Lipid droplets and the compartmentalization of subcellular metabolism

脂滴（LDs）是负责脂质储存的细胞器，也是大多数细胞类型中最大的能量储备， 是非酒精性脂肪肝发展的特征和病因 （NAFLD）。此外，LD被认为在NAFLD与更多系统性合并症的偶联中发挥核心作用 如2型糖尿病和心血管疾病等。LD与许多细胞器相互作用， 特别是ER和线粒体，它们被认为是协调从头LD生物合成和脂肪酸（FA）的 转移/氧化。然而，我们实验室和其他人发表的研究结果质疑了这一点。 一个公认的教条，即脂肪酸从LD直接转移到线粒体是其氧化的主要途径 在禁食期间。使用涉及细胞器蛋白质组学，同位素示踪和许多 超分辨率显微镜的方法，我们首次表明，在肝脏，蛋白质组和 附着于LD的线粒体（peridrolet mitochondrial，PDM）的代谢支持脂质合成代谢途径， 而不附着于LD的线粒体（胞质线粒体，CM）适于增强FA氧化， OXPHOS。此外，我们的数据指出了一个重要的作用，神经相关膜（MAMs）， 与线粒体紧密相互作用的ER结构域，作为调节LD-线粒体的关键组分 互动和动态。总的来说，这些数据表明，与LD的相互作用深刻地影响了细胞器， 动态和功能。基于这些数据，本应用程序的目的是定义LD如何相互作用 影响脂质代谢和传感，以协调ER和线粒体功能， 病理生理条件。我们假设LD与ER和线粒体的相互作用是至关重要的 MAM脂质传感和线粒体功能的调节剂，其控制肝脏脂质和能量代谢。 为了验证这一假设，我们提出了以下三个具体目标：目标1 -全面定义LD- 线粒体相互作用及其对FA运输的影响;目的2 -确定 MAM亚群对线粒体生物能量学和脂质代谢的影响不同; 确定NAFLD如何影响NAFLD中的LD/MAM/线粒体动态。为了实现这些目标，我们将 采用各种先进的超分辨率成像方法，蛋白质组学和RNA测序， 同位素示踪和其他细胞生物学和生物化学方法在细胞，小鼠模型和人类肝脏 活组织检查在完成这些研究后，我们将期望我们将揭示新的机制， LD可以改变NAFLD病因学基础的细胞功能/功能障碍。我们预计这项工作将 开辟了细胞内信号动力学的新研究领域，这将推动治疗方法的发展 针对NAFLD和相关合并症。

Lipid droplets (LDs), the organelles responsible for lipid storage and the largest energy reserve in most cell types, are the defining characteristic and etiological factor in the development of non-alcoholic fatty liver disease (NAFLD). Moreover, LDs are recognized to play central roles in coupling NAFLD to more systemic comorbidities such as Type 2 Diabetes and cardiovascular disease among others. LDs interact with numerous organelles, especially ER and mitochondria, which are thought to coordinate de novo LD biogenesis and fatty acid (FA) transfer/oxidation, respectively. However, published work from our laboratory and others have questioned the established dogma that direct transfer of FAs from LDs to mitochondria is the primary route of their oxidation during fasting. Using a multifaceted approach involving organelle proteomics, isotope tracing, and numerous super resolution microscopy approaches, we show for the first time that in the liver, the proteomes and metabolism of mitochondria attached to LDs (peridroplet mitochondria, PDM) support lipid anabolic pathways, whereas mitochondria unattached to LDs (cytosolic mitochondria, CM) a geared for enhanced FA oxidation and OXPHOS. Moreover, our data point to an important role for mitochondrial-associated membranes (MAMs), an ER domain that tightly interacts with mitochondria, as a key component in regulating LD-mitochondria interactions and dynamics. Collectively, these data suggest that interactions with LDs profoundly affect organelle dynamics and function. Based upon these data, the objective of this application is to define how LD interactions affect lipid metabolism and sensing to coordinate ER and mitochondrial function under physiological and pathophysiological conditions. We hypothesize that interactions of LDs with ER and mitochondria are critical modulators of MAM lipid sensing and mitochondrial function that govern hepatic lipid and energy metabolism. To test this hypothesis, we propose the following three specific aims: Aim 1 - To comprehensively define LD- mitochondria interactions and their impact on FA trafficking; Aim 2 - To determine the mechanisms through which subpopulations of MAM differentially impact mitochondrial bioenergetics and lipid metabolism; and Aim 3 - To determine how NAFLD impacts LD/MAM/mitochondria dynamics in NAFLD. To complete these aims, we will employ a wide range of advanced super resolution imaging approaches, proteomics and RNA sequencing, isotope tracing, and other cell biology and biochemical approaches in cells, mouse models and human liver biopsies. Upon completion of these studies, we will expect that we will have revealed novel mechanisms through which LDs can alter cellular function/dysfunction that underlie NAFLD etiology. We anticipate that this work will open new areas of research into intracellular signaling dynamics, which will advance therapeutic approaches targeting NAFLD and related comorbidities.