Precision formulation, a new concept to improve dietary amino acid absorption based on the study of cationic amino acid transporters.

Amino acid (AA) transporters (AAT) control AA cellular fluxes across membranes, contributing to maintain cellular homeostasis. In this study, we took advantage of rainbow trout metabolic feature, which highly relies on dietary AA, to explore the cellular and physiological consequences of unbalanced diets on AAT dysregulations with a particular focus on cationic AAs (CAA), frequently underrepresented in plant-based diets. Results evidenced that 24 different CAAT are expressed in various trout tissues, part of which being subjected to AA- and CAA-dependent regulations, with y+LAT2 exchanger being prone to the strongest dysregulations. Moreover, CAA were shown to control two major AA-dependent activation pathways (namely mTOR and GCN2) but at different strength according to the CAA considered. A new feed formulation strategy has been put forward to improve specifically the CAA supplemented absorption in fish together with their growth performance. Such "precision formulation" strategy reveals high potential for nutrition practices, especially in aquaculture.

Chaperone-mediated autophagy protects against hyperglycemic stress.

Chaperone-mediated autophagy (CMA) is a major pathway of lysosomal proteolysis critical for cellular homeostasis and metabolism, and whose defects have been associated with several human pathologies. While CMA has been well described in mammals, functional evidence has only recently been documented in fish, opening up new perspectives to tackle this function under a novel angle. Now we propose to explore CMA functions in the rainbow trout (RT, Oncorhynchus mykiss), a fish species recognized as a model organism of glucose intolerance and characterized by the presence of two paralogs of the CMA-limiting factor Lamp2A (lysosomal associated membrane protein 2A). To this end, we validated a fluorescent reporter (KFERQ-PA-mCherry1) previously used to track functional CMA in mammalian cells, in an RT hepatoma-derived cell line (RTH-149). We found that incubation of cells with high-glucose levels (HG, 25 mM) induced translocation of the CMA reporter to lysosomes and/or late endosomes in a KFERQ- and Lamp2A-dependent manner, as well as reduced its half-life compared to the control (5 mM), thus demonstrating increased CMA flux. Furthermore, we observed that activation of CMA upon HG exposure was mediated by generation of mitochondrial reactive oxygen species, and involving the antioxidant transcription factor Nfe2l2/Nrf2 (nfe2 like bZIP transcription factor 2). Finally, we demonstrated that CMA plays an important protective role against HG-induced stress, primarily mediated by one of the two RT Lamp2As. Together, our results provide unequivocal evidence for CMA activity existence in RT and highlight both the role and regulation of CMA during glucose-related metabolic disorders.Abbreviations: AREs: antioxidant response elements; CHC: α-cyano -4-hydroxycinnamic acid; Chr: chromosome; CMA: chaperone-mediated autophagy; CT: control; DMF: dimethyl fumarate; Emi: endosomal microautophagy; HG: high-glucose; HMOX1: heme oxygenase 1; H2O2: hydrogen peroxide; KFERQ: lysine-phenylalanine-glutamate-arginine-glutamine; LAMP1: lysosomal associated membrane protein 1; LAMP2A: lysosomal associated membrane protein 2A; MCC: Manders' correlation coefficient; Manders' correlation coefficient Mo: morpholino oligonucleotide; NAC: N-acetyl cysteine; NFE2L2/NRF2: NFE2 like bZIP transcription factor 2; PA-mCherry: photoactivable mCherry; PCC: Pearson's correlation coefficient; ROS: reactive oxygen species; RT: rainbow trout; siRNAs: small interfering RNAs; SOD: superoxide dismutase; Tsg101: tumor susceptibility 101; TTFA: 2-thenoyltrifluoroacetone; WGD: whole-genome duplication.

Diving into the Evolutionary History of HSC70-Linked Selective Autophagy Pathways: Endosomal Microautophagy and Chaperone-Mediated Autophagy.

