内质网应激参与DHA促进草鱼脂肪细胞脂解过程的机制研究

DHA could decrease triacylglycerol accumulation in fish. Endoplasmic reticulum stress (ER stress) induced-UPR signaling provides an essential mechanism to sense dietary fatty acids and regulate lipid metabolism. Our laboratory found that the expression levels of lipolytic enzymes and ER stress markers were significiantly increased in DHA-induced grass carp adipocytes lipolysis. Previous research has already determined the lipolysis response to ER stress in rat adipocytes. Hence, we hypothesize that lipolysis induced by DHA may be mediated by ER stress in grass carp adipocytes. In this project, grass carp, the unique carp species in China, would be applied as the experimental animal, the molecular biological and cell biological techniques, including cell culture, lentiviral-mediated overexpression and RNA interference, co-immunoprecipitation, as well as feeding experiment would be adopted to investigate whether DHA regulate lipolysis through “ER stress- cAMP/PKA” pathway to adjust the interaction among perilipin1, CGI-58, HSL and ATGL, aiming to construct the molecular regulatory network of transcriptional mechanism that regulates lipolysis by n-3 HUFA in fish. The results will give a new theoretical foundation and research approach for the study of lipid metabolism in freshwater fish.

DHA具有促进脂解而降低鱼类体脂蓄积的作用。内质网是体内营养素的敏感感受器，其应激可激活UPR从而调控脂代谢。前期研究中，申请人发现DHA促进草鱼脂肪细胞脂解的同时，上调关键脂酶及UPR标志基因的表达。结合内质网应激促进鼠脂肪细胞脂解的报道，申请人提出内质网应激可能参与了DHA促草鱼脂肪细胞脂解过程的假说。本项目拟以草鱼为试验材料，采用离体原代脂肪细胞培养、激动剂与抑制剂处理、慢病毒介导的过表达与RNA干扰、免疫共沉淀等细胞分子生物学技术，结合在体饲喂，探究内质网应激在DHA促草鱼脂肪细胞脂解过程中的作用及机制，着重研究DHA诱导内质网应激情况下，cAMP/PKA信号通路参与调控脂酶ATGL、HSL及调控因子CGI58、Perilipin共同介导脂肪细胞脂解的机理。研究结果将有助于阐明DHA降低草鱼脂质蓄积的分子调控机制，为淡水鱼脂质代谢调控原理及技术研究提供参考资料和研究思路。

在水产养殖生产中，由于摄入营养成分不均衡或者摄入能量过多，养殖鱼类会出现鱼体脂质过度积累等问题。DHA有降低鱼类体脂蓄积的作用，但相关机制尚不明确。内质网是营养素的敏感感受器，其应激状态下发生的未折叠蛋白反应可影响细胞脂代谢稳态。本项目以草鱼为试验材料,克隆获得6个草鱼脂质代谢基因（perilipin, PKA, IRE1,CGI58, GSK3β和ATG4）的全长CDS，在此基础上结合在体饲喂与离体细胞处理开展研究，旨在探究内质网应激（ER stress）在DHA促草鱼脂肪细胞脂解过程中的作用及机制，着重研究了DHA诱导ER stress情况下，cAMP/PKA信号通路参与调控脂酶ATGL、HSL及调控因子CGI58、Perilipin共同介导脂肪细胞脂解的机理。结果表明：① 日粮添加1% DHA饲喂草鱼8周，可有效降低腹腔脂肪指数；用200μM DHA处理分化末期脂肪细胞48h，脂解和凋亡关键基因表达升高，细胞甘油释放量增加，且 ER stress被激活。与离体条件下单独添加TM 激活ER stress，增强脂肪细胞脂解的效果一致，表明ER stress 可能参与了DHA诱导的脂肪细胞脂解和凋亡；②离体添加4-PBA（ER stress抑制剂），可有效缓解DHA诱导的脂解和凋亡，说明ER stress介导了DHA诱导的脂肪细胞的脂解和凋亡；③靶向cAMP/PKA信号轴，添加H89（PKA的抑制剂），DHA和TM诱导的cAMP的含量和PKA的酶活性下降，且PKA信号通路关键因子表达量下降，水解酶ATGL和HSL的酶活性下降。研究表明，DHA通过诱导ER stress激活cAMP/PKA信号通路，提高ATGL和HSL的转录水平和酶活性促进草鱼脂肪细胞脂解。研究结果将有助于阐明DHA降低草鱼脂质蓄积的分子调控机制，为淡水鱼脂质代谢调控原理及技术研究提供参考资料和研究思路。