Visfatin经AMPK信号通路调控肉仔鸡食欲的分子机制

At present, insufficiency of active feed intake is an important factor of restricting high yield animal performance, especially in high intensive poultry production. Exploiting new orexia regulatory factors and researching their mechanisms that control appetite and energy balance are crucial for poultry health and efficient production.Visfatin is known as a newly identified adipocytokine.The newest researches have indicated that visfatin played key roles in animal orexia reglulation.However,the mechanisms underlying its action remain unclear. In the current study, regarding AMPK signal pathway regulating Appetite and energy balance as breakthrough spot,we will firstly investigate effects of visfatin on orexia regulatory factors From CNS and peripheral tissue and key enzymes and mesostates in AMPK pathway in chickens to analyze core position of AMPK pathway by means of intracerebroventricula injection,qRT-PCR,gene microarray and NMR metabonomics analysis.Then, we will investigate effects of visfatin and repressors on gene expression of up-down stream factors in AMPK signal pathway to confirm molecular targets of visfatin acting through AMPK pathway using qRT-PCR and Western-blotting. The objective is to elucidate its functional mechanism of visfatin regulating appetite in chickens, which may lay the foundation for perfecting the theory of orexia regulation and energy balance in poultry.

现代家禽生产中，主动采食量不足是影响生产性能的最大限制因素。发掘新的食欲调控因子并研究其机制，对于家禽健康、高效生产至关重要。Visfatin 是新发现的脂肪细胞因子，最新研究表明，其在动物食欲调控中扮演关键作用，但其作用机制还不明确。本项目拟以调节食欲和能量平衡的AMPK信号通路为突破口，首先采用脑室注射、基因芯片、代谢组学和qRT-PCR等技术手段，研究Visfatin对肉仔鸡中枢和外周组织食欲调控信号因子及AMPK信号通路关键酶和代谢产物的影响，解析AMPK通路在食欲调控中的核心地位；然后采用qRT-PCR和Western-blotting技术，结合动物试验和神经元培养试验，研究Visfatin和阻断剂对AMPK信号通路上下游基因表达的影响，确定Visfatin作用AMPK信号通路的分子靶点，以揭示Visfatin调控肉仔鸡食欲的作用机制，为完善家禽的食欲调控和能量平衡理论奠定基础。

为阐明Visfatin 促食的分子机制，完善家禽的食欲调控和能量平衡理论。本项目采用脑室注射、转录组学、代谢组学和qRT-PCR 等技术手段，结合体内和体外试验，研究Visfatin 对雏鸡中枢和外周组织食欲调控信号因子及信号通路关键酶和代谢产物的影响，结果如下:. 1.构建了鸡Visfatin克隆和原核表达系统，完成了鸡Visfatin基因的克隆和重组蛋白可溶性表达和纯化。活性鉴定结果表明，鸡Visfatin重组蛋白可以显著促进3T3-L1脂肪细胞的葡萄糖摄取。. 2.脑室注射Visfatin能显著提高雏鸡的采食量，且Visfatin参与血清中的糖类物质和脂类物质的调节。在转录水平上，Visfatin发挥促食作用是通过降低POMC、GHRH、GHR、SST、CCK、GLP-1R等抑食因子mRNA的表达，提高潜在促食因子HCRT、VIP、ABAT和NPFFR2等mRNA的表达而实现的。在代谢水平上，脑室注射Visfatin可显著影响下丘脑中营养物质代谢，下丘脑营养物质代谢产物含量降低，改变动物机体内的能量稳态平衡是Visfatin对雏鸡发挥促食作用的主要途径。. 3.培养下丘脑神经元细胞和肝原代细胞，通过体外试验研究了Visfatin调控食欲的关键因子和信号通路。Visfatin发挥促食作用的主要机制不是通过激活LKB1-AMPK信号通路，而是通过抑制PI3K-AKT- mTOR信号通路而实现。