Fatty acid transport protein expression and fatty acid transport across Human Brain Microvessel Endothelial Cells (HBMEC) and the regulation of Cardiolipin synthesis by Fatty Acid Transport Protein-1 (FATP-1)

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Mitchell, Ryan
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The blood-brain barrier (BBB) formed by the brain capillary endothelial cells provides a protective barrier between the systemic blood and the extracellular environment of the central nervous system. Since most fatty acids in the brain enter from the blood, we examined the mechanism of permeability of various fatty acids across primary human brain microvessel endothelial cells (HBMEC). Cardiolipin (CL), a major mitochondrial phospholipid involved in energy metabolism in mammalian mitochondria, and fatty acid transport protein-1 (FATP-1) may regulate the intracellular level of fatty acyl-Coenzyme A’s. Since fatty acids are required for oxidative phosphorylation via mitochondrial oxidation, we also examined the effect of altering FATP-1 levels on CL biosynthesis. The permeability of radiolabeled fatty acids was determined using confluent cells grown on Transwell® inserts following inhibition of various fatty acid transporters. The passage of [1-14C]oleate across confluent HBMEC monolayers was significantly enhanced when fatty acid free albumin was present in the basolateral media. Knockdown of FATP-1, FATP-4, fatty acid translocase/CD36, or fatty acid binding protein 5 significantly decreased permeability of a number of radiolabeled fatty acids across the HBMEC monolayer from either apical as well as basolateral sides. The findings indicate that transport of some fatty acids across HBMEC is, in part, a transcellular process mediated by fatty acid transport proteins. Next, HEK 293 cells were used as a model to determine the effect of altering FATP-1 levels on CL. HEK-293 mock- and FATP-1 siRNA-transfected cells or mock and FATP-1 expressing cells were incubated for 24 h with 0.1 mM oleate bound to albumin (1:1 molar ratio) then incubated for 24 h with 0.1 mM [1,3-3H]glycerol and radioactivity incorporated into CL determined. FATP-1 siRNA-transfected cells exhibited reduced FATP-1 mRNA and increased incorporation of [1,3-3H]glycerol into CL (2-fold, p<0.05) compared to controls indicating elevation in de novo CL biosynthesis. In contrast, expression of FATP-1 resulted a reduction in incorporation of [1,3-3H]glycerol into CL (65%, p<0.05) indicating reduced CL synthesis. In addition, in vitro cytidine-5’-diphosphate-1,2-diacyl-sn-glycerol synthetase (CDS) activity was reduced by exogenous addition of oleoyl-Coenzyme A. The data indicate that CL de novo biosynthesis may be regulated by FATP-1 through CDS-2 expression in HEK 293 cells.
Pharmacology, Lipid, Blood-Brain-Barrier, Cardiolipin
Mitchell RW, Edmundson CL, Miller DW, Hatch GM. (2009) On the mechanism of oleate transport across human brain microvessel endothelial cells. J Neurochem. 110(3):1049-57
Mitchell RW, Hatch GM. (2009) Regulation of cardiolipin biosynthesis by fatty acid transport protein-1 IN HEK 293 cells. Biochim Biophys Acta. 1788(10):2015-21