生物钟基因Per2调控细胞氧代谢的分子机制

The homeostasis of aerobic metabolism and anaerobic metabolism has precise mechanism of regulation. The unbalanced oxygen metabolism results in a significant change of muscular endurance and coordination, as well as in the abnormality of cardiac reserve, liver glycogen reserves and breathe ability, which can induce many chronic diseases. Circadian clock is closely related to basal metabolism and oxygen metabolism. However, the mechanism of circadian clock gene Per2 regulates oxygen metabolism remains unclear. The project will use the knockout mice and transgenic mice to study the synchronization of circadian clock gene Per2 rhythm and oxygen metabolism rhythm, the effect of the Per2 gene on the body oxygen metabolism on the overall level can be confirmed. Moreover, we will use wild-type mice，Per2-null mice and Per2 over-expression mice to investigate metabonomics assay with glucose stimulation. This can validate the metabolism of Per2 gene regulates aerobic and anaerobic metabolism. These will illuminate the mechanisms of Per2 gene regulate aerobic and anaerobic metabolism is through regulating the expression of PDK-associated gene. Additionally, we use the aging mice to compensate Per2 gene expression to investigate the reversal of oxygen metabolism, which can ascertain the regulation of Per2 gene on oxygen metabolism. These results will enrich our understanding of the relationship between aging and circadian clock, provide new insights on the mechanism of circadian clock regulating metabolic rate.

细胞有氧代谢和无氧代谢稳态有精确的调控机制。氧代谢的失衡会导致肌肉耐力和协调性的变化，心力储备、肝糖元储备和呼吸能力异常等各种慢性疾病。生物钟与机体的基础代谢和氧代谢有紧密联系，但是生物钟如何调控氧代谢的分子机制依然是一个未知的领域。本项目将利用基因敲除和转基因小鼠模型系统研究生物钟基因Per2节律与细胞的氧代谢节律的同步性，确定Per2基因在机体整体水平上对氧代谢的调控；通过对野生型小鼠、Per2基因缺失小鼠和Per2过表达小鼠在葡萄糖刺激下的代谢组学分析，进一步验证生物钟基因Per2对有氧代谢和无氧代谢的影响，阐明Per2通过调控PDK等相关基因的表达来影响机体氧代谢的分子机制。通过研究在衰老的小鼠中补偿Per2基因表达对小鼠氧代谢水平的恢复，进一步确定Per2基因对氧代谢调控的证据。其研究结果将丰富我们对衰老与生物钟关系的认识，从而在生物钟系统调控机体代谢的分子机制方面有新的突破。

生物节律广泛存在于生物界，生物钟基因Period2（Per2）是Period基因家族中的一个重要组成部分。一些研究证实Per2与机体的基础代谢紧密联系，但是Per2如何影响机体的氧代谢仍未知。我们发现生物节律蛋白PER2在红细胞应对外界压力的过程起到了一定作用。利用基因Per2敲除小鼠，我们证明Per2缺失导致全身氧代谢水平下降。通过Wright-Giemsa染色切片以及扫描电镜发现Per2-/-小鼠的畸形红细胞明显增加。Per2缺乏的小鼠红细胞对急性应激更敏感，苯肼、渗透和H2O2处理后溶血明显。红细胞寿命实验显示Per2-/-小鼠红细胞的寿命显著缩短。基于代谢组学的方法研究了Per2缺失小鼠红细胞中代谢物变化，发现Per2-/-小鼠红细胞中乳酸显著性升高，而葡萄糖及ATP水平显著性下降。Per2缺失不影响红细胞糖酵解过程，但是会导致红细胞膜的ATPase活性上升。免疫共沉淀（Co-IP）结果表明PER2作为转录共激活因子招募转录因子SP1形成二聚体，结合到Atp1a1启动子增强SP1的转录抑制作用，从而调节ATPase活性。向TF-1细胞中导入Per2的cDNA可以抑制ATPase表达。Per2缺失导致以甘油和丙酮酸为前体的血糖显著上升，阻断了以乳酸为前体的糖异生过程，表明Per2在有氧代谢和无氧代谢的转化过程中有重要作用。随着年龄的增长，机体的氧代谢能力显著降低。对比分析了8周龄小鼠和64周龄小鼠肝脏中生物钟基因的转录，结果显示在64周龄小鼠肝脏中Per2表达减少了40%，其他的生物钟基因的转录没有削弱。与8周小鼠相比，64周龄小鼠肝脏中铁的含量增加了约50%。检测喂食高铁食物的小鼠肝脏中的生物钟基因的表达，实验发现铁过量累积小鼠Per2的表达下降了60%，其他的生物钟基因的表达没有减少。此外，高脂饮食下，Per2-/-小鼠体重和体脂含量较WT小鼠显著地增多，肝脂和血脂的含量也显著性的上升，小鼠Per2的缺失上调脂肪合成基因Dgat1和Mgat2在小肠的表达，促进脂肪在小肠合成，导致高脂脂肪积累增多。研究结果证实了生物钟基因Per2通过调控红细胞的携氧来维持氧代谢稳态，衰老通过铁积累削弱Per2基因的表达，拓展了生物钟系统与基础代谢调控机制的认识。