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Journal of Lipid Research, Vol. 46, 597-602, March 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

Methods

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

Jinfang Liao^*, Richard Sportsman^*, Jeff Harris^* and Andreas Stahl^1,,

^* Molecular Devices Corporation, Sunnyvale, CA 94089
Palo Alto Medical Foundation Research Institute, Palo Alto, CA 94301
Division of Gastroenterology/Hepatology, Stanford University School of Medicine, Stanford, CA 94305

¹ To whom correspondence should be addressed. e-mail: astahl{at}stanford.edu

Uptake of nonesterified long-chain fatty acids (LCFAs) into many cell types and organs such as 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, represent significant therapeutic targets for the treatment of metabolic disorders, including type 2 diabetes. However, currently available methods for the quantification of LCFA uptake neither allow for real-time measurements of uptake kinetics nor are ideally suited for the development of LCFA uptake inhibitors in high-throughput screens. To address both problems, we developed a LCFA uptake assay using a fluorescently labeled fatty acid and a nontoxic cell-impermeable quenching agent that allows fatty acid transport to be measured in real time using fluorescence plate readers or standard fluorescence microscopy. With this assay, we faithfully reproduced known differentiation- and hormone-induced changes in LCFA uptake by 3T3-L1 cells and determined 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.

Supplementary key words long-chain fatty acids • fatty acid uptake assay • quencher dyes

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