Development and Validation of a High-Throughput Screening Assay for Human Long-Chain Fatty Acid Transport Proteins 4 and 5 (original) (raw)
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Real-time quantification of fatty acid uptake using a novel fluorescence assay
Journal of Lipid Research - J LIPID RES, 2004
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.
A live-cell high-throughput screening assay for identification of fatty acid uptake inhibitors
Analytical Biochemistry, 2005
We developed a live-cell high-throughput assay system using the bakerÕs 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, fat1Dfaa1D. 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.
Identification and characterization of small compound inhibitors of human FATP2
2010
Fatty acid transport proteins (FATPs) are bifunctional proteins, which transport long chain fatty acids into cells and activate very long chain fatty acids by esterification with coenzyme A. In an effort to understand the linkage between cellular fatty acid transport and the pathology associated with excessive accumulation of exogenous fatty acids, we targeted FATP-mediated fatty acid transport in a high throughput screen of more than 100,000 small diverse chemical compounds in yeast expressing human FATP2 (hsFATP2). Compounds were selected for their ability to depress the transport of the fluorescent long chain fatty acid analogue, C(1)-BODIPY-C(12). Among 234 hits identified in the primary screen, 5 compounds, each representative of a structural class, were further characterized in the human Caco-2 and HepG2 cell lines, each of which normally expresses FATP2, and in 3T3-L1 adipocytes, which do not. These compounds were effective in inhibiting uptake with IC(50)s in the low micromo...
Immunology, Endocrine & Metabolic Agents - Medicinal Chemistry (formerly Current Medicinal Chemistry - Immunology, Endocrine & Metabolic Agents), 2009
One principal process driving fatty acid transport is vectorial acylation, where fatty acids traverse the membrane concomitant with activation to CoA thioesters. Current evidence is consistent with the proposal that specific fatty acid transport (FATP) isoforms alone or in concert with specific long chain acyl CoA synthetase (Acsl) isoforms function to drive this energy-dependent process. Understanding the details of vectorial acylation is of particular importance as disturbances in lipid metabolism many times lead to elevated levels of circulating free fatty acids, which in turn increases fatty acid internalization and ectopic accumulation of triglycerides. This is associated with changes in fatty acid oxidation rates, accumulation of reactive oxygen species, the synthesis of ceramide and ER stress. The correlation between chronically elevated plasma free fatty acids and triglycerides with the development of obesity, insulin resistance and cardiovascular disease has led to the hypothesis that decreases in pancreatic insulin production, cardiac failure, arrhythmias, and hypertrophy are due to aberrant accumulation of lipids in these tissues. To this end, a detailed understanding of how fatty acids traverse the plasma membrane, become activated and trafficked into downstream metabolic pools and the precise roles provided by the different FATP and Acsl isoforms are especially important questions. We review our current understanding of vectorial acylation and the contributions by specific FATP and Acsl isoforms and the identification of small molecule inhibitors from high throughput screens that inhibit this process and thus provide new insights into the underlying mechanistic basis of this process.
The American journal of clinical nutrition, 2000
Classic fat balance studies detect fat malabsorption but do not discriminate between the potential causes of malabsorption, such as impaired intestinal lipolysis or reduced uptake of fatty acids. We aimed to validate a novel test for the specific, sensitive detection of impaired intestinal uptake of long-chain unesterified fatty acids in an appropriate rat model of fat malabsorption. The absorption and appearance in plasma of [(13)C]palmitic acid were determined in control rats and in rats with fat malabsorption due either to chronic bile deficiency (permanent bile diversion) or to oral administration of the lipase inhibitor orlistat (200 mg/kg diet). [(13)C]Palmitic acid results were compared with the percentage absorption of ingested dietary fat determined by fat balance. Between 1 and 6 h after intraduodenal administration, plasma [(13)C]palmitate concentrations in control rats were 4-10-fold higher than in bile-deficient rats (P < 0.05) but were not significantly different be...
Journal of Lipid Research
Membrane-bound proteins have been proposed to mediate the transport of long-chain fatty acid (LCFA) transport through the plasma membrane (PM). These proposals are based largely on reports that PM transport of LCFA can be blocked by a number of enzymes and purported inhibitors of LCFA transport. Here, using the ratiometric pH indicator (2’,7’-bis-(2-carboxyethyl)-5-and-6-)-carboxyfluorescein (BCECF) and acrylodated intestinal fatty acid-binding protein (ADIFAB)-based dual fluorescence assays, we investigated the effects of nine inhibitors of the putative FA transporter protein CD36 on the binding and transmembrane movement of LCFA. We particularly focused on sulfosuccidimidyl oleate (SSO), reported to be a competitive inhibitor of CD36-mediated LCFA transport. Using these assays in adipocytes and inhibitor-treated protein-free lipid vesicles, we demonstrate that rapid LCFA transport across model and biological membranes remains unchanged in the presence of these purported inhibitor...
Biochemical and Biophysical Research Communications, 2013
In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4, for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4hr. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The trafficking of exogenous C16:0 and C22:6 into PA was significant where there was 6.9-and 5.3-fold increased incorporation, respectively, over the control; C18:3 and C20:4 also trended to increase in the PA pool while there were no changes for C18:1 and C18:2. The trafficking of C18:3 into PC and PI trended higher and approached significance. In the case of C20:4, expression of FATP2 resulted in increases in all four classes of phospholipid, indicating little selectivity. In the case of C22:6, there were significant increases of this exogenous fatty acids
Journal of Biological Chemistry, 2011
The trafficking of fatty acids across the membrane and into downstream metabolic pathways requires their activation to CoA thioesters. Members of the fatty acid transport protein/ very long chain acyl-CoA synthetase (FATP/Acsvl) family are emerging as key players in the trafficking of exogenous fatty acids into the cell and in intracellular fatty acid homeostasis. We have expressed two naturally occurring splice variants of human FATP2 (Acsvl1) in yeast and 293T-REx cells and addressed their roles in fatty acid transport, activation, and intracellular trafficking. Although both forms (FATP2a (M r 70,000) and FATP2b (M r 65,000 and lacking exon3, which encodes part of the ATP binding site)) were functional in fatty acid import, only FATP2a had acyl-CoA synthetase activity, with an apparent preference toward very long chain fatty acids. To further address the roles of FATP2a or FATP2b in fatty acid uptake and activation, LC-MS/MS was used to separate and quantify different acyl-CoA species (C14 -C24) and to monitor the trafficking of different classes of exogenous fatty acids into intracellular acyl-CoA pools in 293T-REx cells expressing either isoform. The use of stable isotopically labeled fatty acids demonstrated FATP2a is involved in the uptake and activation of exogenous fatty acids, with a preference toward n-3 fatty acids (C18:3 and C22:6).
Membrane transport of long-chain fatty acids: evidence for a facilitated process
Journal of lipid research, 1998
In mammalian cells, membrane uptake of long-chain fatty acids is mediated by two separate components; a passive component that is a linear function of the concentration of free fatty acid in the extracellular medium and a saturable component that exhibits the characteristics of a protein-facilitated process. This review summarizes the body of work that has accumulated related to the mechanism of fatty acid transport. Evidence in support of a facilitated uptake process is presented with relation to the different cell types or membrane systems where it was collected. The evidence includes saturation kinetics, competition between different substrates, and sensitivity to a variety of inhibitors. Recent knowledge related to membrane proteins thought to be implicated in the uptake process is reviewed. Factors that may modulate uptake or alter the relative contribution of passive versus facilitated components are briefly discussed. These include the molar ratio of fatty acid to its physiol...