout to deal with a glucose load and to improve glucose utilization in fish receiving a chronic administration of insulin plus metformin treatments. Fish received (implanted miniosmotic pumps) saline, insulin, metformin, and insulin plus metformin solution for 4 days and then were subjected to a glucose challenge (intraperitoneal injection) to study glucose homeostasis, analyzing plasma glycemia, mRNA levels of glucose metabolism-related proteins, insulin signaling, and glycogen levels in liver and muscle. Control fish received a saline pump implantation and saline intraperitoneal injection. We found no evidence that the 'glucose intolerance' in this species could be linked to any of the molecular markers of metabolism in the tissues analyzed. By contrast, very interestingly, we show for the first time, that metformin is not only unable to improve glucose homeostasis in trout, but, in fact, its counter-acts the effects of insulin, creating an 'insulin resistance,' especially in the muscle. These results make trout an attractive original model to study both insulin and metformin effect on biological systems. fish; insulin; glucose metabolism; liver; muscle; metformin doi: 10.1152/ajpregu.00369.2009

Camivorous fish are poor users of dietary carbohydrates and are considered to be glucose intolerant. In this context, we have tested, for the first time in rainbow trout, metformin, a common anti-diabetic drug, known to modify muscle and liver metabolism and to control hyperglycemia in mammals. In the present study, juvenile trout were fed with very high levels of carbohydrates (30% of the diet) for this species during 10 days followed by feeding with pellets supplemented with metformin (0.25% of the diet) for three additional days. Dietary mefformin led to a significant reduction in postprandial glycemia in trout, demonstrating unambiguously the hypoglycemic effect of this drug. No effect of metformin was detected on mRNA levels for glucose transporter type 4 (GLUT4), or enzymes involved in glycolysis, mitochondrial energy metabolism, or on glycogen level in the white muscle. Expected inhibition of hepatic gluconeogenic (glucose-6-phosphatase, fructose1,6-bisphosphatase, and phosphoenolpyruvate carboxykinase) mRNA levels was not found, showing instead paradoxically higher mRNA levels for these genes after drug treatment. Finally, metformin treatment was associated with higher mRNA levels and activities for lipogenic enzymes (fatty acid synthase and glucose-6-phosphate dehydrogenase). Overall, this study strongly supports that the induction of hepatic lipogenesis by dietary glucose may permit a more efficient control of postprandial glycemia in carnivorous fish fed with high carbohydrate diets. glucose; anti-diabetic drug; muscle; liver; carnivorous fish

Based on the concept of nutritional programming in mammals, we tested whether an acute hyperglucidic-hypoproteic stimulus during first feeding could induce long-term changes in nutrient metabolism in rainbow trout. Trout alevins received during the five first days of exogenous feeding either a hyperglucidic (40% gelatinized starch + 20% glucose) and hypoproteic (20%) diet (VLP diet) or a high-protein (60%) glucose-free diet (HP diet, control). Following a common 105- day period on a commercial diet, both groups were then challenged (65 days) with a carbohydrate-rich diet (28%). Short- and long-term effects of the early stimuli were evaluated in terms of metabolic marker gene expressions and intestinal microbiota as initial gut colonisation is essential for regulating the development of the digestive system. In whole alevins (short term), diet VLP relative to HP rapidly increased gene expressions of glycolytic enzymes, while those involved in gluconeogenesis and amino acid catabolism decreased. However, none of these genes showed persistent molecular adaptation in the liver of challenged juveniles (long term). By contrast, muscle of challenged juveniles subjected previously to the VLP stimulus displayed downregulated expression of markers of glycolysis and glucose transport (not seen in the short term). These fish also had higher plasma glucose (9 h postprandial), suggesting impaired glucose homeostasis induced by the early stimulus. The early stimulus did not modify the expression of the analysed metabolism-related microRNAs, but had short- and long-term effects on intestinal fungi (not bacteria) profiles. In summary, our data show that a short hyperglucidic-hypoproteic stimulus during early life may have a long-term influence on muscle glucose metabolism and intestinal microbiota in trout. [ABSTRACT FROM AUTHOR]

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., (© 2024. The Author(s).)

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., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

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 acid n-6 (DPA n-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., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

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 C β , 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.

