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

Abstract

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 differentiationand 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. -Liao, J., R. Sportsman, J. Harris, and A. Stahl. Real-time quantification of fatty acid uptake using a novel fluorescence assay. J. Lipid Res. 2005. 46: 597-602.Supplementary key words long-chain fatty acids • fatty acid uptake assay • quencher dyes Uptake of long-chain fatty acids (LCFAs) plays an important role in the absorption of dietary lipids as well as the delivery of metabolic energy to a variety of tissues. Besides their function as substrates for ␤ -oxidation, fatty acids also contribute to membrane synthesis, protein modifications, immune responses, and activation of protein kinases and nuclear hormone receptors (1, 2). Recent findings have also directly implicated increased intracellular levels of LCFAs in the obesity-associated insulin desensitization of skeletal muscle and liver (1, 3).LCFA uptake in adipocytes is mainly facilitated by membrane proteins, particularly members of the fatty acid transport protein (FATPs/SLC27) family. Mammalian genomes have been shown to contain six FATP genes (4). The identification of this fatty acid transporter family and other fatty acid uptake-enhancing proteins such as CD36 has allowed a better understanding of the mechanisms and regulation of LCFA transport on a cellular level, yielding insight into the control of energy homeostasis and its dysregulation in diseases such as diabetes and obesity. In addition, these cell surface proteins represent new targets for the inhibition of LCFA uptake.To gain a better understanding of the molecular dynamics of fatty acid uptake and the development of small molecular inhibitors of this process, robust and physiologically relevant measurements of LCFA uptake kinetics are required. Here, we report the development of a fluorescence assay bas...

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