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

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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.