（-）-HCA调控肉鸡脂肪代谢的差异代谢产物和效应蛋白的筛选及其机制

For a long time, the aim of poultry production was to increase the growth rates, but excessive fat deposition in the abdomen had been neglected. This trait represents a major drawback for the industry, as it reflects reduced feed efficiency and carcass quality. Thus, how best to control excess fat and improve carcass quality are critical areas to poultry production and the research community. The key metabolic intermediate or its structural analogues, as a chemical signal, ccould alter the direction of nutrient metabolism. Using key metabolic intermediate or its structural analogues to regulate the nutrients metabolism pathway not only could improve the animal growth performance, but also ensure the quality and safty of animal products. This project aim to study the effect of (-)-hydroxycitric acid, an analogues of citric acid which play an important role in the carbohydrate metabolic pathway, on the lipid metabolism and its mechanism involved in broilers chiken. In this project, we applied the technology of metabolomics and proteomics to analysis the changes of serum intermediate and final metabolite and construction the proteins profiles of liver in broiler chickens with (-)-hydroxycitric acid treated, and then screening out of the differential metabolites and effectors protein. In chicken embryo, we first to verify the differential metabolites in serum and effectors protein in liver of chicken embryo with (-)-hydroxycitric acid treated, and then to determin the key metabolic pathway and dominant effector protein by bioinformatics methods. Subsequently, we confirmed the key metabolic pathway and effectors protein of (-)-hydroxycitric acid regulated fat metabolism throuth the methods of inhibiting the expression of dominant effector protein. Finaly, study the effect of (-)-hydroxycitric acid on the cAMP-PKA and PI3/Akt signaling pathways in cultured primary chicken hepatocytes and elucidated the relation of those signals pathway with dominant effectors protein, then depth reveals the cell biological mechanisms of (-)-hydroxycitric acid regulated on the fat metabolism. The results of this project would comprehensive and systematically clarify the effect of (-)-hydroxycitric acid on the fat metabolism and its mechanisms involved in broiler chicken, also it could provide theoretical basis for the development of metabolic intermediate biological function.

控制家禽脂肪过度沉积、改善肉品质已成为肉鸡养殖业亟待解决的重要问题。关键性代谢中间体的结构类似物作为化学信号对代谢途径发生影响，从而定向地改善动物的生产性能。本研究应用代谢组学和蛋白质组学的方法，探讨碳水化合物代谢途径中间物柠檬酸的类似物-（-）-羟基柠檬酸（HCA）对家禽脂代谢的影响及机制。通过分析血清代谢产物的变化和构建肝脏蛋白表达的精细图谱，筛选(-)-HCA调控的差异代谢产物和效应蛋白；在胚胎水平上对差异代谢产物和主要效应蛋白进行验证，利用生物信息学的方法确定(-)-HCA调节脂代谢的关键代谢途径和主导效应蛋白，并通过抑制主导效应蛋白的表达加以验证；进而在细胞水平上深入阐明(-)-HCA调控主导效应蛋白、继而影响关键代谢途径的细胞信号转导通路。研究结果将系统地、全面地阐明（-）-羟基柠檬酸对家禽脂肪代谢的调节作用及机制，同时也为天然安全的代谢中间体的开发利用提供科学依据和理论指导。

控制家禽腹部脂过度沉积已成为肉鸡生产经营者和科研工作者共同亟需解决的问题之一。作为碳水化合物代谢中重要的代谢中间体--柠檬酸的结构类似物，(-)-羟基柠檬酸具有调节脂肪酸合成、抑制食欲等众多生物学功能，但其机制还不是十分清楚。因此，本项目在动物整体水平上探讨(-)-羟基柠檬酸对家禽脂肪代谢的影响，并通过“组学”的方法分析血清中差异代谢产物和肝脏中差异蛋白表达的变化；在鸡胚验证差异代谢产物和效应蛋白因子变化的基础之上，利用生物信息学方法确定(-)-羟基柠檬酸调节肉鸡脂肪代谢的关键代谢途径和主导效应蛋白，继而从鸡胚原代肝细胞上阐明其调节主导效应蛋白的细胞信号转导机制。动物整体研究结果表明，(-)-羟基柠檬酸通过抑制脂肪酸合成相关因子的表达和下调NAD依赖型苹果酸酶的表达减少用于脂肪酸合成的辅酶NADPH而抑制脂肪酸的生物合成，同时通过升高烯酰辅酶A水合酶、肉碱棕榈酰转移酶等表达水平而加速脂肪分解；胎胚期研究结果发现，(-)-羟基柠檬酸通过升高果糖2,6-二磷酸的含量和葡萄糖-6-磷酸异构酶、异柠檬酸脱氢酶、NADH脱氢酶等表达水平以及降低磷酸果糖激酶、L-乳酸脱氢酶B链等表达水平而增强柠檬酸循环效率和促进能量代谢，最终通过减少脂肪酸合成前体-乙酰CoA的含量而降低肉鸡腹部脂肪沉积；细胞生物学研究证实，(-)-羟基柠檬酸通过AMPK-PGC1α-NRF-1这一主旨信号通路影响肝脏中糖、脂肪和能量代谢进程中关键效应因子而调控家禽体内营养物质的代谢流向，最终发挥其降脂效应。研究结果不仅深入揭示了(-)-羟基柠檬酸对家禽脂肪代谢的调节作用及生物化学机制，为天然、安全的代谢中间体—(-)-羟基柠檬酸作为一种营养补充剂用于降低畜禽胴体脂肪含量和提高动物产品质量提供重要理论基础，同时该结果对于促进动物健康养殖以及营养代谢病的防控也具有一定的科学指导性意义。