AMPK/ACC通路调控草鱼糖代谢的研究

Excessive intake of dietary carbohydrates in grass carp (Ctenopharyngodon idellus) usually results in excessive fat accumulation in liver. This phenomenon is closely related to the carbohydrate metabolism disorder of grass carp. In mammals, carbohydrate metabolism is regulated by the AMP-activated protein kinase (AMPK) / acetyl-CoA carboxylase (ACC) pathway, and ACC can be used as active target for drug discovery of metabolic syndrome. However, whether or not fish carbohydrate metabolism is regulated by the AMPK/ACC pathway is still unkown. Therefore, the purpose of this study is to investigate the roles of AMPK/ACC pathway in carbohydrate metabolism and determine whether ACC could be used as active target for its metabolic syndromes of grass carp. Since ACC1 has already been reported, we will clone ACC2 gene, analyze the transcription and translation patterns of ACC2 in different tissues, and observe sub-cellular localization in grass carp. Furthermore, based on the experiments of feeding, growth and isolated hepatocytes culture, the changes of key target protein expression levels of AMPK/ACC pathway (in liver, brain and heart), key genes mRNA expression of glucose and lipid metabolism, metabolic composition and structure in liver, serum physiological and biochemical parameter, and glycogen and fat content in different tissues, are investigated when ACC is activated by high carbohydrate and inhibited by CP-640186. This study will not only establish foundation to reveal the molecular carbohydrate metabolism mechanisms, but also provide some theoretical insight into preventing the metabolic syndrome of grass carp. Furthermore, based on the experiments of feeding and isolated hepatocytes culture, the changes of key target protein expression levels of AMPK/ACC pathway (in liver, brain and heart), key genes mRNA expression of glucose and lipid metabolism, metabolic composition and structure in liver, serum physiological and biochemical parameter, and glycogen and fat content in different tissues, are investigated when ACC is activated by high carbohydrate and inhibited by CP-640186. Through this study, it will not only establish foundation to reveal the molecular carbohydrate metabolism mechanisms, but also provide some theoretical insight into prevent the metabolic syndrome of grass carp.

草鱼摄入过多糖会导致其肝脏脂肪过度蓄积，这与草鱼糖代谢发生障碍密切相关。在哺乳动物上发现糖转化为脂肪受腺苷酸激活蛋白激酶（AMPK）/乙酰CoA羧化酶(ACC)通路调控，且ACC可作为治疗糖代谢障碍类疾病的活性位点，在鱼类上是否亦如此目前尚不完全清楚。因此本项目拟克隆草鱼ACC2基因（ACC1已有报道），并研究其转录、翻译模式和细胞定位；通过摄食生长和离体肝细胞培养实验，深入研究ACC被高糖激活和CP-640186抑制后，该通路关键靶蛋白表达水平（肝脏、心脏、脑），肝脏糖和脂肪代谢关键基因的mRNA表达水平、代谢物成分和组织结构，组织糖原和脂肪含量及血液生理生化指标等的变化，初步探明AMPK/ACC通路调控草鱼糖代谢的机制，并分析ACC作为防治草鱼糖代谢障碍类疾病活性位点的可行性。本研究将为进一步揭示草鱼糖代谢的分子机制奠定基础，为草鱼糖代谢障碍类疾病防治提供理论指导。

在现代水产养殖中， 为了满足养殖品种快速生长的需求， 通常会采取在较短时间内给予养殖对象以过高能量的营养策略。蛋白质、碳水化合物和脂质这三种大营养素是水产动物主要的能量来源。草鱼是我国淡水养殖产量第一的鱼类，其肝脏代谢障碍（俗称 “脂肪肝”）发生普遍，这制约了草鱼朝优质健康方向发展。摄入过多糖是导致草鱼发生脂肪肝的最主要因素之一，但其代谢机制尚不清楚。在哺乳动物上发现糖和脂肪的代谢与转化受腺苷酸激活蛋白激酶（AMPK）/乙酰辅酶A羧化酶(ACC)通路调控，且该通路可作为治疗脂肪肝的活性位点，在鱼类上未见相关报道。因此，本项目结合已有的研究结果，克隆了草鱼ACC2基因，其cDNA全长8535个碱基，ORF共7146个碱基，编码2382个氨基酸，没有信号肽。ACC2 mRNA主要在脑和肝脏中表达，蛋白主要在肝脏和肌肉中表达。AICAR慢性激活AMPK后，可降低草鱼血糖含量，抑制ACC和SREBP-1活性，降低肝脏和肌肉脂肪生成，AMPK可作为草鱼糖脂代谢综合征的活性靶点。细胞培养液中添加40nM的CP-640186，在葡萄糖浓度为0-40nM时，显著降低了肝细胞中的葡萄糖和甘油三酯含量；饲料含过高的脂肪（13.5%）和蛋白质（41.8%）并不影响草鱼生长性能和肝脏脂肪含量，而饲料中含过高的糖（48.65%）显著降低了草鱼的生长性能，导致草鱼肝脏脂肪过度沉积，即形成脂肪肝。高脂肪和高糖饲料通过上调磷酸戊糖途径和脂肪合成的关键酶表达增加肌肉脂肪沉积，而高能饲料通过上调糖异生酶表达水平增加肌肉糖原含量。高脂饲料激活肝脏AMPK/ACC通路，高蛋白饲料激活肝脏TOR/ SREBP -1通路，高能量饲料主要影响肝脏糖脂代谢，而非蛋白质代谢，饲料脂肪水平的影响主要与脂肪分解代谢酶活性和mRNA表达有关。草鱼通过促进肝脏糖酵解和磷酸戊糖途径，抑制糖异生途径来调控饲料糖摄入。通过激活AMPK，抑制ACC,可以显著提高草鱼对饲料糖的利用。本项目从饲料大营养素（蛋白、脂肪、碳水化合物）水平、AMPK/ACC通路、ACC抑制等角度系统研究草鱼脂肪沉积机制，为草鱼脂肪肝的防治提供理论指导。