The formulation of sustainable fish feeds based on plant ingredients supplemented by alternative ingredients to plant (insect, micro-algae, yeast) and genetic selection of fish for plant-based diets were tested on rainbow trout in two separate experiments. Plant-based diets and corresponding diets supplemented with an ingredient mix: insect, micro-algae and yeast in Experiment A, and insect and yeast in Experiment B were compared to commercial-like diets. In experiment A, the mix-supplemented diet was successful in compensating the altered growth performance of fish fed their respective plant-based diet compared to those fed the commercial diet, by restoring feed conversion. In experiment B, the selected line demonstrated improved growth performances of fish fed mix-supplemented and plant-based diets compared to the non-selected line. Metabolomics demonstrated a plasma compositional stability in fish fed mix-supplemented and basal plant-based diets comprising an amino acid accumulation and a glucose depletion, compared to those fed commercial diets. The selected line fed mix-supplemented and commercial diets showed changes in inositol, ethanol and methanol compared to the non-selected line, suggesting an involvement of microbiota. Changes in plasma glycine-betaine content in fish fed the mix-supplemented diet suggest the ability of the selected line to adapt to alternative ingredients., (© 2023. The Author(s).)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Physical enrichment is known to improve living conditions of fish held in farming systems and has been shown to promote behavioral plasticity in captive fish. However, the brain's regulatory-mechanism systems underlying its behavioral effects remain poorly studied. The present study investigated the impact of a three-month exposure to an enriched environment (EE vs. barren environment, BE) on the modulation of brain function in rainbow trout ( Oncorhynchus mykiss ) juveniles. Using high-throughput RT-qPCR, we assessed mRNA genes related to brain function in several areas of the trout brain. These included markers of cerebral activity and plasticity, neurogenesis, synaptogenesis, or selected neurotransmitters pathways (dopamine, glutamate, GABA, and serotonin). Overall, the fish from EE displayed a series of differentially expressed genes (neurotrophic, neurogenesis, and synaptogenesis markers) essentially localized in the telencephalon, which could underpin the beneficial effects of complexifying the environment on fish brain plasticity. In addition, EE significantly affected blood plasma c-miRNA signatures, as revealed by the upregulation of four c-miRNAs (miR-200b/c-3p, miR-203a-3p, miR-205-1a-5p, miR-218a-5p) in fish blood plasma after 185 days of EE exposure. Overall, we concluded that complexifying the environment through the addition of physical structures that stimulate and encourage fish to explore promotes the trout's brain function in farming conditions.

Background: The broodstock diet, and in particular the lipid and fatty acid composition of the diet, is known to play a key role in reproductive efficiency and survival of the progeny in fish. A major problem when replacing both fish meal and fish oil by plant sources is the lack of n-3 long chain polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). To address this problem, we studied the effect of the plant-based diet supplemented with Schizochytrium sp. microalgae, source of DHA, compared to a conventional commercial diet rich in fish meal and fish oil on reproductive performance and egg quality and the consequences on progeny, in female rainbow trout broodstock., Results: The results demonstrated that DHA-rich microalgae supplementation in a plant-based diet allowed for the maintenance of reproductive performance and egg quality comparable to a conventional commercial feed rich in fish meal and fish oil and led to an increased significant fry survival after resorption. Moreover, when females were fed a plant-based diet supplemented with micro-algae, the 4-month-old progenies showed a significant higher growth when they were challenged with a similar diet as broodstock during 1 month. We provide evidence for metabolic programming in which the maternal dietary induced significant protracted effects on lipid metabolism of progeny., Conclusions: The present study demonstrates that supplementation of a plant-based diet with DHA-rich microalgae can be an effective alternative to fish meal and fish oil in rainbow trout broodstock aquafeed., (© 2022. The Author(s).)

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., (© 2021. The Author(s).)

