Aquaculture Europe 2023

September 18 - 21, 2023


Add To Calendar 21/09/2023 17:00:0021/09/2023 17:15:00Europe/ViennaAquaculture Europe 2023ARGININE AND LYSINE ABSORPTION IN RAINBOW TROUT Oncorhynchus mykiss: UNDERSTAND THEIR ABSORPBTION AND FATE IN CELLS TO COPE WITH AQUACULTURE STAKESStrauss 2The European Aquaculture Societywebmaster@aquaeas.orgfalseDD/MM/YYYYaaVZHLXMfzTRLzDrHmAi181982



Guillaume Morin *,  Karine Pinel, Soizig Le Garrec, Cécile Heraud, Akintha Ganot, Karine Dias, Iban Seiliez and Florian Beaumatin


 Université de Pau et des Pays de l’Adour, E2S UPPA, INRAE, NUMEA, Saint-Pée-sur-Nivelle, France

 E-mail :



The use of p lant  proteins (PP) in fish feeds has considerably increased,  at the expense of fishmeal (FM) inclusion, over the last decades  to  better  cope with aquaculture sustainability. Nonetheless, after decades of research,  100% plant-based diets (PBD) still disturb fish physiology owing to multifactorial reasons among which an imbalance of  PP amino acids (AA) profiles with some essential AA (EAA), mainly Methionine (Met), Lysine (Lys) and less frequently Arginine (Arg), being underrepresented in PP compared to FM . To overcome this issue, those EAA are  usually added in free form to the PBD to ful fil requirements of fish determined  previously  using FM-based diets as standards. Although supplementation in these EAA improves fish growth, it  does not rescue the full growing potential of fish fed FM-based diets. This could be explained, at least partially , by the fact that free AA are  differentially absorbed compared to protein-bound AA, leading  to  differences in their bioavailabilities

 . Interestingly, we noticed in two independent studies that some intestinal  AA transporters (AAT), crucial for Arg, Lys and potentially Met absorption, and described in mammals to work by exchang ing Arg and Lys with neutral AA

 , were overexpressed in fish fed PBD

 . Thus,  we wondered if  the  difference in bioavailabilities of protein-bound neutral AA  versus free Arg, Lys and Met added to PBD could  compromise their absorption, leading to dysregulations of these AAT and subsequent negative outcomes on fish physiology and growth. Thus, our study aimed to challenge this hypothesis by i ) identifying and characterising the whole sub-family of cationic AAT (in charge of Arg and Lys uptake) in response to cationic AA starvation, ii) studying the consequences of cationic AA starvation on two major AA-sensing dependent pathways called GCN2 (General Control Nonderepressible 2) and mTOR (mechanistic Target Of Rapamycin) pathways and iii) proposing, through fundamental knowledge gathered using rainbow trout (RT) cell lines as models to study AA absorption, a new formulation strategy that promotes the absorption of cationic AA supplemented in their free forms to 100% PBD in vivo to improve the zootechnical parameters of fish fed such PBD.

Materials and methods

Cell culture experiments were performed at 18°C using RTH-149 and RTgutGC cell lines. Cells lines were subjected to various nutritional challenge (e.g. AA starvation or specific cationic amino acid (CAA) starvations…) prior to determine cellular absorption of AA in cell lines, using UPLC-FL as well as the activation of AA-dependent signalling pathways, such as GCN2 and mTOR, involved in growth and stress through RT-qPCR and western blot analyses.  For  the presented in vivo results , RT were fed at libitum for 21 weeks with a full PBD supplemented with free Arg and Lys (RK) or the same PBD also supplemented with free Glycine (RKG). Plasma  were sampled from the caudal vein 5 hours after the last meal  (during the post-prandial amino academia peak)  and AA levels were analysed by UPLC-FL to assess the effect of Gly supplementation on absorption of Arg and Lys .


 Using in silico analysis we identified 42 orthologous  genes of  the 16 human cationic amino acid transporters  (CAAT) in the rainbow trout (RT) genome. At least 28 of them are expressed in trout tissues. Both RTH-149 and RTgutGC cell lines express the same 16  CAAT which are also expressed in their tissues of origin, liver and intestine respectively.  Of the 16 CAAT expressed in the cell lines, 6 of them were systematically shown to be-upregulated upon nutritional challenges in AA- and CAA-dependent manners, very likely through the activation of the GCN2 pathway. Interestingly, one of them,  called y+ LAT2 which is  an important CAA exchanger (notably in the intestine)  is one of the most upregulated by Arg and Lys starvation in cells  while it was also shown to be upregulated  in previous in vivo studies where fish were fed a 100% PBD supplemented with free CAA

. Therefore we supposed that the y+LAT2 upregulation observed in fish fed PBD  could be  due to an activation of the GCN2 pathway  caused by a  compromised  ability of fish to absorb  the supplemented free CAAs and therefore leading to  decrease  fish growth  performances. Knowing from our in vitro assays that part of the cellular absorption of CAAs relies on the presence of non-essential neutral AA, among which glycine certainly exchanged by y+LAT2, we therefore build a working hypothesis according which the  known difference of bioavailability of free AA versus  protein-bound AA, could considerably dampens the absorption of supplemented CAAs in PBD . Consequently, we challenged this hypothesis by comparing zootechnical parameters of fish fed  a PBD supplemented with Arg and Lys (RK) to those fed with the same diet also supplemented with Gly (RKG). Following a 21 weeks feed trial, we first could observed that such Gly supplementation improves significantly the feeding efficiency (as already observed in broilers). Very strikingly, we could also noticed that such Gly supplementation allows a better absorption of CAA as demonstrated by the significant increase in post-prandial AA levels of Arg and Lys (see figure) while, with the exception of Gly and Serine, no other AA levels was observed to be significantly increased.  These results demonstrate that free Gly supplementation increases free Arg and Lys absorption that in turns could improve feed efficiency.


 In this study, we first identified the CAAT expressed in RT and showed with the use of RT cell lines that some of them are upregulated through activation of the GCN2 pathway during Arg and Lys starvation. One of them, y+LAT2, is also upregulated in fish fed with PBD supplemented with free Arg and Lys. Because y+ LAT2 is  1) a strict exchanger of CAA against neutral AA and 2) an essential intestinal  CAA transporter  in mammals, we supposed that bioavailability difference between protein-bound neutral AA and free CAA affect absorption of the latter. Thus , we demonstrated with combined in vitro and in vivo approaches, that most of this working hypothesis was correct by showing that free CAA absorption can be improved by supplementing PBD with free neutral AA like Gly. Altogether, this study shed a new light on the importance considering AAT functions and activities in the biology of fish of agronomic interest and open new exciting avenues to develop news feed formulation that better meet the sustainable developmental goals of the aquaculture industry.


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