Autophagy is a pleiotropic and evolutionarily conserved process in eukaryotes that encompasses different types of mechanisms by which cells deliver cytoplasmic constituents to the lysosome for degradation. Interestingly, in mammals, two different and specialized autophagic pathways, (i) the chaperone-mediated autophagy (CMA) and (ii) the endosomal microautophagy (eMI), both rely on the use of the same cytosolic chaperone HSPA8 (also known as HSC70) for targeting specific substrates to the lysosome. However, this is not true for all organisms, and differences exist between species with respect to the coexistence of these two autophagic routes. In this paper, we present an in-depth analysis of the evolutionary history of the main components of CMA and eMI and discuss how the observed discrepancies between species may contribute to improving our knowledge of these two functions and their interplays.

Are the Main Methionine Sources Equivalent? A Focus on DL-Methionine and DL-Methionine Hydroxy Analog Reveals Differences on Rainbow Trout Hepatic Cell Lines Functions.

The replacement of fishmeal by plant proteins in aquafeeds imposes the use of synthetic methionine (MET) sources to balance the amino acid composition of alternative diets and so to meet the metabolic needs of fish of agronomic interest such as rainbow trout (RT-Oncorhynchus mykiss). Nonetheless, debates still exist to determine if one MET source is more efficiently used than another by fish. To address this question, the use of fish cell lines appeared a convenient strategy, since it allowed to perfectly control cell growing conditions notably by fully depleting MET from the media and studying which MET source is capable to restore cell growth/proliferation and metabolism when supplemented back. Thus, results of cell proliferation assays, Western blots, RT-qPCR and liquid chromatography analyses from two RT liver-derived cell lines revealed a better absorption and metabolization of DL-MET than DL-Methionine Hydroxy Analog (MHA) with the activation of the mechanistic Target Of Rapamycin (mTOR) pathway for DL-MET and the activation of integrated stress response (ISR) pathway for MHA. Altogether, the results clearly allow to conclude that both synthetic MET sources are not biologically equivalent, suggesting similar in vivo effects in RT liver and, therefore, questioning the MHA efficiencies in other RT tissues.

Myostatin gene inactivation increases post-mortem calpain-dependent muscle proteolysis in mice.

Myostatin deficiency leads to extensive skeletal muscle hypertrophy, but its consequence on post-mortem muscle proteolysis is unknown. Here, we compared muscle myofibrillar protein degradation, and autophagy, ubiquitin-proteasome and Ca2+-dependent proteolysis relative to the energetic and redox status in wild-type (WT) and myostatin knock-out mice (KO) during early post-mortem storage. KO muscles showed higher degradation of myofibrillar proteins in the first 24 h after death, associated with preserved antioxidant status, compared with WT muscles. Analysis of key autophagy and ubiquitin-proteasome system markers indicated that these two pathways were not upregulated in post-mortem muscle (both genotypes), but basal autophagic flux and ATP content were lower in KO muscles. Proteasome and caspase activities were not different between WT and KO mice. Conversely, calpain activity was higher in KO muscles, concomitantly with higher troponin T and desmin degradation. Altogether, these results suggest that calpains but not the autophagy, proteasome and caspase systems, explain the difference in post-mortem muscle protein proteolysis between both genotypes.

The autophagy response during adipogenesis of primary cultured rainbow trout (Oncorhynchus mykiss) adipocytes.

