PITP缺失的肝脏细胞中脂肪酸代谢流的时空网络调控机制研究

Our recent study revealed that PITP deficient mice exhibited elevated energy expenditure, insulin sensitivity, and a lean phenotype. It was also observed that the PITP deficient hepatocytes expressed less neutral lipid storage and smaller lipid droplets (LDs). We suppose there are two possibilities namely, decreased lipid synthesis and increased lipolysis, which caused due to a defective multiple-organelles interaction network, for example LD-endoplasmic reticulum-mitochondria (LD-ER-Mito) network. In this project, we will constantly monitor the spatiotemporal fatty acid flow network in the PITP deficient mice and hepatocytes by using super-resolution imaging of molecular localization, organelle overlapping interactome method, and an imaging quantification of dynamic lipolysis rates for individual LDs. Furthermore, we will screen out unreported key proteins or phospholipids, which involved in the LD-ER-Mito interactions, by using biochemistry, LC-MS/MS proteomic spectroscopy, and proximity-tagging technologies. The study will elucidate the systematically regulated mechanism of fatty acid flow network connected by organelles and provide potential therapeutic targets to fatty liver disease and lipid metabolic disorders.

我们过去的研究发现PITP敲除小鼠表现出能量代谢加速，胰岛素敏感性增加以及瘦型表型；肝脏细胞呈现中性脂含量减少、脂滴减小。我们推测多细胞器互作网络的缺陷，如脂滴-内质网-线粒体的细胞器网络，导致了脂合成积累减少或脂降解氧化增加的两个可能原因。因此本研究拟用PITP敲除小鼠及其肝脏细胞，借助超分辨显微成像定位技术、多细胞器互作组学影像重迭法与单一脂滴动态降解的影像定量法，实现追踪脂肪酸代谢流的时空动态网络。进一步，我们将从细胞器互作的建立和维持中，结合一些常规的生化、质谱与临近分子标记技术来筛选出脂滴、内质网与线粒体之间未被报导的新型细胞器互作蛋白或磷脂分子。本研究将阐明脂肪酸代谢流在细胞器间的协同作用机制，以期为脂肪肝和脂代谢紊乱疾病提供潜在的分子治疗靶点。

本研究通过Pitpnc1全敲小鼠（Pitpnc1-/-）模型鉴定了在正常喂养或高脂喂养下，Pitpnc1-/-小鼠与野生型小鼠相比并没有显着的代谢特征变化，但是在急性4℃冷刺激下，Pitpnc1-/-小鼠表现出低温不耐受表型，生化检测和代谢组学研究鉴定出Pitpnc1基因缺失导致棕色脂肪组织（BAT）利用脂肪酸产热能力受损。进一步病理机制研究发现Pitpnc1蛋白定位在脂滴上并且往返于脂滴与线粒体之间，其磷脂结合功能位点是脂滴定位的关键结构域；Pitpnc1蛋白缺失会导致棕色脂肪细胞内线粒体的磷脂酰胆碱（PC）、鞘磷脂（SM）和神经酰胺（Ceramides）含量过度积累，导致了线粒体的磷脂调控紊乱。通过本项目研究，我们首次阐明了Pitpnc1蛋白通过磷脂转运、调控线粒体的磷脂稳态来维持线粒体产热功能，并表现出小鼠在低温下维持体温的生理重要性，此发现对于机体调控产热作用具有重要理论意义，且提供一个潜在靶点用于开发防治肥胖等代谢性疾病的新策略。