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 l...

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Bibliographic Details
Published in:PloS one 2011-08, Vol.6 (8), p.e22868-e22868
Main Authors: Rae, James, Fontaine, Frank, Salim, Angela A, Lo, Harriet P, Capon, Robert J, Parton, Robert G, Martin, Sally
Format: Article
Language:English
Subjects:
1-Butanol - chemistry
Accumulation
Alkynes - chemistry
Alkynes - isolation & purification
Alkynes - pharmacology
Animals
Aquatic Organisms - chemistry
Australia
Biocompatibility
Biological activity
Biological Products - chemistry
Biological Products - isolation & purification
Biological Products - pharmacology
Biology
Butanol
Cell Line
Cell Line, Tumor
Cells (Biology)
Chemical Fractionation - methods
Chemistry
Cholesterol
Chromatography, High Pressure Liquid
Computer programs
Cytoplasmic Granules - drug effects
Cytoplasmic Granules - metabolism
Cytotoxicity
Diabetes
Embryo, Nonmammalian - drug effects
Embryo, Nonmammalian - metabolism
Embryos
Endoplasmic reticulum
Fatty acids
Fatty Acids - metabolism
Fatty Acids, Unsaturated - chemistry
Fatty Acids, Unsaturated - isolation & purification
Fatty Acids, Unsaturated - pharmacology
Feature extraction
Fish
Fluorescence
Fractionation
Hepatocytes
Hepatocytes - drug effects
Hepatocytes - metabolism
High-performance liquid chromatography
High-throughput screening
Homeostasis
Humans
Lipid Metabolism - drug effects
Lipids
Lipids - analysis
Liquid chromatography
Liver
Mammalian cells
Marine organisms
Mass Spectrometry
Medical screening
Medicine
Metabolism
Metabolites
Microscopy, Fluorescence
Monounsaturated fatty acids
Morphology
NMR
Nuclear magnetic resonance
Obesity
Oleic acid
Oleic Acid - pharmacology
Open source software
Organelles
Physiological aspects
Porifera - chemistry
Proteins
Santalaceae
Source code
Spectroscopic analysis
Storage
Toxicity
Trends
Triglycerides
Triglycerides - metabolism
Zebrafish
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Summary: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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0022868