Adipogenesis is a tightly regulated process, and the involvement of autophagy has been recently proposed in mammalian models. In rainbow trout, two well-defined phases describe the development of primary cultured adipocyte cells: proliferation and differentiation. Nevertheless, information on the transcriptional profile at the onset of differentiation and the potential role of autophagy in this process is scarce. In the present study, the cells showed an early and transient induction of several adipogenic transcription factors genes' expression (i.e., cebpa and cebpb) along with the morphological changes (round shape filled with small lipid droplets) typical of the onset of adipogenesis. Then, the expression of various lipid metabolism-related genes involving the synthesis (fas), uptake (fatp1 and cd36), accumulation (plin2) and mobilization (hsl) of lipids, characteristic of the mature adipocyte, increased. In parallel, several autophagy markers (i.e., atg4b, gabarapl1 and lc3b) mirrored the expression of those adipogenic-related genes, suggesting a role of autophagy during in vitro fish adipogenesis. In this regard, the incubation of preadipocytes with lysosomal inhibitors (Bafilomycin A1 or Chloroquine), described to prevent autophagy flux, delayed the process of adipogenesis (i.e., cell remodelling), thus suggesting a possible relationship between autophagy and adipocyte differentiation in trout. Moreover, the disruption of the autophagic flux altered the expression of some key adipogenic genes such as cebpa and pparg. Overall, this study contributes to improve our knowledge on the regulation of rainbow trout adipocyte differentiation, and highlights for the first time in fish the involvement of autophagy on adipogenesis, suggesting a close-fitting connection between both processes.

Autophagy in farm animals: current knowledge and future challenges.

Autophagy (a process of cellular self-eating) is a conserved cellular degradative process that plays important roles in maintaining homeostasis and preventing nutritional, metabolic, and infection-mediated stresses. Surprisingly, little attention has been paid to the role of this cellular function in species of agronomical interest, and the details of how autophagy functions in the development of phenotypes of agricultural interest remain largely unexplored. Here, we first provide a brief description of the main mechanisms involved in autophagy, then review our current knowledge regarding autophagy in species of agronomical interest, with particular attention to physiological functions supporting livestock animal production, and finally assess the potential of translating the acquired knowledge to improve animal development, growth and health in the context of growing social, economic and environmental challenges for agriculture.Abbreviations: AKT: AKT serine/threonine kinase; AMPK: AMP-activated protein kinase; ASC: adipose-derived stem cells; ATG: autophagy-related; BECN1: beclin 1; BNIP3: BCL2 interacting protein 3; BVDV: bovine viral diarrhea virus; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CMA: chaperone-mediated autophagy; CTSB: cathepsin B; CTSD: cathepsin D; DAP: Death-Associated Protein; ER: endoplasmic reticulum; GFP: green fluorescent protein; Gln: Glutamine; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; IF: immunofluorescence; IVP: in vitro produced; LAMP2A: lysosomal associated membrane protein 2A; LMS: lysosomal membrane stability; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MDBK: Madin-Darby bovine kidney; MSC: mesenchymal stem cells; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; NBR1: NBR1 autophagy cargo receptor; NDV: Newcastle disease virus; NECTIN4: nectin cell adhesion molecule 4; NOD1: nucleotide-binding oligomerization domain 1; OCD: osteochondritis dissecans; OEC: oviduct epithelial cells; OPTN: optineurin; PI3K: phosphoinositide-3-kinase; PPRV: peste des petits ruminants virus; RHDV: rabbit hemorrhagic disease virus; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy.

Lighting chaperone-mediated autophagy (CMA) evolution with an ancient LAMP: the existence of a functional CMA activity in fish.

Chaperone-mediated autophagy (CMA), as one of the main pathways of lysosomal catabolism, plays essential roles for the maintenance of cellular homeostasis. To date, the absence of any identifiable LAMP2A - the necessary and limiting protein required for CMA - in non-tetrapod lineages, led to the paradigm that this cellular process was restricted to mammals and birds. The recent findings of Lescat et al., demonstrating the existence of a CMA activity in fish, now reshuffle the cards regarding how the entire evolution of CMA function should be considered and appreciated across metazoans. Hence, beyond challenging the current tetrapod-centered accepted view, the work of Lescat et al. tackles the possibility - or the compelling need - of using complementary and powerful genetic models, such as zebrafish or medaka, for studying this fundamental function from an evolutionary perspective.

RTH-149 Cell Line, a Useful Tool to Decipher Molecular Mechanisms Related to Fish Nutrition.

