Adiponectin及其信号通路调控青鱼糖脂代谢的分子机制研究

The glucose and lipid metabolic disorder and adiponectin inhibition were seriously induced in carnivorous Black carp fed with high level of carbohydrate, which limited the effective use of carbohydrate from plant resources. The adiponectin fragment was obtained and identified from the transcriptome databases of live, brain and kidney in Black carp. The active adiponectin’s recombinant protein was successfully expressed in Pichia pastoris X-33 and then injected into Black carp fed with different content of dietary carbohydrate. And these untreated groups were used as negative control groups. Meanwhile, adiponectin’s dsRNA was synthesized and injected into Black carp fed with different content of dietary carbohydrate using the RNA interference technique. The different samples were collected and used to compare the differences between the growth indices, activities of enzymes involved in glucose and lipid metabolism, hormone levels, protein phosphorylations, effector protein’s translocations and relative genes’ expression profiles. And the test methods contain the Western blotting, enzyme assay, immunohistochemistry, proteomics and gene chip technique, etc. Specific adaptor proteins were screened and identified using the Yeast two-hybrid system. These key regulated kinases involved in the downstream of adiponectin signaling pathway were screened and determined using specific signal inhibitors through series of interference experiments. Our results will find the root causes of glucose and lipid metabolic disorder and adiponectin inhibition induced by higher content of dietary carbohydrate. And the effective and targeted nutrients will also be determined to activate adiponectin signaling pathway through the fish feeding tests. And our results will reveal the molecular mechanism of glucose and lipid metabolism regulated by adiponectin and its signaling pathway in Black carp after comprehensive analysis of these indexes at cell, protein and gene levels, respectively. In addition, our results will fill the area of fish molecular nutrition, and supply the scientific basis for the effective use of carbohydrate in Black carp and other carnivorous fish.

本项目针对肉食性青鱼摄食高糖饲料后出现典型糖脂代谢紊乱和Adiponectin（脂联素）抑制的科学问题，在已完成其肝、脑和肾脏转录组测序基础上，对其注射重组表达Adiponectin蛋白或进行RNA干扰，采用Western blot、酶活性测定、免疫组化、蛋白质组学、基因芯片检测等技术，研究Adiponectin对青鱼生长、糖脂代谢关键酶、激素、蛋白磷酸化、效应蛋白转位以及相关基因表达的影响。通过酵母双杂交鉴定Adiponectin受体的接头蛋白，利用特定信号抑制剂干扰试验确定Adiponectin通路中的下游关键调控激酶，从细胞、蛋白与基因水平上研究Adiponectin及其信号通路调控青鱼糖脂代谢的分子机制。结合养殖试验明确靶向Adiponectin信号通路的外源营养素种类，揭示Adiponectin及其信号通路对青鱼糖脂代谢的调控机制，为有效利用高糖及改善青鱼饲料配方提供科学依据。

本项目针对肉食性青鱼摄食高糖饲料后出现典型糖脂代谢紊乱和Adiponectin（脂联素）抑制的科学问题，选择典型的肉食性鱼类--青鱼为材料，完成青鱼Adipo 信号通路关键激酶和蛋白，包括APPL1、LBK1、AMPKs、PI3K、AKTs、PPARs、IκB、TORC2 以及Glut4 等基因cDNA 全长序列；并建立上述基因实时荧光定量PCR 检测方法。分析和鉴定适量糖（25%）和高糖（糊精）（40%）处理后青鱼差异表达的Adipo 信号通路关键激酶和蛋白基因。利用蛋白质组学分析筛选获得翻译量具有差异性的Adipo信号通路关键激酶和蛋白；完成AdipoR的重组表达载体构建，确定2个Adipo信号通路关键激酶或蛋白的相关结合或接头蛋白（AMPK、APPL），完成Adipo信号通路下游关键激酶的磷酸化水平以及IRS-1磷酸化位点（如Ser、Tyr等）检测和鉴定；筛选出5个磷酸化修饰差异的Adipo信号通路关键激酶（AMPK、PI3K、AKT、PPAR、mTOR）。完成不同糖含量处理过的青鱼肝脏和肌肉组织进行切片工作，获得核转录因子和促进葡萄糖吸收的Glut4在细胞内的定位；得到了核转录因子和Glut4在细胞内不同部位相对含量的检测。完善高糖饲料中加入2200IU/Kg维生素A或800IU/Kg维生素D等对核转录因子和Glut4在青鱼不同组织细胞内不同部位相对含量的检测结果。获得了高糖（糊精）（40%）条件下外源营养素（如0.9% DHA, 200mg/Kg 硫辛酸等）对青鱼幼鱼生长性能指标检测。结合生化检测结果筛选出种能够靶向激活Adipo信号通路中关键激酶和蛋白的外源营养素及其使用剂量，如亮氨酸(2.19%)、精氨酸(2.92%)、泛酸(20mg/Kg)、烟酸(40mg/Kg)等。综合分析提出Adipo信号通路对青鱼糖脂代谢的营养调控机制，并比较其与哺乳动物的异同，构建青鱼细胞内Adipo信号调控通路。为有效利用高糖代谢、避免糖脂代谢紊乱及优化青鱼饲料配方提供科学依据。