低氧胁迫下大口黑鲈AMPK-Glut/Mct对乳酸转运调控机制

Dissolved oxgyen (DO) is the most important aquatic environment factor affecting the survival of fish, and hypoxia often causes the metabolim change to produce lactic acid on fish. The study of lactic acid removal is the key to promot the culture of high largemouth bass at intensive density. The AMPK-Glut/Mct signaling pathway plays an important role in the transport of sugar and lactate, however, up to date, it is not clear for the regulatory mechanism, especial removal of lactate We found that there was a significant difference in lactic acid content in the liver, heart, brain and other important tissues of the largemouth bass after hypoxia exposure. According to the related research reports, we speculate that AMPK-Glut/Mct signal pathway may control carbohydrate metabolism by regulating glucose and lactate transport. This pathway may be an important mode for fish to respond to hypoxia stress. To confirm this hypothesis, firstly, the key enzymes activities and metabolite content of glucose metabolism and lactate transport in largemouth bass will be detected under different oxygen concentration exposure in artificial aquarium. Then, we try to exhaustedly clone sequence and quantitate their expression patterns of key genes and proteins in AMPK-Glut / Mct signaling pathway and setup the correlation among glucose, lactate concentration and gene espression. Finialy. to verify the relationship, by vitro experiment (tissue culture with different glucose concentration under oxygen partial pressure) and vivo injection with siRNA inhibitor, we will elucidate the regulation relationship between the transfer of glucose and lactate and the key genes in the AMPK-Glut / Mct signaling pathway. Therefore, the expected results of this project could not only improve our understanding of responce to hypoxia in fish, but also could provide the basic information for exploring the regulation mechanism of AMPK-Glut/Mct signal pathway in energy metabolism under hypoxia stress in largemouth bass.

溶氧（DO）是影响鱼类生存最重要的环境因子，低氧（hypoxia）会造成鱼类代谢转变，产生乳酸。研究低氧胁迫下乳酸的去除是推动大口黑鲈高密度集约化养殖的关键。AMPK-Glut/Mct信号通路在糖及乳酸转运中起重要作用，但其调控机制尚未阐明。本课题组发现低氧胁迫后大口黑鲈肝脏、心脏及脑等重要组织中，乳酸含量明显不同，且存在显著的时序差异。根据相关研究提示AMPK-Glut/Mct信号通路通过调节葡萄糖和乳酸转运来调控糖类代谢，这可能是鱼类对低氧应答的重要途径。为证实这一假说，本项目通过对大口黑鲈进行低氧胁迫，测定胁迫后各组织中糖代谢关键酶活和乳酸含量；分析该通路关键基因和蛋白的表达模式并阐明与葡萄糖和乳酸的相关性；并在细胞和活体两个层面进行验证。因此，通过本项目的实施，可丰富鱼类抗逆适应的理论基础，也能够阐明AMPK-Glut/Mct信号通路在大口黑鲈低氧胁迫后的乳酸转运调控机制。

溶氧（DO）是影响鱼类生存最重要的环境因子，低氧（hypoxia）会造成鱼类代谢转变，产生乳酸。研究低氧胁迫下乳酸的去除是推动大口黑鲈高密度集约化养殖的关键。AMPK-Glut/Mct信号通路在糖及乳酸转运中起重要作用，但其调控机制尚未阐明。本项目通过对大口黑鲈进行低氧胁迫，测定胁迫后各组织中糖代谢关键酶活和乳酸含量；并结合转录组技术分析该通路关键基因和蛋白的表达模式并阐明与葡萄糖和乳酸的相关性；并在细胞和活体两个层面进行验证，结果表明：急性低氧胁迫下，加速大口黑鲈向厌氧代谢转变，且肝脏在低氧胁迫的供能起着重要作用，肝糖原的分解显著增强。无氧糖酵解是急性低氧应激下的主要能量来源，通过上调AMPK-Glut/Mct 信号通路中关键基因及蛋白调控乳酸转运适应低氧供能，明确了miRNAs在葡萄糖代谢调控中起着重要作用，且miR-124通过靶向MCT1调节肌肉乳酸运输。同时，有趣的发现，低氧应激早期（缺氧4h后）肝脏脂质的动员和激活增强，脂质β-氧化也可能参与供能，而复氧可以重塑糖、脂的代谢模式。发现了两个潜在的低氧耐受信号转导通路，即MAPK signaling pathway和Phosphatidylinositol signaling system，并预测了参与调控的miRNAs；低氧改变了肝脏糖脂代谢的利用模式，miRNA在调节糖酵解和脂质动员中起重要作用，构建了急性重度低氧胁迫下代谢的潜在调控网络；miR-124和miR-205下调肝脏CPT-1基因的表达降低CPT-1酶的活性来抑制脂肪酸的氧化；而miR-124参与低氧胁迫下多组织多途径的协同调节。高温和低氧对大口黑鲈鳃和肝脏的有免疫的交互作用。本研究为探索大口黑鲈低氧响应机制提供理论支持,有利于大口黑鲈耐低氧品系的选育。