Nowadays, aquaculture provides more than 50% of fish consumed worldwide but faces new issues that challenge its sustainability. One of them relies on the replacement of fish meal (FM) in aquaculture feeds by other protein sources without deeply affecting the whole organism's homeostasis. Multiple strategies have already been tested using in vivo approaches, but they hardly managed to cope with the multifactorial problems related to the complexities of fish biology together with new feed formulations. In this context, rainbow trout (RT) is particularly concerned by these problems, since, as a carnivorous fish, dietary proteins provide the amino acids required to supply most of its energetic metabolism. Surprisingly, we noticed that in vitro approaches considering RT cell lines as models to study RT amino acid metabolism were never previously used. Therefore, we decided to investigate if, and how, three major pathways described, in other species, to be regulated by amino acid and to control cellular homeostasis were functional in a RT cell line called RTH-149-namely, the mechanistic Target Of Rapamycin (mTOR), autophagy and the general control nonderepressible 2 (GCN2) pathways. Our results not only demonstrated that these three pathways were functional in RTH-149 cells, but they also highlighted some RT specificities with respect to the time response, amino acid dependencies and the activation levels of their downstream targets. Altogether, this article demonstrated, for the first time, that RT cell lines could represent an interesting alternative of in vivo experimentations for the study of fish nutrition-related questions.

New insights into methylmercury induced behavioral and energy-related gene transcriptional responses in European glass eel (Anguilla anguilla).

The effect of methylmercury (MeHg) was investigated in glass eel migration behavior and metabolism. To migrate up estuary, glass eels synchronize their swimming activity to the flood tide and remain on or in the substratum during ebb tide. Following seven days of exposure to MeHg (100 ng L-1), glass eels migration behavior was expressed by their swimming synchronization to the water current reversal every 6.2 h (mimicking the alternation of flood and ebb tides) and their swimming activity level. In relation to their behavior, we then analyzed the energy-related gene expression levels in individual head, viscera and muscle. Results showed that MeHg decreased the number of glass eels synchronized to the change in water current direction and their swimming activity level. This last effect was more pronounced in non-synchronized fish than in synchronized ones, supporting the idea that non-synchronized glass eels could be more vulnerable to stress. As regard the expression of energy-related genes, no significant difference was observed between control and MeHg-exposed fish. In contrast, when the swimming activity levels were plotted against transcriptional responses, positive correlations were evidenced in viscera and especially in the head of exposed glass eels but not in control. Finally, it is noteworthy that non-synchronized glass eels displayed lower expression level of metabolism genes than their synchronized counterpart, but only in the head. Altogether, these results support the interest of focusing on the head to investigate the facultative migration behavior in glass eels and the effect of environmental stressors on this rhythmic behavior.

Chaperone-Mediated Autophagy in the Light of Evolution: Insight from Fish.

Chaperone-mediated autophagy (CMA) is a major pathway of lysosomal proteolysis recognized as a key player of the control of numerous cellular functions, and whose defects have been associated with several human pathologies. To date, this cellular function is presumed to be restricted to mammals and birds, due to the absence of an identifiable lysosome-associated membrane protein 2A (LAMP2A), a limiting and essential protein for CMA, in nontetrapod species. However, the recent identification of expressed sequences displaying high homology with mammalian LAMP2A in several fish species challenges that view and suggests that CMA likely appeared earlier during evolution than initially thought. In the present study, we provide a comprehensive picture of the evolutionary history of the LAMP2 gene in vertebrates and demonstrate that LAMP2 indeed appeared at the root of the vertebrate lineage. Using a fibroblast cell line from medaka fish (Oryzias latipes), we further show that the splice variant lamp2a controls, upon long-term starvation, the lysosomal accumulation of a fluorescent reporter commonly used to track CMA in mammalian cells. Finally, to address the physiological role of Lamp2a in fish, we generated knockout medaka for that specific splice variant, and found that these deficient fish exhibit severe alterations in carbohydrate and fat metabolisms, in consistency with existing data in mice deficient for CMA in liver. Altogether, our data provide the first evidence for a CMA-like pathway in fish and bring new perspectives on the use of complementary genetic models, such as zebrafish or medaka, for studying CMA in an evolutionary perspective.

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.