Within the context of a growing aquaculture production coupled with a plateau of the production in the main components of aquafeeds (fish oil and fishmeal), recent studies have typically focused on replacing these feedstuffs with terrestrial plant ingredients for cultured carnivorous aquatic species, such as rainbow trout ( Oncorhynchus mykiss ). Substitution rates without adverse effects have, however, reached their limit. One potential way forward would be to take advantage of the genetic variability that exists in the salmonid population. However, to date, little is known about the underlying molecular mechanisms responsible for this genetic variability. The aim of the present research was to understand why some genotypes are better able to utilize plant-based diets devoid of marine resources. In this regard, three isogenic lines of rainbow trout (R23h, AB1h, and A22h), with similar growth when fed marine resources-based diets and which differ greatly in their responses to a plant-based diet, were fed with either a complete plant-based diet (V diet) or a marine resources-based diet (M diet) since first-feeding. Fish traits and the hepatic transcriptome of these three genotypes were compared after 5 months of feeding. First, differences in the ability to grow with the V diet observed amongst genotypes was not due to higher feed intake, but instead due to differences in feed efficiency. The comparison of transcriptome profiles revealed 575 (R23h vs. AB1h), 1,770 (R23h vs. A22h), and 2,973 (AB1h vs. A22h) probes differentially expressed amongst the three genotypes when fed the V diet. Interestingly, R23h and AB1h fish, which were the least affected by the V diet, exhibited the highest growth. These results demonstrate that these fish were able to maintain a high level of energy production and protein synthesis. Moreover, these genotypes were also able to activate pathways linked to lipid and cholesterol metabolisms, such as the biosynthesis of long-chain polyunsaturated fatty acids. Finally, as previously, immunity seems to also play an important role in the ability of fish to use the V diet, and further studies are needed to understand the mechanisms by which immunity interacts with growth., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Callet, Dupont-Nivet, Danion, Burel, Cluzeaud, Surget, Aguirre, Kerneis, Labbé, Panserat, Quillet, Geurden, Skiba-Cassy and Médale.)

Background: Farmed fish food with reduced fish-derived products are gaining growing interest due to the ecological impact of fish-derived protein utilization and the necessity to increase aquaculture sustainability. Although different terrestrial plant proteins could replace fishmeal proteins, their use is associated with adverse effects. Here, we investigated how diets composed of terrestrial vegetal sources supplemented with proteins originating from insect, yeast or terrestrial animal by-products affect rainbow trout (Onchorynchus mykiss) gut microbiota composition, growth performance and resistance to bacterial infection by the fish pathogen Flavobacterium psychrophilum responsible for frequent outbreaks in aquaculture settings., Results: We showed that the tested regimes significantly increased gut bacterial richness compared to full vegetal or commercial-like diets, and that vegetal diet supplemented with insect and yeast proteins improves growth performance compared to full vegetal diet without altering rainbow trout susceptibility to F. psychrophilum infection., Conclusion: Our results demonstrate that the use of insect and yeast protein complements to vegetal fish feeds maintain microbiota functions, growth performance and fish health, therefore identifying promising alternative diets to improve aquaculture's sustainability.

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., (Copyright © The Author(s) on behalf of the American Society for Nutrition 2020.)

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., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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 transcriptional 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., Competing Interests: The authors have declared that no competing interests exist.

Nutrition of high trophic species in aquaculture is faced with the development of sustainable plant-based diets. Insects seem particularly promising for supplementing plant-based diets. However, the complex effect of whole insect meal on fish metabolism is not well understood, and even less is known about insect meal extracts. The purpose of this work was to decipher the metabolic utilization of a plant-based diet supplemented with the gradual addition of an insect protein extract (insect hydrolysate at 0%, 5%, 10% and 15%). 1 H-NMR profiling was used to assess metabolites in experimental diets and in fish plasma, liver and muscle. A significant dose-dependent increase in growth and feed efficiency with increasing insect extract amounts was observed. The incremental incorporation of the insect extract in diet had a significant and progressive impact on the profile of dietary soluble compounds and trout metabolome. The metabolites modulated by dietary insect extracts in plasma and tissues were involved in protein and energy metabolism. This was associated with the efficient metabolic use of dietary free amino acids toward protein synthesis through the concomitant supply of balanced free amino acids and energy substrates in muscle. The findings provide new insights into how the dietary food metabolome affects fish metabolism.

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.

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 C m CGG 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 C m CGG 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 C m CGG DNA methylation level in muscle of trout juveniles, showing that the first feeding stage is efficient for programming the glucose metabolism in fish., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s). 2019.)

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. 1 H 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 1 H 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.

Rainbow trout ( Oncorhynchus mykiss ) is recognized as a typical "glucose-intolerant" fish, and the limits of dietary carbohydrate utilization have been investigated for many years. In this study, the objective was to test the molecular effects of dietary carbohydrates on intermediary metabolism in two major metabolic tissues, liver and muscle. Another objective was also to study if the response to carbohydrate intake depended on the genetic background. We fed two isogenic lines of rainbow trout (named A22h and N38h) with high carbohydrate diet (carbohydrate, 22.9%) or low carbohydrate diet (carbohydrate, 3.6%) for 12 weeks. Carbohydrates were associated with higher feed utilization owned by the well-known protein-sparing effect, with better fish growth performance. However, atypical regulation of glycolysis and gluconeogenesis in liver and absence of hk and glut4 induction in muscle, were also observed. Regarding the effects of carbohydrates on other metabolism, we observed an increased, at a molecular level, of hepatic cholesterol biosynthesis, fatty acid oxidation and mitochondrial energy metabolism. Genetic variability (revealed by the differences between the two isogenic lines) was observed for some metabolic genes especially for those involved in the EPA and DHA biosynthetic capacity. Finally, our study demonstrates that dietary carbohydrate not only affect glucose metabolism but also strongly impact the lipid and energy metabolism in liver and muscle of trout.

