adiponectin/AdipoR对草鱼肝脏糖代谢的调控及机制研究

The pancreas in most teleosts is underdeveloped, and it has not formed as a dense endocrine organ. Furthermore, the regulation of blood glucose homeostasis by insulin/glucagon antagonism seems to be imperfect in fish. Adiponectin is one of the key adipokines in mammals, and it has significant effects of promoting glucose uptake and attenuating insulin resistance. This study takes adiponectin in grass carp (Ctenopharyngodon idella) as key point, based on the Pichia pastoris expression system, recombinant proteins (adiponectin A and B) will be produced and used for functional experiments. The major studies are as follows: ①The effects of adiponectin on glucose metabolism in the liver of grass carp by detecting the mRNA levels and activities of key enzymes involved in glucose metabolism, the glucose transporter (Glut2) mRNA expression, the levels of glucose metabolic intermediates, hepatic glycogen and blood glucose, and glucose uptake. ②Whether adiponectin activates AMPK or PI3K/Akt signal pathway to mediate the regulation of glucose metabolism in the liver of grass carp. ③The roles of AdipoR1-A, AdipoR1-B and AdipoR2 in adiponectin-stimulated regulation of glucose metabolism in the liver of grass carp. This study is helpful to replenish the regulatory network of glycometabolism in fish, and has important significance to understand the low utilization of carbohydrates by fish.

大多数硬骨鱼类胰脏不发达，没有形成致密型的内分泌器官，且鱼类调控血糖稳态的拮抗机制不够完善。脂联素（adiponectin）是哺乳类的关键脂肪细胞因子，具有促进葡萄糖摄取、改善胰岛素抵抗等生理效应。本研究拟通过毕赤酵母表达系统制备草鱼两种脂联素的重组蛋白（adiponectin A和adiponectin B），研究：①脂联素对草鱼肝脏糖代谢的调控作用，包括糖代谢关键酶活性及其mRNA表达、葡萄糖转运蛋白（Glut 2）mRNA表达、葡萄糖代谢中间产物、葡萄糖摄取、肝糖原以及血糖水平；②阐明脂联素对草鱼肝脏糖代谢调控的信号通路（AMPK、PI3K/Akt）；③通过RNAi和基因过表达技术，研究草鱼脂联素受体（AdipoR1-A、AdipoR1-B和AdipoR2）在介导脂联素调控草鱼肝脏糖代谢中的作用。本研究有助于完善鱼类糖代谢的内分泌调控网络，有利于进一步揭示鱼类糖利用率低的原因。

脂联素（adiponectin）在哺乳动物中是调节能量稳态的关键脂肪细胞因子，具有促进葡萄糖摄取、改善胰岛素抵抗等生理效应。然而，脂联素在鱼类糖代谢中调控作用尚未阐明。本项目以草鱼为研究对象，首先获得了两种脂联素基因以及其受体基因的cDNA序列，并对其组织分布特性以及在葡萄糖耐量、胰岛素和胰高血糖素刺激以及免疫应激过程中的表达特性进行了研究；其次，我们制备并获得了重组草鱼脂联素蛋白（adiponectin A和adiponectin B），通过在体和离体实验，研究了这两种脂联素对草鱼肝脏糖和脂质代谢以及AMPK、PI3K/Akt信号通路的影响。结果结果如下：（1）草鱼adiponectin A和adiponectin B分别编码258和290个氨基酸，均包括22个Gly-X-Y（X和Y为任意氨基酸）重复；（2）adiponectin A主要表达于心脏和肌肉，adiponectin B则在脂肪组织和肌肉中表达水平高，脂联素受体广泛表达于鱼体各个部位；（3）在糖耐量初期，脂联素基因的表达水平下降，糖耐量后期表达水平升高，胰岛素和免疫应激能够显著上调脂联素受体的表达水平；（4）用两种脂联素进行短期处理，均能降低血糖，提高肝糖原含量，这可能是通过促进糖酵解、糖原合成、糖生脂等途径以及AMPK和PI3K/Akt信号通路来实现的。项目的研究成果有助于完善鱼类糖代谢的内分泌调控网络，有利于进一步揭示鱼类糖利用率低的原因。