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.
1 .Cowey, C. B. & Walton, M. J. Studies on the uptake of (14C) amino acids derived from both dietary (14C) protein and dietary (14C) amino acids by rainbow trout, Salmo gairdneri Richardson. J. Fish Biol. 33, 293–305 (1988).
2 .Bröer, A., Wagner, C. A., Lang, F. & Bröer, S. The heterodimeric amino acid transporter 4F2hc/y+LAT2 mediates arginine efflux in exchange with glutamine. Biochem. J. 349, 787–795 (2000).
3 .Balasubramanian, M. N. et al. Molecular pathways associated with the nutritional programming of plant-based diet acceptance in rainbow trout following an early feeding exposure. BMC Genomics 17, 449 (2016).
4. Callet, T. et al. Detection of new pathways involved in the acceptance and the utilisation of a plant-based diet in isogenic lines of rainbow trout fry. PLOS ONE 13, e0201462 (2018).