牛磺酸影响斜带石斑鱼脂类代谢的分子机制研究

The sulfur-containing β-amino acid, taurine, is a conditionally essential amino acid for fish growth and plays a vital role in several important biological functions such as bile salt conjugation, osmoregulation, membrane stabilization, antioxidation, and early development of visual and neural systems. A comprehensive understanding of taurine in fish biosynthesis, metabolism and its regulation mechanisms will provide a theoretical basis for the efficient use of protein ingredients and the development of feed formulations, thus improving the economic efficiency of fish farming. Our recent studies have shown that dietary taurine deficiency caused an increase in liver fat and whole-body fat deposition in grouper Epinephelus coioides, an economically important aquaculture species, and a perturbation in body energy metabolism and substance metabolism. However, little is known about the molecular regulation mechanism behind the phenomena. In the present study, the species of grouper is selected as the target animal. The gene and protein profiles in the hepatocyte steatosis models of the primary cultured hepatocytes of grouper cultured in L-15 medium wtih different taurine concentrations will be analysed using a transcriptomic approach combined with a comparative proteomic approach, and the differentially expressed genes and proteins will be identified respectively. The metabolic pathways related to lipid metabolism in liver of grouper associated with the administration of taurine in vitro will be investigated, with the aid of bioinformatics analysis tools. Furthermore, the expressions of functional genes and proteins related to lipid metabolism in liver of grouper under the administraton of dietary taurine in vivo will be analysed and validated by the qRT-PCR and western blot methods, and in combination with the analysis of phenotypic index system in tissues. Therefore, the molecular regulation mechanisms of taurin in lipid metabolism of grouper at mRNA and protein levels will be further clarified. The results will improve the understanding of metabolic roles of taurine in fish and provide scientific basis for the rational use of taurine in aquafeed and aquaculture production.

牛磺酸在鱼类渗透调节、营养物质代谢、免疫调节和抗氧化等方面发挥重要生物学作用。我们前期研究发现，饲料牛磺酸缺乏引起斜带石斑鱼肝脂和体脂沉积增加，机体能量代谢和物质代谢均受到扰动，但造成这些现象的分子机制还不清楚。本项目拟通过联合采用转录组学和比较蛋白质组学技术分析牛磺酸对斜带石斑鱼离体肝细胞脂肪变性模型基因和蛋白质的表达情况，结合生物信息学分析，筛选差异表达基因与蛋白质及其共同表达蛋白质，构建肝脂代谢相关基因与蛋白质的调控网络，并通过应用实时荧光定量PCR和免疫印迹法验证牛磺酸干预下斜带石斑鱼在体肝脂代谢相关基因及其对应蛋白质的表达情况，同时结合在体组织脂代谢相关表型指标体系分析，揭示牛磺酸影响鱼类脂类代谢的分子调控机制，成果将丰富鱼类牛磺酸营养学的内容，为牛磺酸在水产饲料配制和水产养殖生产中的合理使用提供科学依据。

为了探明牛磺酸降低石斑鱼肝脂沉积的作用机制，我们首先建立了石斑鱼原代培养肝细胞脂肪变性模型，并进行牛磺酸干预试验，通过转录组学分析构建了牛磺酸介导离体肝细胞脂代谢相关信号通路图，发现牛磺酸主要通过调节PPAR、AMPK、Jak-STAT和MAPK信号通路、初级胆汁酸生物合成、甘油磷脂代谢途经、脂肪酸延伸等脂代谢和免疫相关信号通路来发挥降低肝脂沉积、减轻炎症反应的作用；通过蛋白质组学分析构建了牛磺酸介导离体肝细胞脂代谢相关蛋白质表达网络图，发现牛磺酸通过影响胰岛素信号通路、糖酵解/糖异生、脂肪细胞因子代谢通路、甘油磷脂代谢途径中的蛋白表达，激活AMPK的磷酸化，促进脂肪分解和糖异生等来发挥其降低肝脂沉积作用。其次，我们利用在体试验得到的肝样本进行脂质组学分析，通过比对石斑鱼肝中脂质分子分布和含量，发现牛磺酸可通过减少18:1n-9和18:2n-1附着在肝TG分子sn-2位置的含量以及减少18:2n-1附着在肝TG分子sn-3位置的含量来降低肝脂沉积；肝样转录组学分析发现，胆汁分泌、胰岛素分泌、磷脂酶D信号等相关通路是牛磺酸降低石斑鱼肝脂沉积的关键通路；肝样蛋白质组学分析发现，牛磺酸主要通过影响甘油磷脂、鞘脂代谢、初级胆汁酸生物合成、AMPK信号通路等通路中的蛋白质表达来影响石斑鱼肝脂沉积。通过蛋白质与转录组学结果的关联分析，我们发现牛磺酸组与高脂组比对时磷脂酰肌醇磷脂酶D（PLCD）基因的表达水平显著上调，而PLCD的蛋白表达水平显著下调，说明牛磺酸可通过下调PLCD的表达来减少二酰甘油生成，进而降低TG的合成。此外，饲料牛磺酸还可通过下调磷脂酸磷酸酶（LPIN）的表达降低TG的合成，这一结果进一步支持了脂质组学分析的结果。综上，牛磺酸的降肝脂作用是通过介导PLCD和LPIN蛋白的下调表达来减少TGs在肝中的积累。本研究从多组学层面探索了牛磺酸参与鱼类脂肪代谢的调控机制，丰富了鱼类牛磺酸营养学的内容，为牛磺酸在水产饲料配制和水产养殖生产中的合理使用提供科学依据。