低氧适应下AMPK对宰后牦牛肉能量代谢影响机理研究

Under low oxygen environment, unique energy metabolism in yak can maintain muscle energy metabolic balance. AMP-activated protein kinase (AMPK) has critical roles in regulating energy balance of mammals, and has recently been connected to postmortem energy metabolism and meat qualities. Nonetheless, few researchers have clarified the fundamental mechanism of AMPK in regulating energy metabolism postmortem. To study postmortem energy metabolism pathways of yak meat under hypoxic adaptation, metabolomics, phosphoproteomics, bioinformatics, immunohistochemistry and RT-PCR methods were used. Furthermore, to determine the regulation of AMPK activity pathways, AMP and AMPKK regulation of AMPK, the role of phosphorylation level in AMPK regulation and Sirt1 pathway to AMPK were studied. In this study, key factors in energy metabolism regulated by AMPK were proven, and then meat quality traits influenced by different energy metabolic pathways were researched. Eventually, the mechanism of AMPK in regulating postmortem energy metabolism and meat quality under hypoxic adaptation was revealed. The purpose of the study was to develop theoretical system of postmortem energy metabolism in yak meat, and provide scientific basis for improving and reasonable utilization of yak meat.

低氧环境下牦牛机体存在特有的代谢机制，以维持肌肉能量代谢平衡，腺苷酸活化蛋白激酶（AMPK）是维持动物机体能量平衡的调节器，与宰后能量代谢及肉质相关。目前对AMPK调控宰后肉类能量代谢的机理缺乏研究。本项目拟利用代谢组学、磷酸化蛋白质组学、生物信息学、免疫组化、RT-PCR等方法，解析低氧适应下宰后牦牛肉能量代谢途径，通过AMP、AMPKK、AMPK磷酸化修饰及AMPK/Sirt1信号通路对AMPK的调节，阐明AMPK活性调节机理，探明AMPK对能量代谢关键因子的影响，研究不同能量代谢途径与肉质的关系，揭示低氧适应下AMPK对宰后牦牛肉能量代谢及肉质影响的机制，为丰富牦牛肉能量代谢理论体系，改善肉质及合理利用提供科学依据。

本项目以不同海拔牦牛肉为研究对象，采用磷酸化iTRAQ、RT-PCR、免疫印迹、酶联免疫、扫描电镜等技术，揭示了低氧适应下维持牦牛肉能量供需平衡的代谢机制，阐明了宰后不同海拔牦牛肉的AMPK活性调节通路；明确了AMPK调节通路、能量代谢途径、以及肉品质变化的级联调控关系，明晰了AMPK对宰后牦牛肉品质变化的调控途径。主要研究结果如下：1. 高海拔牦牛AMPKα2、HIF-1、GLUT4 mRNA以及蛋白质表达量均高于低海拔牦牛。宰后成熟过程中，高海拔牦牛肉AMPK活性、糖酵解、能量代谢水平、L*值、b*值以及嫩度均高于低海拔牦牛肉，但a*值低于却低于低海拔牦牛肉。2. 牦牛肉宰后，AMPK可直接被AMP、CaMKK以及活化的SIRT1激活，主要体现在AMPKα2的磷酸化。3. 通过iTRAQ蛋白组技术共检测胞质蛋白2333个，发现高海拔牦牛与低海拔牦牛存在差异的蛋白质主要都集中在糖酵解、肌动蛋白连接、线粒体、氧化还原反应、血液循环等方面；涉及的代谢通路主要是肌肉收缩、糖酵解、心脏发育和HIF1信号通路。这些代谢途径均与低氧适应有关，进一步验证上述结果。4. CaCl2处理可通过CaMKKβ级联激活AMPK，进而促进AMPK依赖性糖酵解，加快牦牛肉成熟进程，改善肌肉品质。5. 牦牛肉宰后NO结合sGC产生cGMP，cGMP通过PKG激活PLC并诱导产生IP3，IP3诱导内质网释放Ca2+，使CaMKKβ依赖性AMPK激活。因此，NO-AMPK途径可以加快肌肉糖代谢水平，促进肌糖原分解和pH值降低。6. ROS可通过诱导内质网钙泵功能障碍促进了Ca2+释放，Ca2+激活CaMKKβ进一步使AMPK激活。活化的AMPK通过磷酸化增加了PFK、PK活性以以及糖酵解代谢，从而使牦牛肉嫩度得到改善。7. 牦牛肉宰后成熟过程中，活化的AMPK通过诱导细胞质中Bax向线粒体膜易位，导致线粒体膜上Bax/Bcl-2值增加，促进了牦牛肉线粒体膜的通透性增加。这进一步增强了线粒体功能障碍和细胞色素c释放，促进了caspase-3激活和细胞凋亡并且改善了牦牛肉的嫩度。综上，本研究从细胞层面、蛋白质分子层面及牦牛肉品质层面，深入探究了牦牛低氧适应以及AMPK介导牦牛肉能量代谢及肉质变化级联反应机制，为揭示牦牛肉宰后成熟机制提供了新的理论依据。