PGC-1在应激调控肉鸡骨骼肌线粒体能量代谢中的作用研究

Mitochondrial function is associated with feed efficiency, and plays an important role in muscle development. Broiler chickens have been reported to have impaired skeletal muscle mitochondrial respiratory function under stress. Recent interest has focused on a possible role of mitochondrial dysfunction in the ectopic fat deposition and in particular the development of skeletal muscle insulin resistance. Peroxisome proliferator-activated receptor (PPAR) γ coactivator 1 (PGC-1) is the master transcription regulator that stimulates mitochondrial biogenesis, by up-regulating nuclear respiratory factors (NRF-1, 2) and mitochondrial transcription factor A (mtTFA), which leads to increased mitochondrial DNA replication and gene transcription. Other targets of PGC-1 are the PPARs, which regulate mitochondrial fatty acid oxidation. A key question is the role of PGC-1 co-activators in skeletal muscle mitochondrial energy metabolism of broilers under stress..To address this, the expression pattern of PGC-1 family members (PGC-1α, PGC-1β and PRC) and the development rule of mitochondria will be investigated first to confirm the association of PGC-1 with stress response, followed by an analysis of the effect of over-expressing exogenous and RNA-interfered endogenous PGC-1 on skeletal muscle mitochondrial energy metabolism, aiming to clarify the regulatory role of PGC-1 in stress response. And then the relationship between PGC-1 and a sensor of celluar energy state (AMPK, mTOR or Sirt1) will be assessed, to reveal the underlying mechanism of PGC-1 responding to stress. The obtained results will not only provide the scientific basis for truly understanding stress-response mechanism and effectively guiding production, but also offer some reference value to the study of obesity and diabetes in humans.

线粒体功能影响肉鸡饲料转化率，介导骨骼肌生长发育。应激损害其功能，导致脂肪异位沉积和胰岛素耐受。过氧化物酶体增殖物激活受体γ辅助活化因子1（PGC-1）影响线粒体生成和功能，参与能量代谢。申请人前期研究发现，糖皮质激素处理降低肉鸡胸肌和成肌细胞PGC-1α蛋白表达，抑制线粒体数量和活性。由此提出的科学问题是：PGC-1在应激调控肉鸡骨骼肌线粒体能量代谢中的作用是什么？本项目拟采用基因干涉、蛋白质组学等关键技术，以线粒体的生成和功能变化为考察重点，体内和体外试验相结合，首先测定肉鸡骨骼肌PGC-1表达和线粒体发育规律，确定PGC-1与应激的相关性；然后研究PGC-1干扰或过表达对线粒体能量代谢的影响，阐明PGC-1对应激的调控；最后剖析能量感受器AMPK、mTOR、Sirt1与PGC-1的联系，揭示PGC-1对应激的响应。本项目所获成果有望为家禽应激反应的防控以及人类相关医学研究提供支持。

为探明过氧化物酶体增殖物激活受体γ（PPARγ）辅助活化因子1（PGC-1）在应激调控肉鸡骨骼肌线粒体能量代谢中的作用，本项目以外源糖皮质激素导入的方法建模，一方面观察线粒体形态、结构和数量，评价线粒体生物合成和氧化磷酸化功能，测定PGC-1表达丰度；另一方面利用重组腺病毒载体和shRNA干扰质粒，考察PGC-1β基因敲减或过表达后的影响。结果发现：（1）在生长早期（5-10日龄）和后期（30-37日龄），皮质酮暴露（CORT，30 mg/kg饲料）使肉鸡胸肌和腿肌线粒体肿胀、变形，数量减少（10日龄腿肌除外），结构遭受不可逆性损伤（如嵴断裂等）。CORT抑制（P < 0.05）胸肌、腿肌PGC-1β蛋白表达，降低（P < 0.05）线粒体膜电位、呼吸控制率、电子传递链酶复合体活性或丰度。（2）在离体条件下，CORT暴露（1000 nM，24 h）降低（P < 0.05）鸡胚源成肌细胞线粒体膜电位和ATP含量，下调（P < 0.05）PGC-1β、NRF-1（核呼吸因子1）、ATP5A（ATP合酶α亚基）和Cyt C（细胞色素C）蛋白表达。（3）在鸡胚源成肌细胞上，通过重组腺病毒载体和shRNA干扰质粒分别过表达和敲减PGC-1β基因，发现在正常状态下，高丰度PGC-1β促进（P < 0.01）线粒体生成，提高（P < 0.05）线粒体膜电位和细胞ATP含量，上调（P < 0.05）NRF-1、ATP5A和COX IV（细胞色素C氧化酶IV亚基）蛋白表达；PGC-1β敲降的作用恰好相反。（4）蛋白质组学分析表明，PGC-1β的生理作用非常广泛，参与能量代谢、蛋白质泛素化、抗氧化等多个过程。（5）在鸡胚源成肌细胞上，通过重组腺病毒载体过表达PGC-1β，发现在应激状态下，高丰度PGC-1β消除了（P < 0.05）糖皮质激素（CORT，1000 nM, 24 h）对细胞活力、ATP含量和线粒体膜电位、DNA拷贝数的不利影响，使ATP5A和COX IV的蛋白表达恢复（P < 0.05）至正常水平。以上结果表明，PGC-1β调控线粒体生物合成和氧化磷酸化，参与肉鸡骨骼肌能量代谢。糖皮质激素通过降低PGC-1β蛋白表达，破坏线粒体结构、数量和功能完整性，抑制骨骼肌生长发育。本项目提出了家禽应激干预的新靶点，具有广阔的开发和应用前景。