J. Lipid Res. Neurobiology of Lipids (ISSN1683-5506)
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A more recent version of this article appeared on March 1, 2005

Papers In Press, published online ahead of print November 16, 2004
J. Lipid Res., doi:10.1194/jlr.D400023-JLR200
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Submitted on September 7, 2004
Revised on November 5, 2004
Accepted on November 14, 2004

Real-time quantification of fatty acid uptake using a novel fluorescence assay

Jinfang Liao, Richard Sportsman, Jeff Harris, and Andreas Stahl

PAMF Research Institute, Stanford University, Palo Alto, CA 94301

Corresponding Author: astahl{at}stanford.edu

Uptake of non-esterified long-chain fatty acids (LCFA) into many cell-types and organs such as the liver, heart, intestine, and skeletal muscle, occurs primarily through a saturable, protein mediated mechanism. Membrane proteins that increase the uptake of LCFAs, such as FAT/CD36 and fatty acid transport proteins (FATPs), represent significant therapeutic targets for the treatment of metabolic disorders including type-2 diabetes. However, currently available methods for quantification of LCFA uptake neither allow for real-time measurements of uptake kinetics nor are they ideally suited for the development of LCFA uptake inhibitors in high throughput screens. To address both problems we have developed a LCFA uptake assay using a fluorescently-labeled fatty acid and a non-toxic cell impermeable quenching agent, that allows fatty acid transport to be measured in real time using fluorescent plate readers or standard fluorescent microscopy. With this assay, we could faithfully reproduce known differentiation-and hormone-induced changes in LCFA uptake by 3T3-L1 cells and determine LCFA uptake kinetics with previously unobtainable temporal resolution. Applications of this novel assay should facilitate new insights into the biology of fatty acid uptake and provide new means for obesity related drug discovery


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