鱼类新型细胞因子Adiponectin和受体的分子克隆及其对糖脂代谢的调控机制

With low utilization ratio of saccharides, fish is considered as the "natural diabetic", and it's highly dependent on protein as source of energy. Saccharides and fat are cheaper sources of energy than protein, and in addition of this, their use in feed can save the expenditure of protein as energy, and decrease the water-soluble nitrogen excretion of fish into the aquicultural water, thereby reduce the degrees of water pollution. So it becomes pretty important to develop more researches about the special molecular mechanism of fish glucose and lipid metabolism. This project will carried out studies on the regulation to glucose and lipid metabolism in fish by a novel cytokine adiponectin and its analogue CTRP-9, C1q and TNF related protein-9). First of all, we will clone the cDNA of adiponectin, adiponectin receptors and CTRP-9 from orange-spotted grouper (Epinephelus coioides), after the analysis of these genes and their expression levels in different tissues and different stages of embryonic development, the recombinant proteins of adiponectin and CTRP-9 will be prepared for the following studies using eukaryotic (Pichia pastoris) and prokaryotic (bacterial) expression systems. With in vivo and in vitro experiments, we will explore the regulation mechanism to glucose and lipid metabolism and their cell signal transduction mechanisms at molecular level in teleosts that mediated by adiponectin and its analogues CTRP-9. These studies will add new contents to fish nutriology and physiology, and provide a new theoretical foundation for thorough researches about the endocrine mechanism of energy metabolism balance in fish. Furthermore, this project will offer some guidance for the controlling of food intake more effectively, the improving of feed utilization, reducing of farming costs and water pollution, and promote the shifting of fish breeding from traditional industry to green industry.

鱼类被视为天生的"糖尿病患者"，对糖类的利用率低下，而糖类和脂肪作为成本较低廉的能量来源，可以替代部分作为能源消耗的蛋白质，具有降低饲料中蛋白质原料使用量的作用，从而减少鱼体对环境中可溶性氮的排放，减少对养殖水体的污染。因此，对鱼类糖脂代谢的特殊分子机制的研究显得尤为重要。本项目将开展鱼类新型细胞因子adiponectin及其类似物CTRP-9对鱼类糖脂代谢的调控机理研究，在对其基因进行分子克隆、序列分析和时空表达特性研究的基础上，制备adiponectin及CTRP-9的重组蛋白。通过在体和离体试验，从分子水平上探索鱼类adiponectin和CTRP-9对糖脂代谢的调控机理和信号传导机制，为鱼类营养学和生理学提供新的内容，为深入阐明鱼类的物质与能量代谢平衡的内分泌机理、更有效控制摄食、提高饵料利用率，降低养殖成本，减少对水质的污染，促进鱼类养殖产业向绿色产业发展提供新的理论基础。

脂联素(adiponectin)在哺乳动物中是主要由脂肪组织分泌的脂肪细胞因子，具有调节葡萄糖和脂质代谢等生物学功能。Adiponectin通过与其受体(AdipoRs)相结合，触发其下游各种信号通路进而介导上述的各种生物学功能。CTRP9作为脂联素同源性最高的同家族成员，具有相似的功能。我们的研究取得的主要成果：1) 首次获得了斜带石斑鱼CTRP9、AdipoR1和AdipoR2的全长cDNA序列。跨膜区预测结果显示，AdipoR1和AdipoR2均为七次跨膜受体。时空分布特性表明，斜带石斑鱼AdipoR1和AdipoR2在成鱼各种组织中均有表达而CTRP9在肾脏中表达量最高。在胚胎发育过程中，CTRP9、AdipoR1和AdipoR2的表达水平随着发育进程逐渐上升。2）成功制备了石斑鱼CTRP9的全长和CTRP9球状区的重组蛋白及多克隆抗体，这是目前鱼类中的首次报道，有助于对CTRP9在鱼类中功能的挖掘。3）通过禁食和再投喂实验表明adiponectin受体和CTRP9参与鱼体的摄食与能量调节。4）腹腔注射gCTRP9的结果表明gCTRP9参与斜带石斑鱼的糖脂代谢和摄食基因的调控。其中，gCTRP9会抑制肝脏和肌肉组织中糖异生和脂肪合成相关基因的表达，促进脂肪代谢相关基因的表达；在下丘脑中，促进抑摄食相关基因的表达而抑制促摄食相关基因的表达；在血清中，Glu、TG和LDL-C的水平降低，HDL-C的含量升高。5）原代肝脏细胞和原代脂肪细胞的实验结果表明adiponectin和CTRP9抑制鱼体的脂肪合成。在原代肝细胞实验中，gCTRP9和adiponectin都能抑制糖异生和脂肪合成相关基因的表达，促进脂肪代谢相关基因的表达，还降低原代肝细胞中葡萄糖和TG的含量；在原代脂肪细胞实验中，gCTRP9能够抑制脂肪合成相关基因的表达并能够降低原代脂肪细胞中TG的水平。6）首次证明斜带石斑鱼的AdipoR1和AdipoR2以C端位于细胞膜外N端则位于膜内的形式定位于细胞膜上并且可以介导CTRP9的信号通路。.综上所述，通过本研究首次得到斜带石斑鱼CTRP9和AdipoRs，制备重组CTRP9蛋白，证明CTRP9和AdipoRs参与斜带石斑鱼的糖脂代谢和摄食基因的调控。这些结果首次揭示了CTRP9在鱼类代谢中的功能。.