蛋白激酶AMPK介导高脂抑制团头鲂肝脏脂肪酸β-氧化的分子机制研究

Fatty liver of cultured fish often results in the poor growth. It also can cause immune suppression, make fish vulnerable to pathogen infection then lead to disease outbreak, which seriously affect the healthy development of aquaculture. Therefore, studies for mechanism and prevention of fatty liver are urgent. In the previous study, we have studied fatty liver induced by high-fat diets in blunt snout bream (Megalobrama amblycephala), which showed that high-fat could suppress hepatic β-oxidation via decreasing CPT I activity and mitochondrial content. However, the molecular mechanism is still unknown. Revealing the mechanism of high-fat suppressing hepatic β-oxidation is helpful to illustrate the formation mechanism of fatty liver. Thus, the present study will give deeper understanding of regulation of β-oxidation, taking blunt snout bream as research object. Firstly, we will study the regulation to CPT I activity and mitochondrial content of AMPK, a protein kinase playing important roles in both lipid metabolism and mitochondrial function, to confirm the regulation of AMPK to β-oxidation in vitro. Then, we will study effects of high-fat on the pathway of AMPK regulating β-oxidation in vivo and in vitro. The results will reveal the mechanism of high-fat suppressing hepatic β-oxidation, illustrate the formation mechanism of fatty liver, and provide targets for nutritional regulation to fatty liver.

养殖鱼类的脂肪肝不仅会导致鱼体生长缓慢，还会降低鱼体免疫力使其易受病原感染而爆发疾病，严重影响养殖业的健康发展，因此开展鱼类脂肪肝发生机制及预防其发生的研究尤为迫切。申请人前期通过研究高脂诱导的团头鲂脂肪肝，发现高脂饲料可通过降低CPT I活性和线粒体含量而抑制肝脏脂肪酸β-氧化速率，但具体机制尚不清楚。揭示高脂降低β-氧化速率的分子机制有助于阐明鱼类脂肪肝的发生机制。为此，本项目在前期研究基础上，以团头鲂为研究对象，采用肝细胞离体培养的方法，研究对脂肪代谢和线粒体功能均有重要调控作用的蛋白激酶--AMPK对CPT I活性和线粒体生成的调控作用及途径，从而明确AMPK对脂肪酸β-氧化的调控作用；在此基础上，分别通过离体和活体实验，研究高脂条件下，AMPK调控肝脏脂肪酸β-氧化通路的变化，揭示高脂降低脂肪酸β-氧化速率的分子机制，阐明鱼类脂肪肝的发生机制，为鱼类脂肪肝的营养调控提供靶点。

AMPK对调控脂代谢和线粒体功能有重要作用。在本项目的资助下，申请人专注研究AMPK对团头鲂脂肪酸β-氧化的调控作用。首先，构建AMPK激活与抑制模型，在此基础上研究AMPK对CPT I活性与线粒体生成的调控作用；进而研究了高脂饲料对AMPK活性及其调控通路的影响。经实施，取得如下主要研究成果：1）采用胰蛋白酶消化法分离肝细胞，通过密度梯度离心和percoll分离法进行纯化，用15% FBS的L-15培养基于25℃恒温培养，得到状态稳定的原代肝细胞；4mM二甲双胍显著激活肝细胞AMPK，400 mM油酸显著抑制AMPK活性；2）激活AMPK能够提高CPT I活性及β-氧化速率，反之亦然；且AMPK/PPARα/CPT I与AMPK/ACC2/CPT I是调控CPT I活性的重要通路；3）激活AMPK提高线粒体数量，反之亦然；发现团头鲂PGC-1α第176位为脯氨酸，丧失磷酸化位点，无法受AMPK直接调控；4）团头鲂摄入高脂饲料会导致肝脏AMPK活性降低、调控CPT I活性与线粒体生成的基因下调，最终导致肝脏脂肪沉积；高脂饲料中添加羟基酪醇可激活AMPK通路而缓解脂肪沉积。综上所述，本项目完成了计划任务，但AMPK调控线粒体生成的分子机制还需进一步研究；实施过程，项目资助发表SCI论文3篇，待发表2篇；培养硕士生1名（已毕业）。