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

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

The Autophagic Flux Inhibitor Bafilomycine A1 Affects the Expression of Intermediary Metabolism-Related Genes in Trout Hepatocytes.

Autophagy is an evolutionarily conserved process of cellular self-eating which emerged these last years as a major adaptive metabolic response to various stresses such as fasting, hypoxia, or environmental pollutants. However, surprisingly very few data is currently available on its role in fish species which are directly exposed to frequent environmental perturbations. Here, we report that the treatment of fasted trout hepatocytes with the autophagy inhibitor Bafilomycine A1 lowered the mRNA levels of many of the gluconeogenesis-related genes and increased those of genes involved in intracellular lipid stores. Concurrently, intracellular free amino acid levels dropped and the expression of the main genes involved in the endoplasmic reticulum (ER) stress exhibited a sharp increase in autophagy inhibited cells. Together these results highlight the strong complexity of the crosstalk between ER, autophagy and metabolism and support the importance of considering this function in future studies on metabolic adaptation of fish to environmental stresses.

DNA methylation of the promoter region of bnip3 and bnip3l genes induced by metabolic programming.

BACKGROUND: Environmental changes of biotic or abiotic nature during critical periods of early development may exert a profound influence on physiological functions later in life. This process, named developmental programming can also be driven through parental nutrition. At molecular level, epigenetic modifications are the most likely candidate for persistent modulation of genes expression in later life. RESULTS: In order to investigate epigenetic modifications induced by programming in rainbow trout, we focused on bnip3 and bnip3l paralogous genes known to be sensitive to environmental changes but also regulated by epigenetic modifications. Two specific stimuli were used: (i) early acute hypoxia applied at embryo stage and (ii) broodstock and fry methionine deficient diet, considering methionine as one of the main methyl-group donor needed for DNA methylation. We observed a programming effect of hypoxia with an increase of bnip3a and the four paralogs of bnip3l expression level in fry. In addition, parental methionine nutrition was correlated to bnip3a and bnip3lb1 expression showing evidence for early fry programming. We highlighted that both stimuli modified DNA methylation levels at some specific loci of bnip3a and bnip3lb1. CONCLUSION: Overall, these data demonstrate that methionine level and hypoxia stimulus can be of critical importance in metabolic programming. Both stimuli affected DNA methylation of specific loci, among them, an interesting CpG site have been identified, namely - 884 bp site of bnip3a, and may be positively related with mRNA levels.

Dietary methionine deficiency affects oxidative status, mitochondrial integrity and mitophagy in the liver of rainbow trout (Oncorhynchus mykiss).

The low levels of methionine in vegetable raw materials represent a limit to their use in aquafeed. Methionine is considered as an important factor in the control of oxidative status. However, restriction of dietary methionine has been shown to reduce generation of mitochondrial oxygen radicals and thus oxidative damage in liver. Here, we aim to evaluate the effect of dietary methionine deficiency in hepatic oxidative status in rainbow trout and identify the underlying mechanisms. Fish were fed for 6 weeks diets containing two different methionine concentrations: deficient (MD, Methionine Deficient diet) or adequate (CTL, control diet). At the end of the experiment, fish fed the MD diet showed a significantly lower body weight and feed efficiency compared to fish fed the CTL diet. Growth reduction of the MD group was associated to a general mitochondrial defect and a concomitant decrease of the oxidative status in the liver. The obtained results also revealed a sharp increase of mitochondrial degradation through mitophagy in these conditions and emphasized the involvement of the PINK1/PARKIN axis in this event. Collectively, these results provide a broader understanding of the mechanisms at play in the reduction of oxidant status upon dietary methionine deficiency.

CMA restricted to mammals and birds: myth or reality?

