ins1和ins2对罗非鱼糖代谢的调控作用及分子机制

Taking advantage of its available genome background, omnivorous tilapia is used as the research object to explore the phenomenon of postprandial persistent hyperglycemia in fish. Using insulin (INS, the only hormone to reduce blood glucose level) as the breakthrough point, this project separates specific metabolic ways related with cellular glucose uptake and utilization from INS signaling pathway. Through glucose tolerance test, growth trial and gene knockout experiments, dynamic change of blood glucose level and the expression of ins1 and ins2 in the Brockmann Body (BB) after a glucose load, a comparison of liver, muscle and adipose tissue in the efficacy of glucose uptake (glucose transporter 4, glut4), glucose storage (glycogen synthase, gs), glycolysis and anabolism (phosphofructokinase-1, pfk1; isocitrate lyase, acly; sterol regulatory element binding protein-1, srebp-1; Acetyl-CoA carboxylase, acc), and catabolism and gluconeogenesis (hormone sensitive lipase, hsl; glucose-6-phosphatase, g6p), and the specific role of ins2 and ins1 in glucose metabolism in tilapia will be investigated. The underlying mechanism of INS resistance in peripheral tissue will be uncovered, and the regulatory role of metformin (mammalian antidiabetic drugs) in INS resistance will also be investigated. The implementation of this project will make great contributions to clarifying the molecular mechanisms of insulin to regulate glucose metabolism of fish, revealing the underlying mechanism of poor glucose utilization in fish and enhancing the capacity of fish to tolerate higher dietary carbohydrate level, which will not only enrich the knowledge of fish nutrition and physiology, but also provide many practical applications in aquaculture.

本项目针对鱼类餐后的持续性高血糖现象，选择杂食性的罗非鱼为研究对象，充分发掘其基因组已公布的优势，从降低血糖的唯一激素胰岛素（INS）出发，解析INS信号通路引发组织细胞摄取利用葡萄糖的代谢路径，通过葡萄糖耐受实验、生长实验、基因敲除实验，研究葡萄糖负载后罗非鱼血糖和BB（Brockmann Body）ins1和ins2表达的动态变化规律，比较肝脏、肌肉和脂肪组织在葡萄糖吸收转运（glut4）、储存（gs）、分解利用（pfk1、acly、srebp-1、acc）、糖异生（hsl、g6p）等环节的效率差异，揭示ins1和ins2在罗非鱼糖代谢中扮演的角色，阐明罗非鱼外周组织发生INS抵抗的根本原因，并使用降糖药物二甲双胍进行调控。本项目的执行有利于阐明INS调节鱼类糖代谢的分子机制，揭示鱼类糖利用能力低下的根本原因，调控鱼类对饲料糖的耐受能力，不但能丰富鱼类营养生理学理论知识，还具有重要的应用价值。

本项目针对不同食性鱼类糖利用能力迥异这一现象，以杂食性的罗非鱼为研究对象，执行了餐后营养状态试验、葡萄糖耐受实验、摄食生长试验和胰岛素1（ins1）基因敲除试验。在营养状态试验中，评价了餐后营养状态从合成代谢过渡到分解代谢过程中，罗非鱼肝脏、前肠、脂肪组织葡萄糖代谢的动态变化规律，筛选了这些组织对营养状态敏感且能有效反映糖代谢的关键同源基因。急性腹腔注射葡萄糖耐受试验结果表明：葡萄糖是罗非鱼有效的胰岛素促分泌素；肝脏可通过促进葡萄糖的吸收转运、糖酵解、糖原合成以及脂肪生成等代谢途径，在清除葡萄糖负载中起到了重要作用；红肌组织对于清除糖负载亦发挥了积极作用，但白肌组织所起的作用则十分有限。摄食生长试验结果表明，实用饲料淀粉水平从13%增加至32%会导致罗非鱼生长受损、体脂肪蓄积、肝脏和肌肉组织葡萄糖储存与分解利用增强和糖耐受能力上升，在32%淀粉组饲料中添加0.15%的二甲双胍并不能改善罗非鱼的生长和糖耐受能力。通过CRISPR/Cas9技术突变ins1基因后，F0代敲除阳性罗非鱼在孵化出膜后1-7 d内（受精后8-14 d）全部死亡，暗示ins1基因的缺失会导致罗非鱼生长发育受阻而死亡。本项目的执行初步揭示了杂食性罗非鱼葡萄糖代谢的调节机制，为解释不同食性鱼类糖耐受能力差异的分子机制和罗非鱼配合饲料的研发提供了重要参考。