The function of the fatty acyl-CoA synthetase ACSL3 in the dynamic metabolism of lipid droplets

脂滴是细胞内储存细胞器，与脂质稳态和代谢疾病的病理生理学高度相关。它们动态地适应营养供应，相应地生长和萎缩。关于所涉及的机制，以及需要哪些分子参与者，还有许多悬而未决的问题。脂酰辅酶A合成酶家族的酶是合成脂滴核心中的中性脂质和周围膜的磷脂所必需的。当中性脂质合成被诱导时，ACSL 3从内质网易位到出现的脂滴，表明在脂滴的形成和生长中起重要作用。初步数据表明，缺乏ACSL 3的细胞在中性脂质合成和脂滴形成方面受到极大损害，该提议现在要求ACSL 3在哺乳动物细胞中脂质的动态储存中的功能。总体假设是：脂滴定位的ACSL 3显著有助于中性脂质的有效储存和动员。这可以通过提供脂肪酸特别是脂滴的生长，并通过调节生物合成和脂解之间的脂肪酸-脂肪酸循环来实现。主要的实验策略是分析ACSL 3缺失的细胞。阐明ACSL 3是否通过表面磷脂和核心甘油三酯的局部生物合成促进脂滴生长。将评估脂滴的生物合成能力及其脂质组成。体内补充脂肪酸和溶血脂质将显示ACSL 3.2的生理相关性。揭示ACSL 3在调节甘油三酯-脂肪酸循环中的作用。将通过使用代谢标记和脂质滴的显微镜定量来比较ACSL 3-RNAi和对照细胞之间的基础和激素刺激的脂解。评估ACSL 3对脂滴的推定独特功能。其他酰基辅酶A合成酶和一种不能转位到脂滴的ACSL 3变体将用于ACSL 3缺失细胞的拯救实验。研究ACSL 3的功能将产生对脂滴生物学的新见解，这将提高我们对代谢疾病分子细节的理解。

Lipid droplets are intracellular storage organelles highly relevant for lipid homeostasis and the pathophysiology of metabolic diseases. They adapt dynamically to nutrient supply, growing and shrinking accordingly. There are many open questions about the mechanisms involved, and which molecular players are required. Enzymes of the fatty acyl-CoA synthetase family are essential for the synthesis of the neutral lipids in the lipid droplet core, and for the phospholipids of the surrounding membrane.Recently, we investigated the association of the acyl-CoA synthetase ACSL3 with lipid droplet membranes. ACSL3 translocated from the endoplasmic reticulum to emerging lipid droplets when neutral lipid synthesis was induced, suggesting an important role in the formation and growth of lipid droplets. Preliminary data indicate that cells lacking ACSL3 are greatly impaired in neutral lipid synthesis and the formation of lipid droplets.This proposal now asks for the function of ACSL3 in the dynamic storage of lipids in mammalian cells. The overall hypothesis is: Lipid droplet localized ACSL3 contributes significantly to the efficient storage and mobilization of neutral lipids. This may be achieved by providing fatty acids specifically for the growth of lipid droplets, and by regulating the triglyceride-fatty acid cycle between biosynthesis and lipolysis. The main experimental strategy is to analyze cells depleted for ACSL3.The specific aims are:1. to elucidate if ACSL3 contributes to the growth of lipid droplets by the local biosynthesis of surface phospholipids and core triglycerides. Lipid droplets will be assessed for their biosynthetic capacities and their lipid composition. Fatty acid and lysolipid supplementation in vivo will show the physiological relevance of ACSL3.2. to reveal a role of ACSL3 in the regulation of the triglyceride-fatty acid cycle. Basal and hormone stimulated lipolysis will be compared between ACSL3-RNAi and control cells by using metabolic labeling and microscopy quantification of lipid droplets.3. to assess the putative unique function of ACSL3 on lipid droplets. Other acyl-CoA synthetases and an ACSL3 variant which cannot translocate to lipid droplets will be used for rescue experiments of the ACSL3 depleted cells.Investigating the function of ACSL3 will yield novel insights into lipid droplet biology, which will improve our understanding of metabolic diseases in molecular detail.