A live-cell high-throughput screening assay for identification of fatty acid uptake inhibitors

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

We developed a live-cell high-throughput assay system using the baker's yeast Saccharomyces cerevisiae to screen for chemical compounds that will inhibit fatty acid uptake. The target for the inhibitors is a mammalian fatty acid transport protein (mmFATP2), which is involved in the fatty acid transport and activation pathway. The mmFATP2 was expressed in a S. cerevisiae mutant strain deficient in Fat1p-dependent fatty acid uptake and reduced in long-chain fatty acid activation, fat1Δfaa1Δ. To detect fatty acid import, a fluorescent fatty acid analog, 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s- indacene-3-dodecanoic acid (C 1-BODIPY-C 12), was incubated with cells expressing FATP2 in a 96-well plate. The mmFATP2-dependent C 1-BODIPY-C 12 uptake was monitored by measuring intracellular C 1-BODIPY-C 12 fluorescence on a microtiter plate reader, whereas extracellular fluorescence was quenched by a cell viability dye, trypan blue. Using this high-throughput screening method, we demonstrate that the uptake of the fluorescent fatty acid ligand was effectively competed by the natural fatty acid oleate. Inhibition of uptake was also demonstrated to occur when cells were pretreated with sodium azide or Triacsin C. This yeast live-cell-based assay is rapid to execute, inexpensive to implement, and has adequate sensitivity for high-throughput screening. The assay basis and limitations are discussed.

Original languageEnglish (US)
Pages (from-to)11-19
Number of pages9
JournalAnalytical Biochemistry
Volume336
Issue number1
DOIs
StatePublished - Jan 1 2005

Fingerprint

High-Throughput Screening Assays
Assays
Screening
Fatty Acids
Throughput
Yeast
Saccharomyces cerevisiae
lauric acid
Fatty Acid Transport Proteins
Fluorescence
Chemical activation
Cells
Sodium Azide
Chemical compounds
Trypan Blue
Oleic Acid
Cell Survival
Coloring Agents
Yeasts
Ligands

Keywords

  • C -BODIPY-C
  • Fatty acid transport
  • High-throughput screening
  • Lipotoxicity
  • Obesity
  • Saccharomyces cerevisiae
  • Trypan blue

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

A live-cell high-throughput screening assay for identification of fatty acid uptake inhibitors. / Li, Hong; Black, Paul N.; Dirusso, Concetta C.

In: Analytical Biochemistry, Vol. 336, No. 1, 01.01.2005, p. 11-19.

Research output: Contribution to journalArticle

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