罗非鱼对n-6系多不饱和脂肪酸的细胞代谢机制研究

Most of fish use n-3 polyunsaturated fatty acids (PUFA) as their essential fatty acids (EFA) and also specifically accumulate n-3 PUFA in fillet. Interestingly, tilapia has a special fatty acid metabolism pattern using n-6 PUFA as EFA and specifically accumulating n-6 PUFA in fillet. However, so far, the mechanisms of this special n-6 PUFA metabolism in tilapia are still unknown. In the present project, we will focus on the cellular metabolism of 18:2n-6, 20:4n-6 and 18:3n-3 in hepatocytes, muscle cells and head kidney cells in tilapia. In addition, the same work will also be performed in the corresponding cell types in grass carp, a fish using n-3 PUFA as EFA, in order to understand the potential difference of FA metabolism between two fish species. In the recent years, the applicant has developed a series of novel methods to investigate the metabolic details of FA in different cellular pathways, therefore, this allows us have the possibilities to study several important metabolic pathways of PUFA in tilapia cells, including the FA-albumin binding, trans-membrane uptake, mitochondrial/peroxisomal FA catabolism, glycerolipids synthesis, HUFA synthesis, FA-derived eicosanoids synthesis and FA-gene regulation. The main methods used in the present project will be isotope/fluorescence tracking, GC-LC/MS and cellular/molecular operations. As the aim of this project, we hope to realize the details of metabolism of n-6 PUFA in different cell types in tilapia, and understand the metabolic differences between n-6 and n-3 PUFA in tilapia cells, and between tilapia and another fish using n-3 PUFA as EFA.

与大部分鱼类均以n-3多不饱和脂肪酸（PUFA）作为必需脂肪酸并选择性地在体内保留不同，罗非鱼对于n-6 PUFA的特异性需求和保留是鱼类脂类代谢模式中一个奇特的案例，而相关的代谢机制，至今尚无人知晓。本项目着眼于在细胞和分子水平深入探索罗非鱼对n-6和n-3PUFA的代谢机制。本项目将采用一系列由申请人近年在国际上首先创建的脂肪酸细胞代谢研究的新方法，利用同位素/荧光示踪、色谱/质谱和分子调控等现代生物技术，以罗非鱼的肝细胞、肌肉细胞和头肾细胞作为研究对象，以18:2n-6、20:4n-6和18:3n-3作为目标脂肪酸，对这几种典型脂肪酸在罗非鱼细胞中的摄取、跨膜转运、分解、酯化、转化和基因诱导等几个方面进行系统研究。希望能较为清晰地勾勒出n-6/n-3PUFA在罗非鱼几种主要的细胞类型中的代谢模式和代谢差异，并在和草鱼的比较中发现不同脂肪酸代谢模式的鱼类在脂肪酸代谢中的"关键差异点"。

大部分鱼类均以n-3多不饱和脂肪酸（PUFA）作为必需脂肪酸，但罗非鱼对于n-6 PUFA有特异性需求，而相关的代谢机制至今尚无人知晓。本研究以罗非鱼为研究对象，以特异性需求n-3PUFA的草鱼作为比较参考，运用标记示踪、色谱/质谱、RNA-seq和分子调控技术，首次在细胞和活体两个层面，沿着脂肪酸的蛋白亲和、摄取、转运、氧化分解、酯化合成与基因调控等脂肪酸细胞代谢路径，系统而全面地研究了两种鱼对棕榈酸、油酸、亚油酸、亚麻酸和花生四烯酸的细胞代谢特征和差异，并首次对罗非鱼在n-6系脂肪酸摄入背景下的细胞脂代谢策略和代谢调控机制进行了深入研究。结果表明：1）罗非鱼具有和哺乳动物类似的细胞脂代谢路径和调控机制；2）在各条细胞脂代谢路径上，与草鱼相比，罗非鱼细胞对n-6PUFA的摄取、转运和氧化分解能力较高，这是罗非鱼与草鱼细胞脂代谢的“关键差异点”；3）罗非鱼在n-6PUFA摄入不足时，由肝细胞通过提高糖酵解能力增加脂肪合成底物维持脂代谢稳态，而在n-6PUFA摄入过多时则由脂肪细胞通过增加对游离脂肪酸的吸收，并激活PPARγ促进脂肪细胞增殖来增加对过多n-6PUFA的吸收；4）罗非鱼PPARα是细胞脂分解代谢的重要核调控元件，可通过去磷酸化、增加转录和增加蛋白翻译三种方式加以激活，其中去磷酸化是其本底激活机制，只有在激活刺激较大时才启动后两种激活方式；5）贝特配体是罗非鱼PPARα的激活因子，可通过激活PPARα促进细胞线粒体增殖和脂肪酸β-氧化活性来达到降脂效果。本项目成果首次在国际上阐明了罗非鱼作为特异性需求n-6PUFA鱼类在细胞脂代谢机制上与特异性需求n-3PUFA鱼类的“关键代谢差异点”，并首次阐明了罗非鱼在n-6PUFA摄入背景下的系统性脂代谢模式与调控机制。这些结果有助于人们理解不同鱼类的脂肪酸代谢模式，并有助于人们针对鱼类脂代谢的关键过程与调控节点研发高效饲料/饲料添加剂和进行种质改良。本项目相关成果已通过7篇国际期刊论文在BBA-Molecular and Cell Biology of Lipids、Fish and Shellfish Immunology、Aquaculture等权威期刊得以发表，并获得包括上海水产学会优秀论文一等奖在内的诸多奖励，相关成果也被《新民晚报》等媒体所报导，得到了学界和社会民众的广泛关注与认可。