Development and Validation of a High-Throughput Screening Assay for Human Long-Chain Fatty Acid Transport Proteins 4 and 5

Dietary long-chain fatty acid (LCFA) uptake across cell membranes is mediated principally by fatty acid transport proteins (FATPs). Six subtypes of this transporter are differentially expressed throughout the human and rodent body. To facilitate drugs discovery against FATP subtypes, the authors used mammalian cell lines stably expressing the recombinant human FATP4 and 5 and developed a high-throughput screening (HTS) assay using a 96-well fluorometric imaging plate reader (FLIPR). LCFA uptake signal-to-background ratios were between 3- and 5-fold. Two 4-aryl-dihydropyrimidinones, j3 and j5, produced inhibition of FATP4 with a half-maximal inhibitory concentration (IC50) of 0.21 and 0.63 µM, respectively, and displayed approximately 100-fold selectivity over FATP5. The US Drug Collection library was screened against the FATP5. A hit rate of around 0.4% was observed with a Z′ factor of 0.6 ± 0.2. Two confirmed hits are bile acids, chenodiol and ursodiol with an IC50 of 2.4 and 0.22 µM, respectively. To increase throughput, a single time point measurement in a 384-well format was developed using the Analyst HT, and the results are comparable with the 96-well format. In conclusion, the FATP4 and 5 cell-based fluorescence assays are suitable for a primary drug screen, whereas differentiated cell lines are useful for a secondary drug screen.

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PPAR/RXR Regulation of Fatty Acid Metabolism and Fatty Acid -Hydroxylase (CYP4) Isozymes: Implications for Prevention of Lipotoxicity in Fatty Liver Disease

PPAR Research ◽

10.1155/2009/952734 ◽

2009 ◽

Vol 2009 ◽

pp. 1-20 ◽

Cited By ~ 63

Author(s):

James P. Hardwick ◽

Douglas Osei-Hyiaman ◽

Homer Wiland ◽

Mohamed A. Abdelmegeed ◽

Byoung-Joon Song

Keyword(s):

Gene Expression ◽

Fatty Acids ◽

Fatty Acid ◽

Fatty Liver ◽

Transport Proteins ◽

Chain Fatty Acid ◽

Fatty Acid Transport ◽

Acid Transport ◽

Fatty Acid Hydroxylase ◽

Fatty Acid Transport Proteins

Fatty liver disease is a common lipid metabolism disorder influenced by the combination of individual genetic makeup, drug exposure, and life-style choices that are frequently associated with metabolic syndrome, which encompasses obesity, dyslipidemia, hypertension, hypertriglyceridemia, and insulin resistant diabetes. Common to obesity related dyslipidemia is the excessive storage of hepatic fatty acids (steatosis), due to a decrease in mitochondria -oxidation with an increase in both peroxisomal -oxidation, and microsomal -oxidation of fatty acids through peroxisome proliferator activated receptors (PPARs). How steatosis increases PPAR activated gene expression of fatty acid transport proteins, peroxisomal and mitochondrial fatty acid -oxidation and -oxidation of fatty acids genes regardless of whether dietary fatty acids are polyunsaturated (PUFA), monounsaturated (MUFA), or saturated (SFA) may be determined by the interplay of PPARs and HNF4 with the fatty acid transport proteins L-FABP and ACBP. In hepatic steatosis and steatohepatitis, the -oxidation cytochrome P450CYP4Agene expression is increased even with reduced hepatic levels of PPAR. Although numerous studies have suggested the role ethanol-inducibleCYP2E1in contributing to increased oxidative stress,Cyp2e1-null mice still develop steatohepatitis with a dramatic increase inCYP4Agene expression. This strongly implies thatCYP4Afatty acid -hydroxylase P450s may play an important role in the development of steatohepatitis. In this review and tutorial, we briefly describe how fatty acids are partitioned by fatty acid transport proteins to either anabolic or catabolic pathways regulated by PPARs, and we explore how medium-chain fatty acid (MCFA)CYP4Aand long-chain fatty acid (LCFA)CYP4F-hydroxylase genes are regulated in fatty liver. We finally propose a hypothesis that increasedCYP4Aexpression with a decrease inCYP4Fgenes may promote the progression of steatosis to steatohepatitis.

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New concepts of cellular fatty acid uptake: role of fatty acid transport proteins and of caveolae

Proceedings of The Nutrition Society ◽

10.1079/pns2004341 ◽

2004 ◽

Vol 63 (2) ◽

pp. 259-262 ◽

Cited By ~ 47

Author(s):

Jürgen Pohl ◽

Axel Ring ◽

Thomas Herrmann ◽

Wolfgang Stremmel

Keyword(s):

Fatty Acids ◽

Fatty Acid ◽

Lipid Rafts ◽

Transport Proteins ◽

Fatty Acid Transport ◽

Long Chain Fatty Acids ◽

Acid Transport ◽

Fatty Acid Transport Proteins ◽

Lcfa Uptake ◽

Long Chain

Efficient uptake and channelling of long-chain fatty acids (LCFA) are critical cell functions. Evidence is emerging that proteins are important mediators of LCFA-trafficking into cells and various proteins have been suggested to be involved in this process. Amongst these proteins is a family of membrane-associated proteins termed fatty acid transport proteins (FATP). So far six members of this family, designated FATP 1–6, have been characterized. FATP 1, 2 and 6 show a highly-conserved AMP-binding region that participates in the activation of very-long-chain fatty acids (VLCFA) to form their acyl-CoA derivatives. The mechanisms by which FATP mediate LCFA uptake are not well understood, but several studies provide evidence that uptake of LCFA across cellular membranes is closely linked to acyl-CoA synthetase activity. It is proposed that FATP indirectly enhance LCFA uptake by activating VLCFA to their CoA esters, which are required to maintain the typical structure of lipid rafts in cellular membranes. Recent work has shown that the structural integrity of lipid rafts is essential for cellular LCFA uptake. This effect might be exerted by proteins, e.g. caveolin-1 and FAT/CD36, that use lipid rafts as platforms and bind or transport LCFA. The proposed molecular mechanisms await further experimental investigation.

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