Physiological response and mTOR regulation to branched-chain amino acids in Atlantic salmon (Salmo salar)

Abstract

The increasing demand for sustainable aquafeed ingredients has necessitated extensive research into alternative protein sources, reducing reliance on finite marine resources in Atlantic salmon (Salmo salar) aquaculture. Among key dietary components, branched-chain amino acids (BCAAs)- leucine, isoleucine, and valine—play critical roles in protein synthesis, energy metabolism, and cellular signalling pathways. Despite their well-established importance in mammalian systems, research on BCAA metabolism and mechanistic/ mammalian target of rapamycin (mTOR) signalling in Atlantic salmon remains limited. This study investigates the metabolic fate of dietary BCAAs, their potential role in mTOR activation, and the physiological responses of Atlantic salmon to dietary variations and hypoxic stress. Chapter 1 provides a comprehensive review of BCAA metabolism, dietary requirements, and regulatory factors in fish and terrestrial livestock. The role of mTOR as a central cellular hub integrating amino acid (AA) availability, energy status, and environmental cues is explored in relation to nutrient utilisation and growth regulation. Existing knowledge gaps in Atlantic salmon are highlighted, particularly regarding BCAA catabolism and mTOR-mediated metabolic control under varying physiological conditions. Chapter 2 examines the digestibility and uptake kinetics of crystalline amino acids (cAAs) compared to intact proteins in Atlantic salmon post-smolts. Temporal BCAA absorption patterns were analysed in plasma, muscle, and liver tissue, revealing peak uptake at 4 hr post-feeding. The study confirmed that BCAAs primarily bypass first-pass hepatic metabolism, supporting the hypothesis that they serve as direct substrates for protein synthesis and signalling in muscle tissue. Despite significant temporal and tissue-specific differences in BCAA metabolism and mTOR-related gene expression, the observed mRNA abundance changes were not deemed biologically significant, emphasising the need for proteomic validation. Future investigations should assess direct protein synthesis rates and phosphorylation status of mTOR and its downstream targets. Chapter 3 evaluates the impact of varying dietary BCAA levels and leucine enrichment on growth performance, AA homeostasis, haematological parameters, and molecular signalling in Atlantic salmon parr. A near-significant increase in growth performance was observed (p=0.052), with the highest final weight recorded in fish receiving the suboptimal BCAA diet. Stable isotope analysis revealed tissue-specific retention of δ15N-labeled leucine, with greater incorporation in muscle compared to liver, aligning with established models of BCAA metabolism. Despite no significant differences in blood biochemistry, variation between weeks were observed in haemoglobin, packed cell volume, and white blood cell counts suggested potential physiological adaptations. mRNA transcript analysis indicated temporal fluctuations in BCAA catabolism, particularly in BCKDHA mRNA abundance, reinforcing the dynamic regulation of AA metabolism in response to dietary composition and developmental stage. Chapter 4 investigated transcriptomic and metabolic responses to hypoxia, with emphasis on mTOR modulation across liver, brain, and heart tissues of Atlantic salmon post-smolts. RNA-Seq analysis identified 372 differentially expressed genes (DEGs), with liver exhibiting the highest transcriptomic alterations. Notably, the only direct mTOR-associated DEG, E3 UBIQUITIN-PROTEIN LIGASE RNF152-LIKE, was downregulated in liver tissue, suggesting a potential suppression of mTOR activity under hypoxia. Functional enrichment analysis of the fold change (FC) results, >2FC and <-2FC, identified 37 mTOR-related mRNA transcripts interacting with external WNT and TNF-α pathways and internal ubiquitin-mediated proteolysis mechanisms. Downstream effectors of mTOR, including EIF4E2, RHOA, and SI:CH211-195B13.1, exhibited significant repression, possibly implicating translational control mechanisms in the adaptive response to oxygen deprivation. The transcriptomic data identified possible hypoxia related mTOR signalling but not through the REDD1 hypoxia pathway, but rather through both extrinsic (growth factors, inflammatory mediators) and intrinsic (ubiquitin-proteasome system, translational regulation) pathways. The identification of the same mRNA transcripts both up- and downregulated suggested possible annotation complications. Further transcriptomic analyses on the 1FC and -1FC mRNA transcripts identified 831 mTOR associated mRNA transcripts up- and downregulated under hypoxia, suggesting alternative mechanism of regulation of the mTOR pathway. Overall, the transcriptomic data revealed oscillations within the pathways, but no distinct emergent properties were observed. This may support the hypothesis that mTOR is not transcriptionally regulated. Collectively, this research provides critical insights into the metabolic interplay between BCAA bioavailability, mTOR regulatory pathways, and physiological adaptation in Atlantic salmon. The findings underscore the imperative of optimising dietary AA formulations to maximise growth efficiency while elucidating the intricate signaling networks governing protein metabolism under hypoxic stress. Future studies should incorporate multiomic methodologies, encompassing phosphoproteomics and metabolomics, to delineate the molecular underpinnings of mTOR-driven metabolic regulation in aquaculture species. Additionally, advancing dietary strategies to enhance nutrient assimilation while mitigating environmental stressors is pivotal for promoting sustainability in intensive salmon aquaculture.