Abstract
Omics approaches provide more metabolic information to explain the relationship between dietary nutrition and fish growth. This study aimed to explore the metabolome and proteome response of turbot (Scophthalmus maximus) fed diets containing lysine and leucine in free and dipeptide forms by the approaches of integrated liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolomics and isobaric tags for relative and absolute quantification (iTRAQ)-based proteomics. Plant protein-based diets were formulated to contain the equivalent of lysine and leucine in free amino acid [crystalline amino acid (CAA)] and synthetic Lys-Leu (Lys-Leu) forms. The metabolome and proteome profiles of the liver were screened in fish fed either the CAA diet or the Lys-Leu diet after an 8-week feeding trial. Fish fed the Lys-Leu diet showed a significantly higher final body weight and a specific growth rate compared with fish fed the CAA diet. Protein- and amino acid-related metabolic processes in the liver were identified between the Lys-Leu and CAA groups based on differential metabolites and proteins. The proteolytic enzymes and amino acid transporters from differential proteins of the liver showed that the process of protein digestion and absorption may be affected by the different forms of lysine and leucine in the feed. A mechanistic target of rapamycin complex 1 and ubiquitin proteasome pathways were identified by differential proteins, which were involved in the processes of protein synthesis and degradation in the liver. Lysine degradation, tryptophan metabolism, alanine, aspartate, and glutamate metabolism, arginine biosynthesis, arginine and proline metabolism, and glycine, serine, and threonine metabolism were identified based on differential metabolites and proteins, which showed that the metabolism of various amino acids, including lysine, had been affected by both the CAA and Lys-Leu groups. In conclusion, the data of integrated metabonomics and proteomics suggested that different forms of lysine and leucine in the feed may affect liver metabolic processes including protein digestion and absorption, protein synthesis and degradation, and amino acid metabolism. In addition, a good correlation between differential metabolites and proteins was observed in amino acid metabolism by using the approaches of integrated LC-MS/MS-based metabolomics and iTRAQ-based proteomics.
Highlights
With the rising demand and price of fish meal, expanding the amount of plant protein in aquafeeds was an effective strategy to replace fish meal (Daniel, 2017)
Differential metabolites were screened from all identified metabolites based on P < 0.05, fold change (FC) ≥ 1.2, or FC ≤ 0.8333 in the t-test and variable importance for the projection (VIP) ≥ 1 in the partial least squaresdiscriminate analysis (PLS-DA) model (Figure 1)
A total of 1,140 and 661 identified metabolites were obtained between the crystalline amino acid (CAA) and Lys-Leu treatments in the POS and the NEG modes, respectively (Supplementary Tables 1, 2 and the annotation information of metabolites is provided in Supplementary Table 3 based on compound.ID)
Summary
With the rising demand and price of fish meal, expanding the amount of plant protein in aquafeeds was an effective strategy to replace fish meal (Daniel, 2017). Nutritionists proposed that essential amino acids in feed could be supplemented with synthetic peptides or crystalline forms (Dabrowski et al, 2003; Ostaszewska et al, 2013) Those studies in fish species mainly focused on a comparison of dietary amino acid sources in peptides or free amino acids forms using classical nutritional methods, such as the evaluation of growth and feed utilization and the analysis of digestive enzyme activity and amino acid composition of fish whole body (Mamauag et al, 2012; Kim and Lee, 2013; Kamaszewski et al, 2019, 2020). The effect of dietary amino acid in free and peptide forms on fish growth was still controversial in different fish species, such as carp (Cyprinus carpio) (Kamaszewski et al, 2014), red sea bream (Pagrus major) (Mamauag et al, 2012), and olive flounder (Paralichthys olivaceus) (Rahimnejad and Lee, 2014).
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