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 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.
Highlights
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
We report the development of a fluorescence assay based on the extracellular quenching of a fluorescent fatty acid analog that can be quantified in real time by fluorescence plate readers or by standard fluorescence microscopybased systems without a washing step
The Quencher-Based Technology (QBT) Fatty Acid Uptake Assay Kit consisted of a proprietary formulation of the quenching agent Q-Red.1 (Molecular Devices) and 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-S-indacene3-dodecanoic acid (BODIPY-FA; Molecular Probes, Inc.)
Summary
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. 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. 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. This new assay offers both the benefit of detailed real-time observations and measurements of cellular fatty acid uptake and a simple mix-and-read format for high-throughput screens
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