中华绒螯蟹对甘油三酯的分解供能途径及代谢机制研究

It has been well known that different lipid sources result in different effects in growth performance, feed efficiency, immunological activities and products quality of aquatic animals. Considering the basic differences between oils correspond to the differences of chemical properties of triglycerides which constitute more than 98% of common dietary lipid source, the utilization of different triglycerides in animals should be intensively studied. From energy production aspects, the triglycerides which are easily degraded into free fatty acids and those are also easily beta-oxidized for ATP production would be the ideal triglycerides for the formulation of “ideal lipid source”. As compared with fish, crustacean, such as Chinese mitten crab, has no specific adipose tissue, and is hypothesized to have different mechanisms of lipid metabolism from fish. A number of studies have indicated crustaceans have high sensitivity to lipid source and poor lipid sources will cause significant negative effects in crustaceans. Thus, the utilizing mechanisms of different triglycerides in crustacean should be firstly studied. However, very little information is known in this domain. In the present project, we choose Chinese mitten crab as the representative animal to study the catabolic pathway and following energy production of triglycerides. We will focus on two critical steps in the triglyceride catabolism and energy production: 1) release of free fatty acids from triglycerides through hydrolysis; 2) degradation of these free fatty acids to produce energy through beta-oxidation. In details, we will firstly analyze the chemical structures of different representative triglycerides of dietary lipid sources and build correlations between the nutritional values and chemical properties of these triglycerides. In order to clarify the mechanisms of release of free fatty acids from triglycerides, we will study the hydrolysis process of the representative triglycerides in intestine and other organs of mitten crab, and define the key enzymes in this process, and then to investigate the detailed process of degradation, particularly beta-oxidation, of the free fatty acids sourced from triglycerides in mitochondria and peroxisome. Through these work, we would 1) understand the detailed process and key steps of catabolism of triglyceride and related energy production, 2) illustrate a series of representative triglycerides which could be easily utilized for energy production; 3) firstly demonstrate the basic rules for formulation of “ideal lipid source” for crustaceans. These works could be useful for the formulation of proper lipid source and beneficial for improving dietary protein efficiency in aquaculture of crustaceans.

不同饲料脂肪源的营养效能差异，其本质在于水产动物对各种脂肪源所含甘油三酯（TG）的利用差异。与鱼类等较高等的动物相比，甲壳动物缺乏特定的脂肪组织，其对不同脂肪源所含TG的分解和供能代谢差异如何迄今尚无详细报道。本研究拟以中华绒螯蟹为代表，重点研究机体对不同TG的分解供能途径及其代谢机制。拟采用色谱/质谱、代谢组/转录组、细胞培养、分子标记示踪等技术，以常见脂肪源的TG结构特性与构型为切入点，首先探讨不同TG构型与其营养效能之间的关系；继而研究蟹肠道和储脂部位的TG水解过程，查明脂肪酸从TG中解离的过程与关键步骤；再通过研究蟹的线粒体和过氧化物酶体对不同脂肪酸的β-氧化效率，以揭示TG脂肪酸在蟹细胞器中的分解供能途径与关键步骤，最终解析TG在蟹体内被彻底分解供能的过程与关键调控节点。研究结果可为“理想脂肪源”调配原则的设计，提高甲壳动物对脂肪源的利用效能、增加机体蛋白质沉积率提供必备的参考。

不同饲料脂肪源的营养效能差异，其本质在于水产动物对各种脂肪源所含甘油三酯（TG） 的利用差异。与鱼类等较高等的动物相比，甲壳动物缺乏特定的脂肪组织，其对不同脂肪源所 含TG的分解和供能代谢差异如何迄今尚无详细报道。项目以中华绒螯蟹为研究对象，以探讨关键代谢节点和整体代谢网络解析相结合为研究策略，使用代谢组学、转录组学、细胞代谢测定等先进实验技术，由表及里地探究中华绒螯蟹对外源性甘油三酯的分解供能途径及代谢机制。首先利用代谢组学、脂质组学和营养学研究分析了常见脂肪源营养效果差异及其中的根本原因，结果发现由于结构本质和代谢空间构象的差异，富含油酸的脂肪源更利于供能、富含高不饱和脂肪酸的脂肪源易容易出现脂质氧化、富含饱和脂肪酸的脂肪源利于提高河蟹的消化吸收能力；通过活体实验和细胞实验，阐明了河蟹体内脂肪的蓄积部位主要为肝胰腺，以脂滴的形式储存在肝胰腺R细胞中。不同脂肪源代谢途径差异主要体现在三羧酸循环、丙酮酸代谢、磷酸 戊糖途径、嘧啶代谢与丙氨酸、天冬氨酸和谷氨酸代谢等通路。胰脂肪酶 (Pancreatic lipase)，甘油三酯水解酶 (Triacylglycerol lipase)和细 胞内脂肪酶 (Intracellular lipase)，前两种脂肪酶主要作用于饲料中脂质的分解，第 三种主要体现在对细胞内储存的甘油三酯的水解，这三种酶为河蟹脂肪代谢的关键点；甘油三酯水解酶和细胞内脂肪酶主要在肌肉和肝胰腺中发挥作用、胰脂肪酶则在鳃、胃和肝胰腺中起主要作用，但三者发挥作用的主要细胞器均为线粒体；通过本项目的开展证实了河蟹饲料理想脂肪源的脂肪酸组成关键信息、与其他营养素的代谢交互作用、在物质代谢网络中的作用和地位，以及调配脂肪源与其他营养素代谢关系的原则，我们在理想脂肪源的基础上，筛选出适宜的促脂肪分解因子，如肉碱、水飞蓟素、硫辛酸等。