日粮直链/支链淀粉比调控肉鸡肠上皮细胞氨基酸氧化供能的机制

Dietary protein is essential source of amino acid for livestock. However, the production of the dietary protein resources are not enough to meet the demands in China, so huge protein feed is imported from outside of China. Now Chinese are becoming conscious about how to utilize dietary protein in China to fulfill the requirement of livestock. Our previous research demonstrated that increasing the ratio of amylase/amylopectin could lower the protein level in broiler diet without influencing performance, while the mechanism is not clearly understood. The aims of this study are to determine the effect of dietary amylose/amylopectin ratio on broiler performance, starch digestion and the underlying mechanisms. The release of glucose, amino acids transportation and its utilization in intestine and contents of free amino acids in hepatic portal vein will be estimated. TCA metabolism, activation of different enzymes and differential proteins expression will be measured, and the analysis of transcriptomics, proteomics and metabonomics will be used to evaluate the function of glycometabolism and amino acid metabolism in the development and proliferation of enterocyte. Proteins and mRNAs involved in proliferation and differentiation of the intestinal epithelium, will be analyzed by proteomics and transcriptomic analysis, respectively. The metabonomic technology will be utilized to study the TCA cycle. In vivo and in vitro experiments will be performed to study the relationship between the ratio of amylose/amylopectin and the proliferation, amino acid and energy metabolism of enterocyte. This finding of the project will develop novel dietary formula to reduce the protein level for broiler production.

我国蛋白质饲料资源严重不足，每年需进口大量的蛋白质饲料。因此，节约和开发蛋白质资源是动物营养研究中面临的挑战。课题组前期的结果表明，提高直/支链淀粉比可降低肉鸡日粮蛋白水平，实现低蛋白日粮配制技术，但其机理还不清楚。本项目通过配制不同直链/支链淀粉比日粮饲喂肉鸡，分析淀粉消化率，葡萄糖释放量，氨基酸转运率，肠道氨基酸利用和门静脉氨基酸含量等阐明直链/支链淀粉比调控氨基酸利用状况。通过肉鸡肠上皮细胞培养试验，采用组学技术，观察肠绒毛发育，检测黏膜氨基酸、糖代谢差异基因的表达及功能；并观察肠上皮细胞增殖、细胞能量代谢关键产物、限速酶活性变化和差异蛋白，揭示日粮直链/支链淀粉比与肠黏膜增殖、能量代谢和氨基酸代谢的关系，阐明不同直链/支链淀粉比调控肉鸡肠上皮细胞氨基酸氧化供能的机制，为节约蛋白饲料资源提供新的思路。

蛋白质是畜禽重要的营养素，我国蛋白质饲料资源远远不能满足畜禽生产需求。因此，节约蛋白质资源是我国动物营养研究中的重要挑战。研究认为，慢速消化淀粉可以通过延缓肠道葡萄糖的吸收，降低肠黏膜利用氨基酸氧化供能程度，从而提高蛋白质利用。本项目采用动物试验和细胞培养试验相结合的方法，研究了日粮不同直链／支链淀粉比（淀粉消化速率）对肉鸡肠道氨基酸利用、肠道发育、肠上皮细胞增殖及肠道能量代谢影响；并通过细胞试验，研究了葡萄糖与氨基酸作为能量来源对家禽肠上皮细胞增殖和能量代谢的影响，以及葡萄糖缓解家禽肠上皮细胞氨基酸氧化供能条件。研究结果发现：日粮直链/支链淀粉比例为0.40的慢速消化淀粉相对于直链/支链淀粉比例为0.10的快速消化淀粉，以及比例为0.2的中速消化淀粉，可以延长肠道内葡萄糖的释放，显著提高肠粘膜ATP水平和Na+/K+-ATP酶活性，满足肠上皮细胞的能量需求，降低小肠粘膜氨基酸分解代谢，改善低蛋白日粮水平下肉鸡的生产性能，为家禽低蛋白日粮配置技术提供新方案。并探明同氨基酸相比，肠上皮细胞能量来源更依赖于葡萄糖。葡萄糖不足会促进氨基酸的转运，上调氨基酸代谢酶活，刺激氨基酸的分解代谢与氨基酸的转化，产生大量天门冬氨酸来维持细胞蛋白合成，同时下调维持葡萄糖平衡与能量稳态的通路蛋白，破坏肠道功能稳定。肠上皮细胞缺乏葡萄糖时，会引起细胞产生氧化应激，上调氧化还原酶活性，并通过上调Ca2+跨膜转运途径和乙酰化凋亡通路蛋白，诱导肠上皮细胞发生线粒体凋亡。肠上皮细胞处于低氨基酸状态时，细胞会最大程度利用葡萄糖供能，并通过调控糖酵解途径中间代谢产物和酶活性向氨基酸合成通路转化，缓解细胞对氨基酸的需求，维持肠上皮细胞功能稳定。从理论上阐明，提高慢速消化淀粉比例节约肠道氨基酸利用的机制。本研究利用糖节约氨基酸，从而实现肉鸡生长后期日粮粗蛋白质降低2个百分点不影响生产性能，为我国低蛋白日粮配制技术提供了新思路。