不同食性热带海洋鱼类葡萄糖耐量及外周糖代谢调控与利用机制研究

Fish, particularly carnivorous species, are generally thought to have limited ability to utilize carbohydrate, and considered to be glucose intolerant. This limits the amount of use of the carbohydrate, the cheapest source of energy, in fish feed. Until now, the precise explanation for such apparent glucose intolerance in fish is not fully understood. In this project, we will select three different feeding habits of tropical economic marine fishes, Rachycentron canadum (ferocious carnivorous), Trachinotus ovatus (mild carnivorous), and Oreochromis niloticus (omnivorous species) as research species. These species will be reared in indoor circulating system by different nutritional state (fed or fasted) and dietary carbohydrate levels (high, medium, low), respectively. Then, we will test the hypothesis that the ability of these species to clear glucose load is related to feeding habits of species and nutritional state or dietary carbohydrate level, and to the ability of peripheral tissues to transport and phosphorylate circulating glucose, as well as to the balance between hepatic glucose phosphorylation and glucose-6-phosphate hydrolysis. We will focus on the following: ①Ascertaining the glucose tolerance of these species by GTT; ②Cloning, mRNA and protein expression analyses and sutdies on enzymatic reaction kinetics of the peripheral tissue GLUTs, HK, GK, to elucidate the mechanism of peripheral glucose uptake and utilization; ③Cloning, mRNA and protein expression analyses and studies on enzymatic reaction kinetics of the hepatic GK/HK, G6Pase, to find out the coordination mechanism of hepatic glucose utilization and gluconeogenesis; and ④Cloning, mRNA and protein expression analyses of insulin receptor and the downstream signaling molecules (such as IRS, PI3K), to provide a theoretical basis for determining the insulin signaling transduction pathways. These results will be helpful to explain the mechanisms of peripheral glucose metabolic regulation and utilization. Enhancing dietary carbohydrate amount of use of species under aquaculture will decrease demand for fish meal/oil and reduce environmental pollution resulting from high nitrogen/phosphorus meals. All of these will have a positive impact upon the sustainable development of aquaculture. Meanwhile, having elucidated the mechanism of fish hyperglycemia can also provide natural models for the study of human diabetes.

鱼类,特别是肉食性鱼类糖利用能力低下，往往表现为葡萄糖耐受不良，这就限制了糖类作为廉价能源在饲料中的用量。鱼类葡萄糖耐受不良的机制还远未弄清。项目以军曹鱼、卵形鲳鲹、罗非鱼三种不同食性热带海洋经济鱼类为对象，研究在不同营养状态和营养水平下：①不同食性鱼类葡萄糖耐量；②外周组织GLUTs、HK、GK等功能蛋白（酶）基因与蛋白表达以及酶促反应动力学，以探明外周葡萄糖摄取与利用机制；③肝脏GK/HK、G6Pase等功能酶基因与蛋白表达以及酶促反应动力学，以弄清肝脏糖利用与糖异生的协调机制；④胰岛素受体下游关键信号分子（IRS、PI3K等）相关基因克隆与表达分析，为构建胰岛素信号转导通路提供理论基础。研究结果将有利于阐明鱼类外周糖代谢调控与利用机制，提高饲料碳水化合物利用能力，降低鱼粉鱼油的需求，减少氮磷污染，维持水产养殖业健康发展；同时，阐明鱼类高血糖症的机制还可为人类糖尿病提供天然的研究模型。

鱼类特别是肉食性鱼类糖利用能力低下，往往表现为“葡萄糖耐受不良”，这就限制了糖类作为廉价能源在饲料中的用量。鱼类葡萄糖耐受不良的机制仍远未弄清。本项目以军曹鱼、卵形鲳鲹、罗非鱼三种不同食性热带海洋经济鱼类为对象，研究结果发现不同食性鱼类葡萄糖耐受能力具有显著不同，其中以罗非鱼葡萄糖耐受能力最强，卵形鲳鲹次之，军曹鱼葡萄糖耐受能力最低。针对不同食性鱼类葡萄糖利用与糖异生作用的协调机制研究发现鱼类糖异生能力可能与其食性有关。三种不同食性鱼类PK和PEPCK基因同源性和进化关系与食性相关，食性相近的物种相似度更高，进化关系更近。凶猛肉食性军曹鱼糖酵解与糖异生关键功能酶基因在不同饲料糖水平、营养状态及急性血糖负荷下表达与转录组RNA-seq富集图谱相一致。在哺乳动物中被认为对GLUT4从胞内囊泡易位到细胞膜及内吞有重要调控作用的LL功能位点在鱼类GLUT4氨基酸序列中缺失，鱼类GLUT4在FQQI、LL、TELEY功能位点的差异可能导致鱼类GLUT4功能和结构及胞内转运和效率的差异，进而影响鱼类食性和糖代谢能力。饲料中糖对不同食性鱼类组织中GLUT4表达量调控存在不同，可能是由于GLUT4序列中基序功能位点的差异所致。饲料中糖含量的过高和过低都能影响IGF-I基因在肝脏中的正常表达。IGF-1可及时响应血糖的变化，高血糖会抑制罗非鱼胰岛素的分泌， IGF-1可能与胰岛素之间存在某种协同补偿的调控模式。IGF-1R的SUMO化对细胞周期进程和细胞增殖的控制相当重要。SUMO-IGF-1R可干扰LEF1并激活其靶基因，其中一个靶基因就是调节G1-S期的关键周期蛋白D1，因此，nIGF-1R / LEF1 /细胞周期素D1可能是SUMO-IGF-1R活化细胞增殖机制的一条重要信号通路。本项目研究已经按计划全面完成了预期的研究内容和目标。从分子、基因、蛋白质、细胞、组织、器官、个体、群体等多层次、全方位深入系统研究了热带海洋鱼类的糖代谢机制，并探讨了食性、营养状态、营养水平等对糖代谢的影响，初步阐明了不同食性海水养殖鱼类葡萄糖耐受能力。成功克隆并研究了一大批外周组织GLUTs、HK、GK等功能蛋白（酶）的基因及表达，初步探明了鱼类外周组织葡萄糖摄入与利用的机制以及肝脏糖利用与糖异生作用的协调机制。为阐明鱼类外周糖代谢调控与利用机制，提高鱼类对饲料糖类利用能力奠定了坚实的理论基础。