Chaperone-mediated autophagy (CMA) is a major pathway of lysosomal proteolysis essential for the control of intermediary metabolism. So far, the absence of any identifiable LAMP2A - a necessary and limiting protein for CMA - outside of the tetrapod clade, led to the paradigm that this cellular function was (presumably) restricted to mammals and birds. However, after we identified expressed sequences displaying high sequence homology with the mammalian LAMP2A in several fish species, our findings challenge that view and suggest that CMA likely appeared much earlier during evolution than initially thought. Hence, our results do not only shed an entirely new light on the evolution of CMA, but also bring new perspectives on the possible use of complementary genetic models, such as zebrafish or medaka for studying CMA function from a comparative angle/view.

Modeling of autophagy-related gene expression dynamics during long term fasting in European eel (Anguilla anguilla).

Autophagy is an evolutionary conserved cellular self-degradation process considered as a major energy mobilizing system in eukaryotes. It has long been considered as a post-translationally regulated event, and the importance of transcriptional regulation of autophagy-related genes (atg) for somatic maintenance and homeostasis during long period of stress emerged only recently. In this regard, large changes in atg transcription have been documented in several species under diverse types of prolonged catabolic situations. However, the available data primarily concern atg mRNA levels at specific times and fail to capture the dynamic relationship between transcript production over time and integrated phenotypes. Here, we present the development of a statistical model describing the dynamics of expression of several atg and lysosomal genes in European glass eel (Anguilla anguilla) during long-term fasting at two temperatures (9 °C and 12 °C) and make use of this model to infer the effect of transcripts dynamics on an integrated phenotype - here weight loss. Our analysis shows long-term non-random fluctuating atg expression dynamics and reveals for the first time a significant contribution of atg transcripts production over time to weight loss. The proposed approach thus offers a new perspective on the long-term transcriptional control of autophagy and its physiological role.

Distribution of H3K27me3, H3K9me3, and H3K4me3 along autophagy-related genes highly expressed in starved zebrafish myotubes.

The zebrafish (Danio rerio) remains the teleost fish of choice for biological investigations due to the vast array of molecular tools and resources available. To better understand the epigenetic regulation of autophagy, we utilized a primary myotube culture system generated from isolated myogenic precursor cells (MPCs) from zebrafish grown under starvation conditions using a media devoid of serum and amino acids. Here, we report starvation-induced regulation of several autophagy-related genes (atg) expression and profile the distribution of H3K27me3, H3K9me3, and H3K4me3 marks along lc3b, atg4b and p62/sqstm1 loci. These data support epigenetic regulation of autophagy in response to starvation that suggests a level of regulation that can be sustained for chronic conditions via chromatin modification.

miR-210 expression is associated with methionine-induced differentiation of trout satellite cells.

In fish, data on microRNAs (miRNAs) involved in myogenesis are scarce. In order to identify miRNAs involved in satellite cell differentiation, we used a methionine depletion/replenishment protocol to synchronize myogenic cell differentiation. Our results validated that methionine removal (72 h) from the medium strongly decreased myoD1 and myogenin expression, indicating differentiation arrest. In contrast, methionine replenishment rescued expression of myoD1 and myogenin, showing a resumption of differentiation. We performed a miRNA array analysis of myogenic cells under three conditions: presence of methionine for 72 h (control), absence of methionine for 72 h (Meth-) and absence of methionine for 48 h followed by 24 h of methionine replenishment (Meth-/+). A clustering analysis identified three clusters: cluster I corresponds to miRNA upregulated only in Meth-/+ conditions; cluster II corresponds to miRNA downregulated only in Meth-/+ conditions; cluster III corresponds to miRNAs with high expression in control, low expression in Meth- conditions and intermediate expression after methionine replenishment (Meth-/+). Cluster III was very interesting because it fitted with the data obtained for myoD1 and myogenin (supporting an involvement in differentiation) and contained seven miRNAs with muscle-related function (e.g. miR-133a) and one (miR-210) with unknown function. Based on our previously published miRNA repertoire ( Juanchich et al., 2016), we confirmed miR-133a was expressed only in white muscle and showed that miR-210 had strong expression in white muscle. We also showed that miR-210 expression was upregulated during differentiation of satellite cells, suggesting that miR-210 was potentially involved in the differentiation of satellite cells.