High-Throughput Screening of Australian Marine Organism Extracts for Bioactive Molecules Affecting the Cellular Storage of Neutral Lipids

Mammalian cells store excess fatty acids as neutral lipids in specialised organelles called lipid droplets (LDs). Using a simple cell-based assay and open-source software we established a high throughput screen for LD formation in A431 cells in order to identify small bioactive molecules affecting lipid storage. Screening an n-butanol extract library from Australian marine organisms we identified 114 extracts that produced either an increase or a decrease in LD formation in fatty acid-treated A431 cells with varying degrees of cytotoxicity. We selected for further analysis a non-cytotoxic extract derived from the genus Spongia (Heterofibria). Solvent partitioning, HPLC fractionation and spectroscopic analysis (NMR, MS) identified a family of related molecules within this extract with unique structural features, a subset of which reduced LD formation. We selected one of these molecules, heterofibrin A1, for more detailed cellular analysis. Inhibition of LD biogenesis by heterofibrin A1 was observed in both A431 cells and AML12 hepatocytes. The activity of heterofibrin A1 was dose dependent with 20 µM inhibiting LD formation and triglyceride accumulation by ∼50% in the presence of 50 µM oleic acid. Using a fluorescent fatty acid analogue we found that heterofibrin A1 significantly reduces the intracellular accumulation of fatty acids and results in the formation of distinct fatty acid metabolites in both cultured cells and in embryos of the zebrafish Danio rerio. In summary we have shown using readily accessible software and a relatively simple assay system that we can identify and isolate bioactive molecules from marine extracts, which affect the formation of LDs and the metabolism of fatty acids both in vitro and in vivo

Rab18 Dynamics in Adipocytes in Relation to Lipogenesis, Lipolysis and Obesity

Lipid droplets (LDs) are organelles that coordinate lipid storage and mobilization, both processes being especially important in cells specialized in managing fat, the adipocytes. Proteomic analyses of LDs have consistently identified the small GTPase Rab18 as a component of the LD coat. However, the specific contribution of Rab18 to adipocyte function remains to be elucidated. Herein, we have analyzed Rab18 expression, intracellular localization and function in relation to the metabolic status of adipocytes. We show that Rab18 production increases during adipogenic differentiation of 3T3-L1 cells. In addition, our data show that insulin induces, via phosphatidylinositol 3-kinase (PI3K), the recruitment of Rab18 to the surface of LDs. Furthermore, Rab18 overexpression increased basal lipogenesis and Rab18 silencing impaired the lipogenic response to insulin, thereby suggesting that this GTPase promotes fat accumulation in adipocytes. On the other hand, studies of the β-adrenergic receptor agonist isoproterenol confirmed and extended previous evidence for the participation of Rab18 in lipolysis. Together, our data support the view that Rab18 is a common mediator of lipolysis and lipogenesis and suggests that the endoplasmic reticulum (ER) is the link that enables Rab18 action on these two processes. Finally, we describe, for the first time, the presence of Rab18 in human adipose tissue, wherein the expression of this GTPase exhibits sex- and depot-specific differences and is correlated to obesity. Taken together, these findings indicate that Rab18 is involved in insulin-mediated lipogenesis, as well as in β-adrenergic-induced lipolysis, likely facilitating interaction of LDs with ER membranes and the exchange of lipids between these compartments. A role for Rab18 in the regulation of adipocyte biology under both normal and pathological conditions is proposed

Morphologically and Functionally Distinct Lipid Droplet Subpopulations

Lipid droplet (LD), a multi-functional organelle, is often found to associate with other cellular membranous structures and vary in size in a given cell, which may be related to their functional diversity. Here we established a method to separate LD subpopulations from isolated CHO K2 LDs into three different size categories. The subpopulation with smallest LDs was nearly free of ER and other membranous structures while those with larger LDs contained intact ER. These distinct subpopulations of LDs differed in their protein composition and ability to recruit proteins. This method was also applicable to LDs obtained from other sources, such as Huh7 cells, mouse liver and brown adipose tissue, et al. We developed an in vitro assay requiring only isolated LDs, Coenzyme A, and ATP to drive lipid synthesis. The LD subpopulation nearly depleted of ER was able to incorporate fatty acids into triacylglycerol and phospholipids. Together, our data demonstrate that LDs in a given cell are heterogeneous in size and function, and suggest that LDs are one of cellular lipid synthetic organelles