MIB通路影响罗非鱼盐度适应的糖营养调控机制研究

Nutritional regulation of osmoregulation is a direct and effective strategy to improve the salinity adaptability of fish. The target compound or pathway will decide the effect of regulation. The pathways related with osmotic response and osmolyte play crucial roles in the physiology mechanisms used by fish to cope with salinity stress, while the regulation based on these pathways or osmolyte is an unstudied area. Myo-inositol biosynthesis pathway (MIB) is an important response pathway during osmoregulation, by which the glucose 6-phosphate (G-6-P) could be converted to myo-inositol with the catalysis of inositol monophosphatase 1 (IMPA1) and myo-inositol phosphate synthase (MIPS). Myo-inositol can effectively mitigate the adverse effects of osmotic stress, which is an important osmolyte. There was no research about the nutritional regulation of MIB till now. In the present project, we choose Oreochromis mossambicus, which is a representative euryhaline fish, to study the nutritional regulation of osmoregulation by myo-inositol biosynthesis pathway. We will focus on the study of the mechanisms and the development of effective strategy to improve the salinity adaptivity of fish. In details, we will firstly confirm the interaction of MIB and glycometabolism during osmoregulation using the tilapia brain cell line (OmB). In order to clarify the mechanisms of nutritional regulation by carbohydrate, we will use the isotopic glucose and glycometabolism inhibitor to investigate the source of G-6-P and the glucose flow during osmoregulation, and then analyze the critical points of regulation. Based on the above research, we can develop the effective strategy to improve the osmoregulation of fish. Through these work, we would clarify a pathway related with carbohydrate nutrition regulation which would help improve the salinity adaptivity of fish. These investigations are very important for us to further understand the mechanisms of osmoregulation. Meanwhile, they can also help increase our awareness of carbohydrate in aquaculture. This is of great significance in both academic and industrial applications.

在鱼类盐度适应的调控过程中，渗透响应通路或效应物是渗透效应机制的核心部分，而以此为靶目标的营养调控是当前一个全新的研究方向。糖-肌醇转化途径（MIB）是一条重要的渗透响应通路，可将糖代谢中产物葡萄糖-6-磷酸（G6P）大量转化为渗透效应物肌醇，从而有效缓解外界盐度变化带来的不良影响，但目前以MIB为靶目标的营养调控研究尚未见有深入的报道。本项目拟以莫桑比克罗非鱼（广盐性鱼类典型代表）为研究对象，以G6P为切入点，探究鱼类在盐度适应过程中，MIB的糖营养调控机制和策略。首先通过siRNA基因沉默和分子克隆研究手段，在罗非鱼脑细胞模型上确认MIB与糖代谢的关联性；然后通过活体腹腔注射同位素标记物，结合糖代谢关键通路抑制实验，研究糖营养对罗非鱼盐度适应的调控机制，发现MIB营养调控的关键点；基于以上研究基础，再通过养殖实验筛选关键调控因子，开发基于糖营养对鱼类盐度适应的调控策略。

围绕探明MIB与糖代谢之间的相互作用和协同作用、了解MIB上游调控通路和调控关键因子、开发提高鱼类盐度适应能力的营养策略等目标，我们采用生物化学、分子生物学、细胞生物学、蛋白质组学、代谢组学、营养学等方法和技术手段，揭示了在急性渗透压胁迫情况下，罗非鱼会大量动用储存在体内的糖类物质，为渗透压调节提供能量，同时肌醇合成的增加可帮助细胞维持渗透压，有效缓解有机离子毒害作用；长期盐度胁迫情况下，除了维持细胞渗透压外，肌醇可能还会改变糖代谢的模式，同时MIB通路合成的肌醇，后续也可通过磷脂酰肌醇信号转导途径调控胰岛素代谢，继而影响糖代谢。初步的发现随后也在细胞实验中得以验证，结果表明，在高渗条件下，细胞生长减缓，细胞周期停滞；细胞通过渗透调节作用在胞内累积肌醇，应对外界的高渗胁迫；同时磷脂酰肌醇信号通路增强，磷脂酰肌醇含量增加。鉴于MIB对糖代谢模式的改变，我们尝试通过调整饲料中脂肪和糖类的使用，来提高罗非鱼的盐度适应能力。结果发现，在长期盐度胁迫下，饲料中添加肌醇能提升高糖或高脂膳食下的增重率、成活率、粗蛋白含量、肥满度等，缓解肝脏和腹腔中脂肪积聚，提高抗氧化性能。与此同时，肌醇能够调解机体激素水平、肌醇合成通路、糖代谢模式、离子转运，也能够改变鳃中脂肪酸的组成、增加磷脂含量、提高细胞膜的流动性、促进鳃中离子转运能力，继而提高尼罗罗非鱼长期耐盐性。本项目通过以上工作的开展，阐明了两种能够有效提高鱼类渗透压调节能力的营养策略，这些工作对于揭示广盐性鱼类渗透压调节机制、提高鱼类盐度适应能力都具有重要的参考价值。也有助于增加人们对水产动物糖营养的认知。本项目所开发的营养调控策略也通过与饲料企业和交流和合作，得以改良优化，已准备在多家饲料企业的咸淡水养殖或淡化养殖配方设计中加以应用。