National Aquafeed Safety Assessment Center, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, China.
Beijing Animore Sci. & Tech. Co., Ltd, Beijing, 100193, China.
Fish Shellfish Immunol. 2019 Jul;90:338-348. doi: 10.1016/j.fsi.2019.04.294. Epub 2019 May 8.
N-Carbamylglutamate (NCG), an analogue of N-acetylglutamate (NAG), can promote the synthesis of endogenous Arginine (Arg) in mammals, but not well studied in fish. This study was conducted to investigate the capacity of Arg endogenous synthesis by NCG, and the effects of various dietary NCG doses on growth performance, hepatic health and underlying nutrient regulation metabolism on ERK1/2-mTOR-S6K1 signaling pathway in Japanese seabass (Lateolabrax japonicus). Four experimental diets were prepared with NCG supplement levels of 0 (N0), 360 (N360), 720 (N720) and 3600 (N3600) mg/kg, in which N360 was at the maximum recommended level authorized by MOA, China in fish feed, and the N720 and N3600 levels were 2 and 10-fold of N360, respectively. Each diet was fed to 6 replicates with 30 Japanese seabass (initial body weight, IBW = 11.67 ± 0.02 g) in each tank. The results showed that the dietary NCG supplementation had no significant effects on the SGR and morphometric parameters of Japanese seabass, but 360-720 mg/kg NCG inclusion promoted PPV, while the 10-fold (3600 mg/kg) overdose of NCG had remarkably negative effects with significantly reduced feed efficiency, PPV and LPV. We found that Japanese seabass can utilize 360-720 mg/kg NCG to synthesis Arg to improve the amino acid metabolism by increasing plasma Arg and up-regulating intestinal ASL gene expression. Increased plasma GST and decreased MDA indicated the improved antioxidant response. Dietary NCG inclusion decreased plasma IgM and down-regulated the mRNA levels of inflammation (TNF-α and IL8), apoptosis (caspase family) and fibrosis (TGF-β1) related genes in the liver. The immunofluorescence examination revealed significantly decreased hepatic apoptosis and necrosis signals in the NCG groups. The ameliorated liver function and histological structure were closely related to the improved lipid metabolism parameters with decreased plasma VLDL and hepatic TG and NEFA accumulation, down-regulated fatty acid and cholesterol synthesis and simultaneously increased lipolysis gene mRNA levels, which regulated by inhibiting phosphorylation of ERK1/2-mTOR-S6K1 signaling pathway. Consuming 3600 mg/kg of dietary NCG is not safe for Japanese seabass culturing with the significantly increased FCR and decreased protein and lipid retention, and reduced plasma ALB. Accordingly, the observed efficacy and safety level of dietary NCG in the diet of Japanese seabass is 720 mg/kg.
N- 氨甲酰谷氨酸（NCG）是 N- 乙酰谷氨酸（NAG）的类似物，可促进哺乳动物内源性精氨酸（Arg）的合成，但在鱼类中研究甚少。本研究旨在探究 NCG 对 Arg 内源性合成的能力，以及不同饲料 NCG 剂量对生长性能、肝脏健康和 ERK1/2-mTOR-S6K1 信号通路相关营养素调节代谢的影响，该通路与日本真鲈（Lateolabrax japonicus）有关。本研究制备了 4 种实验饲料，NCG 添加水平分别为 0（N0）、360（N360）、720（N720）和 3600（N3600）mg/kg，其中 N360 是中国农业部授权的鱼类饲料中最大允许添加水平，而 N720 和 N3600 水平分别是 N360 的 2 倍和 10 倍。每种饲料投喂 6 个重复，每个水族箱中投喂 30 尾初始体重为 11.67±0.02g 的日本真鲈。结果表明，饲料 NCG 补充对日本真鲈的特定生长率和形态参数没有显著影响，但 360-720mg/kg NCG 可促进肝体比（HSI），而 10 倍（3600mg/kg）的 NCG 过量添加则具有显著的负面影响，显著降低了饲料效率、肝体比和特定生长率。我们发现，日本真鲈可以利用 360-720mg/kg NCG 合成 Arg，通过增加血浆 Arg 和上调肠道 ASL 基因表达来改善氨基酸代谢。血浆 GST 增加和 MDA 减少表明抗氧化反应得到改善。饲料 NCG 添加降低了血浆 IgM 水平，并下调了肝脏中炎症（TNF-α和 IL8）、凋亡（caspase 家族）和纤维化（TGF-β1）相关基因的 mRNA 水平。免疫荧光检查显示 NCG 组肝组织中凋亡和坏死信号明显减少。改善的肝功能和组织学结构与改善的脂质代谢参数密切相关，表现为血浆极低密度脂蛋白（VLDL）和肝甘油三酯（TG）和非酯化脂肪酸（NEFA）积累减少，脂肪酸和胆固醇合成减少，同时脂肪分解基因 mRNA 水平增加，这是通过抑制 ERK1/2-mTOR-S6K1 信号通路的磷酸化来调节的。以 3600mg/kg 的饲料 NCG 投喂日本真鲈并不安全，因为这种投喂方式会显著增加饲料转化效率和降低蛋白质及脂质保留率，同时降低血浆白蛋白水平。因此，在日本真鲈饲料中，NCG 的有效和安全剂量为 720mg/kg。
