Fatty Acid-Binding Protein 5 Facilitates the Blood鈥揃rain Barrier Transport of Docosahexaenoic Acid

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• 作者：Yijun Pan ; Martin J. Scanlon ; Yuji Owada ; Yui Yamamoto ; Christopher J. H. Porter ; Joseph A. Nicolazzo
• 刊名：Molecular Pharmaceutics
• 出版年：2015
• 出版时间：December 7, 2015
• 年：2015
• 卷：12
• 期：12
• 页码：4375-4385
• 全文大小：466K
• ISSN：1543-8392

文摘

The brain has a limited ability to synthesize the essential polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) from its omega-3 fatty acid precursors. Therefore, to maintain brain concentrations of this PUFA at physiological levels, plasma-derived DHA must be transported across the blood鈥揵rain barrier (BBB). While DHA is able to partition into the luminal membrane of brain endothelial cells, its low aqueous solubility likely limits its cytosolic transfer to the abluminal membrane, necessitating the requirement of an intracellular carrier protein to facilitate trafficking of this PUFA across the BBB. As the intracellular carrier protein fatty acid-binding protein 5 (FABP5) is expressed at the human BBB, the current study assessed the putative role of FABP5 in the brain endothelial cell uptake and BBB transport of DHA in vitro and in vivo, respectively. hFAPB5 was recombinantly expressed and purified from Escherichia coli C41(DE3) cells and the binding affinity of DHA to hFABP5 assessed using isothermal titration calorimetry. The impact of FABP5 siRNA on uptake of ¹⁴C-DHA into immortalized human brain microvascular endothelial (hCMEC/D3) cells was assessed. An in situ transcardiac perfusion method was optimized in C57BL/6 mice and subsequently used to compare the BBB influx rate (K_in) of ¹⁴C-DHA between FABP5-deficient (FABP5^{鈥?鈥?/sup>) and wild-type (FABP5+/+) C57BL/6 mice. DHA bound to hFABP5 with an equilibrium dissociation constant of 155 卤 8 nM (mean 卤 SEM). FABP5 siRNA transfection decreased hCMEC/D3 mRNA and protein expression of FABP5 by 53.2 卤 5.5% and 44.8 卤 13.7%, respectively, which was associated with a 14.1 卤 2.7% reduction in 14C-DHA cellular uptake. By using optimized conditions for the in situ transcardiac perfusion (a 1 min preperfusion (10 mL/min) followed by perfusion of 14C-DHA (1 min)), the K_in of 14C-DHA was 0.04 卤 0.01 mL/g/s. Relative to FABP5+/+ mice, the K_in of 14C-DHA decreased 36.7 卤 12.4% in FABP5鈥?鈥?/sup> mice. This study demonstrates that FABP5 binds to DHA and is involved in the brain endothelial cell uptake and subsequent BBB transport of DHA, confirming the importance of this cytoplasmic carrier protein in the CNS exposure of this PUFA essential for neuronal function.}