低肉碱罗非鱼模型的建立及其能量内稳态调控机制研究

The balance of the energy homeostasis is important for the health of organisms, including human, land animals, fish and so on. Now, the impaired energy homeostasis has been regarded as one of the main causes of metabolic syndromes, such as obesity, fatty liver diseases, diabetes, which are mostly characterized with excess lipid deposition. Therefore, energy homeostasis has become a worldwide hot domain in metabolism researches. However, because of the loss of effective animal models, the proceeding of the energy homeostasis study in fish has been blocked for quite long time, thus it is eager to establish an effective fish model for energy homeostasis study. In the present project, the applicant plans to firstly establish low-carnitine tilapia model by using biochemical inhibitor to decrease carnitine content in Nile tilapia or tilapia cells. By following the standard steps in metabolic model establishment, the present project will at first perform “inhibition-rescue” experiments to identify the inhibiting target biochemical reaction in the model fish by independently or jointly using carnitine synthesis inhibitor and/or L/D-carnitine additives. Secondly, the metabolic characteristics of low-carnitine tilapia or tilapia cells will be measured mainly by using metabolomics assay, nutrient metabolism rates assay and molecular measurements for the genes and proteins in nutrient metabolism. Finally, the model fish will be fed with diets containing different energy contents/nutrients composition, and the nutritional physiological functions, nutrient metabolism rates, patch of genes or proteins in nutrients sensing system and the metabolism-related cellular signal pathways will be thoroughly investigated, in order to conclude the energy homeostasis maintaining mechanisms of the model fish when lipid catabolism is lowed. The present project is designed to establish an effective fish model with clear inhibition target, well-known metabolic characteristics and regulatory mechanisms for further energy homeostasis study in fish, and also to provide novel ideas for the theory innovation in the formulation of aquaculture diets.

机体能量内稳态（Energy Homeostasis）失衡是造成人类、畜禽和鱼类中各种以脂肪严重沉积为表征的代谢性疾病的重要发生根源，已成为当前营养代谢研究的全球热点。然而，限于鱼类研究模型的缺乏，经济鱼类的能量内稳态调控机制研究至今进展缓慢。本项目将在多年预研基础上，以尼罗罗非鱼活体和细胞为研究对象，在国际上首次构建基于生化抑制的低肉碱经济鱼类模型，并按照动物代谢模型建立的规范要求，完成“确认代谢控制靶点-描述代谢特征-阐明代谢调控机制”三个研究流程。同时，借助代谢示踪、细胞/细胞器孵育、代谢组学和分子调控等现代生物技术，解析体内不同肉碱含量的罗非鱼在脂代谢受阻情况下应对不同能量/营养素摄入的能量内稳态代谢调控和信号调控机制。本项目力图建立一个靶点清晰、表征全面、机制清晰的经济鱼类营养学研究模型，推动经济鱼类能量内稳态调控机制的深入研究，也为养殖鱼类高效饲料配方的理论创新提供新的思路。

当前的水产鱼类养殖中普遍出现以脂肪严重沉积为表征的代谢性疾病，迫切需要深入了解鱼类能量收支平衡和代谢适应机制。然而，具有稳定生物学特征的、可以人工调控代谢路径的经济鱼类实验模型严重缺乏。因此，构建代谢路径可控的经济鱼类能量内稳态实验模型，是当前鱼类营养学发展的迫切要求。本项目提出构建低肉碱罗非鱼模型，从而通过抑制脂肪分解代谢来探索鱼类的能量内稳态的调控机制。通过四年研究，本研究获得以下研究成果：1）确认低肉碱罗非鱼的代谢控制靶点位于线粒体外膜的CPT-1蛋白，其主要代谢原理为阻断长链脂肪酸的线粒体外膜转运效率。2）在活体和细胞中验证了不同剂量的肉碱合成抑制剂对胞内肉碱含量的控制规律及生理、毒理特性，从而成功构建基于生化抑制的、人为可控的低肉碱鱼类模型。3）借助代谢示踪、细胞和细胞器孵育、代谢组学测定和分子调控等现代生物技术，清晰阐明低肉碱罗非鱼肝脂显著积累、线粒体beta-氧化酶系补偿性激活、过氧化物酶体b-氧化补偿不明显等代谢特征。4）利用低肉碱罗非鱼模型，破解水产业界长期以来对L-/D-型构型肉碱营养功能地位的争议，清晰阐明L-肉碱才能形成具有生理活性的、能成功穿越线粒体膜的脂酰-肉碱，而D-肉碱则导致脂肪酸无法进入线粒体造成脂毒性。5）阐明极低肉碱罗非鱼无法实现对高脂或者高蛋白饲料的代谢适应，造成严重代谢障碍；但是，温和的低肉碱罗非鱼模型却可以通过糖脂代谢的内稳态适应，提高对高糖饲料的适应性。以上工作，最终将低肉碱罗非鱼成功构建为一个靶点清晰、表征全面、机制清晰的经济鱼类营养学研究模型，并已据此衍生出其它低肉碱鱼类模型。更为重要的是，本项目成果使得水产业界和学界真正了解肉碱这一已应用30多年的饲料添加剂的生理功能与潜在的新应用，并决定性地推动肉碱市场的去劣存真，务实有效地推动了水产饲料添加剂产业的健康发展。