microRNA介导胰岛素敏感性调控青鱼糖异生的分子机制研究

The hyperglycemia was highly induced in fish after being fed with high level of carbohydrate, which limited the effective use of carbohydrate from plant resources. Considering the key scientific point, black carp (Mylopharyngodon piceus), one of the cultivated carnivorous fish, was chosen as the experimental animal in this study. After constructing the microRNA library, BLAST and sequence analysis methods were used to screen specific microRNA involving gluconeogenesis, and the microRNA target protein was forecasted using bioinformatics'software, such as miTarget and PicTar. Then the microRNA and its target proteins were identified by the method of Northern blot, western blot and real-time PCR using in vitro cultured cells from the live of black carp. The levels of blood glucose, liver glycogen and insulin were detected in the black carp fed with high content of carbohydrate using HPLC and commercial kits. In addition, the activities and expression levels of enzymes involving gluconeogenesis were detected via Western blot and real-time PCR, etc. Our results will find the root causes of useless of carbohydrate, and reveal the molecular mechanism of gluconeogenesis regulated by insulin sensitivity mediated by microRNA in fish after comprehensive analysis of these indexes. In addition, our results will fill the area of fish molecular nutrition, and supply the scientific basis for the effective use of carbohydrate in carnivorous fish.

本项目针对养殖鱼类在摄食高糖饲料后出现高血糖这一关键科学问题，选择典型的肉食性鱼类- - -青鱼为材料，通过构建青鱼肝脏microRNA文库，获得糖代谢特异性的microRNA分子，利用软件预测microRNA靶蛋白，采用离体细胞培养、Northern 杂交、实时定量PCR等技术筛选和鉴定调控胰岛素敏感特异性的microRNA及其靶蛋白。同时结合HPLC、Western blot及酶活性测定等手段，对高糖条件下青鱼糖异生关键酶活性及其mRNA表达、血糖、肝糖原和胰岛素水平等因子进行综合分析，研究microRNA介导胰岛素敏感性调控青鱼糖异生的分子机制，探明糖代谢缺陷的根本原因，揭示microRNA对青鱼糖异生的调控机制，填补microRNA在鱼类营养代谢研究领域的空白，并为有效利用糖及改善青鱼饲料配方提供科学依据。

本项目针对养殖鱼类在摄食高糖饲料后出现高血糖这一关键问题，选择典型的肉食性鱼类--青鱼（1.5 ± 0.05 g）为材料，投喂等氮等脂饲料（糖含量为0.06%、10.65%、19.43%、28.84%、37.91% 和 47.38%），研究青鱼的血液生理生化指标、microRNA分子种类及其靶蛋白表达模式，糖代谢酶、相关激素和胰岛素信号通路相关蛋白表达模式。利用Solexa测序技术成功构建青鱼肝脏microRNA 文库，获得755个Unique microRNA分子。同时利用软件预测出microRNA 靶蛋白，如adipoR1a、adipoR1b、G6Pase、FBPase、Prx等。 结果显示，随着饲料中糖水平的增加，青鱼增重呈现先增加后降低的趋势，并在糖含量为28.84%达到最大。糖含量显著上调肝脏中GK, HK, PFK, PK和G6PD活性。G6Pase和FBPase呈现先下调后上调的趋势并在19.43%时趋向稳定，而PEPCK酶活性在不同糖含量饲料组间无显著差异。青鱼肝脏中G6Pase、FBPase和PEPCK mRNA表达量在糖含量为10.65%、19.43%、28.84%和37.91%时均呈现出先增加后稳定的趋势。而胰岛素信号通路中的相关靶蛋白，如adipoR1a、adipoR1b、G6Pase、FBPase, Prx 和INSR等 mRNA表达量也呈现出先增加后降低的趋势。基因芯片分析结果显示，在37.91%（过量糖）饲料组中，115个miRNAs的表达量显著上调，99个miRNAs的表达量显著下调，317个miRNAs的表达量无显著变化。在上调的miRNAs中，let-7家族miRNAs占11.3%，而这说明let-7家族在调控青鱼糖代谢中发挥重要作用。而在涉及糖异生相关的miRNAs中，miR-25、miR-363-3p_R+2、miR-122、miR-200b_R+2的表达量无显著变化。此外，miRNA靶向抗氧化酶也会影响胰岛素的敏感性调控作用。上述研究结果从miRNAs角度探索了高糖抑制糖异生酶的原因，初步揭示microRNA 对青鱼糖异生的调控机制。