Multiple Cellular Transport and Binding Processes of Unesterified Docosahexaenoic Acid in Outer Blood–Retinal Barrier Retinal Pigment Epithelial Cells

Abstract

Docosahexaenoic acid (DHA, 22 : 6) is an essential omega-3 long-chain polyunsaturated fatty acid that plays a pivotal role in vision. The purpose of this study was to clarify the cellular uptake and binding processes of free and protein-bound unesterified DHA in retinal pigment epithelial cell (RPE) line ARPE-19 as a model of the human outer blood-retinal barrier and isolated porcine RPE cell fractions. Uptake of free [C]DHA by ARPE-19 cells was saturable with a Michaelis-Menten constant of 283 µM, and was significantly inhibited by eicosapentaenoic acid, arachidonic acid, and linoleic acid, but not by oleic acid. Further, the uptakes of [C]DHA associated with retinol-binding protein ([C]DHA-RBP), [C]DHA associated with low-density lipoprotein ([C]DHA-LDL) and [C]DHA associated with bovine serum albumin ([C]DHA-BSA) in ARPE-19 cells increased time-dependently at 37°C, and were significantly reduced at 4°C, suggesting the involvement of energy-dependent transport processes. [C]DHA-LDL uptake by ARPE-19 cells was significantly inhibited by excess unlabeled LDL, but not by an inhibitor of scavenger receptor B type I. Fatty acid transport protein (FATP) 2 and 4 mRNAs were expressed in ARPE-19 cells, and [C]DHA uptake was observed in FATP2- and FATP4-expressing Xenopus oocytes. Photo-reactive crosslinking and mass spectrometry analyses identified 65-kDa retinal pigment epithelium-specific protein (RPE65) as a DHA-binding protein in porcine RPE cell membrane fractions. Thus, RPE cells possess multiple cellular transport/binding processes for unesterified DHA, involving at least partly FATP2, FATP4, LDL, RBP, and RPE65.

Efficient Enrichment of Retinal DHA with Dietary Lysophosphatidylcholine-DHA: Potential Application for Retinopathies

“…Unlike the brain which acquires DHA predominantly through the Mfsd2a pathway, retina appears to acquire DHA through multiple pathways, since a deficiency of Mfs2a results in only a 45% reduction in retinal DHA, and a 57% reduction in VLCFA [ 47 ]. Thus, the studies by Bazan [ 18 ] showed the importance of Adiponectin receptor for maintaining the DHA levels of retina, whereas the role of FA-binding proteins and lipoprotein receptors have been proposed by others [ 3 , 19 ]. These pathways may account for the small increase in retinal DHA by dietary free DHA and TAG.…”

“…This is because they are all absorbed predominantly in the form of triacylglycerol (TAG) [ 15 , 16 ], whereas enrichment of brain and retina requires a phospholipid form of DHA [ 17 ]. Although previous studies have shown that the uptake of DHA by retina involves lipoprotein receptors [ 3 ], adiponectin receptor [ 18 ], and multiple fatty acid transporters [ 19 ], more recent studies suggest that the predominant pathway for DHA uptake by retina may be through the Mfsd2a (major facilitator superfamily domain-containing protein 2a) pathway [ 20 ]. Since the DHA from the currently used supplements is absorbed mostly as TAG, whereas the Mfsd2a pathway requires a lysophospholipid form of DHA [ 17 ], it is possible that retinal DHA was not appreciably enriched by these supplements.…”

Transcriptome Analyses of lncRNAs in A2E-Stressed Retinal Epithelial Cells Unveil Advanced Links between Metabolic Impairments Related to Oxidative Stress and Retinitis Pigmentosa

“…RPE expresses factors required for lipoprotein intake, cholesterol and lipoprotein synthesis and secretion, as well as reverse cholesterol transport (RCT) [94]. Phagocytosis and degradation of the POSs are the principal lipid source for the RPE [95], as well as processing and recycling back to the photoreceptors of vitamin A and of DHA [96,97]. Moreover, excess cholesterol is loaded on HDLs by ABC transporters and, then, removed, or it is also secreted in VLDL-like lipoproteins basolaterally into Bruch's membrane where it forms drusen [98].…”

Lipid-Binding Proteins in Brain Health and Disease

“…For example, DHA, an essential omega-3 long-chain polyunsaturated fatty acid, is known to have anti-inflammatory properties on glial cells and influences memory and cognitive functions (28). After its transport in blood via the lipocalin retinol-binding protein 4 (RBP4), LDL lipoparticles, or serum albumin (29), it crosses the cell membrane and travels across the brain endothelial cell cytoplasm via FABP5 (30). Lipocalins and FABPs manage lipids not only across the BBB but also between cells in the nervous system (glia-to-glia or glia-to-neuron).…”