热激蛋白hsp72促进草鱼糖代谢效率的分子机理研究

Carbohydrate is rich and inexpensive energy resource of feedstuff in nature, but the relative inability of fish to efficiently utilize dietary glucose, which restricts carbohydrate utilization in fishery feedstuff badly. It is in urgent need of proving the molecular mechanism of carbohydrate metabolism in fishes. Most studies focus on effection of nutriment additions on carbohydrate metabolism in a long time, and pay a little attention to environmental factors. Nowadays some researchers found that higher environmental temperature could enhance dietary carbohydrate utilization and growth performance though the molecular mechanism is still unknown. Proposer have studied heat shock proteins very deeply in fishes, which are regulated by temperature, and found much higher expression of hsp72 after heat shock in grass carp beforehand. Some studies have proved clearly that hsp72 can reduce glucose in blood through regulating insulin signal pathway. so proposer is planning to perform metabolism experiments in grass carp individuals and cell transfection experiments in grass carp cell lines to change hsp72 expression through over-expression and RNA interfere. In metabolism experiments, we will give grass carp samples heat shock weekly for ten consecutive weeks with normal temperature as control, which both have been divided into two groups with medium content carbohydrate and high content carbohydrate separately. Then we could conclude that molecular mechanism of hsp72 regulation carbohydrate metabolism in grass carp, and it will provide good theories for adjusting dietary carbohydrate content seasonly to economize dietary protein, which adapt to water temperature variety.At the same time, we will discuss possibility of developing methods of accurate-temperature-controlling to regulate carbohydrate metabolism of fishes, which has great significance for improving the ability of utilize carbohydrate and reducing feedstuff coefficient in fishes.

糖类是自然界丰富而廉价的饲料能量来源，但鱼类糖代谢效率低下严重制约了水产饲料中糖类的利用，其分子机理亟待探明。长期以来研究多集中在饲料中营养素添加对鱼类糖代谢的影响，对环境因素关注不多。近年有学者发现水温的升高能提高鱼类糖代谢效率和生长率，但其分子机理近乎空白。申请人对鱼类受温度影响的热激蛋白开展了较深入的研究，在前期研究中发现草鱼受热激后hsp72表达显著上调。而哺乳类II型糖尿病致病机理已清晰表明，hsp72能调节胰岛素信号通路，降低血糖含量。因此申请人拟通过草鱼个体在长期间断性热激下对不同糖含量饲料的代谢实验，以及草鱼肝细胞系超表达和干涉草鱼hsp72基因表达的转染实验，研究hsp72对草鱼糖代谢的调控作用及分子机理，为随四季水温变化而调整饲料糖含量以节约蛋白质，提供了良好的理论基础，同时也将探讨通过精确控温提高鱼类糖代谢能力的可行性，在提高糖利用率、降低饲料系数等方面具有重要意义。

鱼类的糖代谢能力相对于哺乳动物要弱得多，尤其是鱼类不能很好地利用饲料中的糖。在哺乳动物中，热休克蛋白Hsp72能够被热激诱导表达，并且阻止压力诱导的JNK （c-Jun amino N-terminal kinase）的激活，从而提高胰岛素的信号通路效率。然而，Hsp72在鱼类糖代谢中的功能还不清楚。我们利用草鱼幼鱼进行实验来研究热激在糖代谢方面的作用。本研究中，草鱼幼鱼分组饲喂高糖饲料和普通低糖饲料，而且一半的幼鱼要在8周的饲喂期经历几次暂时性热激处理。我们对每组幼鱼的生长状况、体成分及幼鱼肝脏中包括Hsp72在内的糖代谢相关基因的表达进行了分析。热激处理导致了草鱼幼鱼生长状况和体成分的改善。我们通过荧光定量PCR和western bolt分析发现Hsp72的表达在经过热激的高糖组（GH组）中显著升高（P < 0.05）。在Hsp72过表达的细胞中也有类似结果。但是JNK和p-JNK在本实验的热激中并没有被抑制。说明Hsp72对糖代谢关键基因的调节并不是和哺乳动物中一样通过JNK途径，这可能是了鱼类糖代谢能力弱于哺乳动物的原因之一。尽管如此，热激能够提高草鱼对糖的代谢能力毋庸置疑。因此，根据四季水温变化调整饲料糖含量确实能够节约蛋白质，而提高饲养温度对提高饲料中糖利用率、降低饲料系数等方面具有重要意义。