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., Competing Interests: The authors have declared that no competing interests exist.

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 , Firmicutes and Actinobacteria are 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.

To meet the growing demand of fish feed for aquaculture, an increasing proportion of marine ingredients are being replaced by blends of plant products. However, the total replacement of marine ingredients in salmonid diets impairs fish performance. This is particularly true during the early fry stage and this stage is therefore considered of particular importance. In rainbow trout (RBT), the existence of a genetic variability to survive and grow with plant-based diets devoid of marine ingredients has now been proved, but the mechanisms behind are little studied especially at early stage. To investigate these, we analysed the whole transcriptome of three isogenic lines of RBT fry, which have similar growth when fed a marine resources-based diet (M diet) but which highly differ in their responses to a plant-based diet (V diet). Analysis of transcriptomes profiles revealed 1740, 1834 and 246 probes differentially expressed among the three genotypes when fed the V diet. The use of these lines led to the discovery of potential molecular markers linked to plant-based diet utilisation, some of them belonging to new pathways, never described before. An important number of genes was related to immunity, but further investigations are needed to better understand the difference between the genotypes in their immune status response to V diet exposure. Finally, differences in expression of genes related to feed intake and sensory perception among genotypes suggested that the mechanisms underlying the differences in growth on plant-based diet are closely linked to diet acceptance. Research on plants components affecting feed intake should be thus further explored., Competing Interests: The authors have declared that no competing interests exist.

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.

In the context of limited marine resources, the exponential growth of aquaculture requires the substitution of fish oil and fishmeal, the traditional components of fish feeds by terrestrial plant ingredients. High levels of such substitution are known to negatively impact fish performance such as growth and survival in rainbow trout (Oncorhynchus mykiss) as in other salmonids. In this respect, genetic selection is a key enabler for improving those performances and hence for the further sustainable development of aquaculture. We selected a rainbow trout line over three generations for its ability to survive and grow on a 100% plant-based diet devoid of both fish oil and fishmeal (V diet) from the very first meal. In the present study, we compared the control line and the selected line after 3 generations of selection, both fed either the V diet or a marine resources-based diet (M diet). The objective of the study was to assess the efficiency of selection and the consequences on various correlated nutritional traits: feed intake, feed efficiency, digestibility, composition of whole fish, nutrient retention and fatty acid (FA) profile. We demonstrated that the genetic variability present in our rainbow trout population can be selected to improve survival and growth. The major result of the study is that after only three generations of selection, selected fish fed the V diet grew at the same rate as the control line fed the M diet, whilst the relative reduction of body weight was 36.8% before the selection. This enhanced performance on the V diet seems to be mostly linked to a higher feed intake for the selected fish.

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., (Copyright © 2016 the American Physiological Society.)

Monitoring autophagic flux in vivo or in organs remains limited and the ideal methods relative to the techniques possible with cell culture may not exist. Recently, a few papers have demonstrated the feasibility of measuring autophagic flux in vivo by intraperitoneal (IP) injection of pharmacological agents (chloroquine, leupeptin, vinblastine, and colchicine). However, the metabolic consequences of the administration of these drugs remain largely unknown. Here, we report that 0.8 mg/kg/day IP colchicine increased LC3-II protein levels in the liver of fasted trout, supporting the usefulness of this drug for studying autophagic flux in vivo in our model organism. This effect was accompanied by a decrease of plasma glucose concentration associated with a fall in the mRNA levels of gluconeogenesis-related genes. Concurrently, triglycerides and lipid droplets content in the liver increased. In contrast, transcript levels of β-oxidation-related gene Cpt1a dropped significantly. Together, these results match with the reported role of autophagy in the regulation of glucose homeostasis and intracellular lipid stores, and highlight the importance of considering these effects when using colchicine as an in vivo "autophagometer."

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., (Copyright © 2015 the American Physiological Society.)

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., (© 2015 S. Karger AG, Basel.)

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., (Copyright © 2014 the American Physiological Society.)