Prostaglandins, Leukotrienes and Essential Fatty Acids

Prostaglandins, Leukotrienes and Essential Fatty Acids ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Prostaglandins, Leukotrienes and EssentialFatty Acids

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The enigmatic membrane fatty acid transporter CD36: New insightsinto fatty acid binding and their effects on uptake of oxidized LDL$

Anthony G. Jay a,b, James A. Hamilton a,n

a Department of Physiology & