Introduction
The development of Rainbow Trout (RT) aquaculture industry in combination with limited availability and high prices of fishmeal has prompted feed producers to include more plant proteins (PP) in aquaculture feed s. However, PP do not meet the necessary nutritional requirements of animals, in particular due to imbalance s in Amino Acid (AA) profiles compared to fishmeal, the historical ingredient. Although representing building blocks of proteins, AA also have many crucial roles as signalling molecules as well as substrates for cellular metabolism. However, t he maintenance of cellular AA homeostasis begins with the absorption and transmembrane traffic of AA within cells, ensured by Amino Acid Transporters (AAT). While characteriz ed in mammals, in which regulation of AAT by nutrients, including AA, has been demonstrated1 , few data exist in fish to date . The aim of this study was to 1) identify fo r the first time all AAT expressed from RT genome and 2) address their regulations by nutrients, and mainly by AA, to evaluate whether AA imbalance profiles could impact AA homeostasis. To reach these objectives, AAT were first identified in RT genome in silico before assessing their expression in different RT tissues, while the study of their regulations by nutrients was conducted by taking advantage of a cellular model: the RTH-149 cell line. Such approach, already validated for the study of AA metabolism in RT2 , allowed us to decipher AAT regulations independently of systemic regulation s that could occur in vivo. Finally, consequences of nutritional AA-induced AAT dysregulations were investigated through their impacts on intracellular AA contents as well as on the mTOR (mechanistic Target Of Rapamycin) signalling pathway, a major regulator of cell metabolism, also described for being controlled by AA availabilities3 . Results presented hereafter, not only allowed us to refine AAT classification according to their nutrient-dependent regulations but also highlight several interdependencies regulated by AA availabilities, AAT and signalling pathways and their potential physiological outcomes in fish fed with alternative protein sources.
Materials and Methods
First, potential AATs in the RT genome were identified by in silico analysis and primers were designed for each AAT, before being tested and validated on a pool of RT tissue s including brain, gut, stomach, liver, ovary, adipose tissue, kidney, spleen and muscle. In vitro experiments were performed using the RT hepatocyte cell line called RTH-149 (ATCC® CRL-1710, LGC standards, Molsheim, France). Cells were subjected to 13 experimental conditions using Hank’s balanced salt solution (HBSS, #14025-092, Gibco) supplemented in combination or not with total AA, Non-essential AA, Essential AA, Foetal Bovine Serum or using MEM deficient in single AA (c4086, Genaxxon Bioscience : -Arginine, -Lysine, -Methionine, -Leucine) as well as 3 conditions with MEM supplemented with pharmacological activators/inhibitors of key signalling pathways (Halofuginone and Tunicamycine, activators of the Integrated Stress Response pathway, and rapamycin, a mTOR inhibitor). Cells were treated for 24h prior being collected and subjected to the following analyses: RTqPCR analyses of AAT expression, UPLC intracellular AA content analyses and Western Blot analyses of 4EBP1 and S6 phosphorylation, two known targets of mTOR. Data gathered from these 3 sets of analyses were cross-analysed (Pearson correlations, generalised linear models) to define correlations and fitting model s that could help to decipher relationship between AA availabilities, AAT dysregulations, AA uptake and mTOR activation.
Results
From the 71 AATs identified in mammals, 212 were found in the RT genome, while at least 116 were expressed in RT tissues. Of the 116 AAT expressed in vivo in RT, 74 are identified in the RTH-149 cell line covering 86% of AAT specifically found to be expressed in the liver. All AAT expressed in RTH-149 were shown to be dysregulated by at least one of the experimental conditions tested, the vast majority of which being under the control, positively or negatively, of AA and/or FBS. Interestingly, our study revealed an antagonistic response between two major classes of AAT: Cationic AA T (CAAT) being predominantly upregulated by starvation , while A nionic AAT (AAAT) were mainly downregulated , which is mainly explained by AA, and more specifically on essential AA. According to the whole set of dysregulations observed in the 13 conditions tested, a hierarchical clustering of AAT expressed in RTH-149 cells has been generated highlighting the existence of 3 main groups of AAT according to their nutritional regulations. Following the analysis of the outcomes of nutritional-induced AAT dysregulations on intracellular AA contents together with effects on the activation of the mTOR signalling pathway, we potentially identified AAT candidates that appear as the main gatekeepers controlling AA homeostasis, and certainly their cellular functions.
Discussion
For the first time, AATs in RT have been identified in RT genome and characteriz ed for their expression in several RT tissues while in vitro assays performed in RTH-149 cells allowed us to refine the current AAT classification by providing new insights in their regulations by nutrients. These results confirm the key role played by AAT in the maintenance of cellular homeostasis, but more significantly illustrate the butterfly effect that a deficiency in a single AA can have . Indeed, we observed that single AA starvations induce dysregulations of large pools of AAT , which consequently strongly influenc e the intracellular content of many AA and thus the global cellular AA homeostasis. More widely, this questions the actual outcomes of diets with imbalanced AA profiles, when compared to fishmeal-based diets, on the physiology of farmed trout. If such observations are to be soon confirmed (work in progress), results gathered from this study provide a wide understanding of AAT regulations by nutrients in RT, but it also has the potential to supply a new method to assess, in a unique so far, comprehensive way to study AAT functions and activities in cells, and that, disregarding the species considered. Thus, such method could be useful not only for the fish nutrition field but for any biological study related to AAT and AA cellular functions which are still deeply required but overlooked due to notably to the lack of tools and methods4.
Bibliography
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