Adiponectin及其信号通路调控青鱼糖脂代谢的分子机制研究

批准号:
31672669
项目类别:
面上项目
资助金额:
63.0 万元
负责人:
吴成龙
依托单位:
学科分类:
C1905.水产动物营养与饲料学
结题年份:
2020
批准年份:
2016
项目状态:
已结题
项目参与者:
叶金云、曹访、于岚、邵仙萍、赵建华、李莉、高俊娥
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中文摘要
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。
英文摘要
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,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。
期刊论文列表
专著列表
科研奖励列表
会议论文列表
专利列表
Molecular cloning, characterization and expression modulation of four ferritins in black carp Mylopharyngodon piceus in response to Aeromonas hydrophila challenge
青鱼青鱼中四种铁蛋白响应嗜水气单胞菌挑战的分子克隆、表征和表达调节
DOI:10.1016/j.aqrep.2019.100238
发表时间:2020-03
期刊:Aquaculture Reports
影响因子:3.7
作者:Chen Shujian;Wu Chenglong;Xie Yuanyuan;Wu Yuancai;Dai Shurong;Wang Xiaowen;Li Ronghua;Ye Jinyun
通讯作者:Ye Jinyun
DOI:10.3969/j.issn.1006-267x.2018.07.019
发表时间:2018-07
期刊:动物营养学报
影响因子:--
作者:陈炼;吴成龙;张易祥;邵仙萍;陈书健;王春琳;叶金云
通讯作者:叶金云
DOI:doi.org/10.1007/s10695-020-00876-8
发表时间:2020
期刊:Fish Physiology and Biochemistry
影响因子:--
作者:Chenglong Wu;Bin Lu;Yunlong Wang;Chuanmin Jin;Yixiang Zhang;Jinyun Ye
通讯作者:Jinyun Ye
Identification, characterization, and expression analysis of adiponectin receptors in black carp Mylopharyngodon piceus in response to dietary carbohydrate
青鱼青鱼脂联素受体对膳食碳水化合物反应的鉴定、表征和表达分析
DOI:10.1007/s10695-018-0501-5
发表时间:2018-04
期刊:Fish Physiology and Biochemistry
影响因子:2.9
作者:Chenglong Wu;Jun’e Gao;Lian Chen;Xianping Shao;Jinyun Ye
通讯作者:Jinyun Ye
Effects of dietary vitamin D3 on growth performance, antioxidant capacities and innate immune responses in juvenile black carp Mylopharyngodon piceus
膳食维生素 D3 对青鱼幼鱼生长性能、抗氧化能力和先天免疫反应的影响
DOI:10.1007/s10695-020-00876-8
发表时间:2020-09
期刊:Fish Physiology and Biochemistry
影响因子:2.9
作者:Chenglong Wu;Bin Lu;Yunlong Wang;Chuanmin Jin;Yixiang Zhang;Jinyun Ye
通讯作者:Jinyun Ye
microRNA介导胰岛素敏感性调控青鱼糖异生的分子机制研究
- 批准号:31202008
- 项目类别:青年科学基金项目
- 资助金额:24.0万元
- 批准年份:2012
- 负责人:吴成龙
- 依托单位:
国内基金
海外基金
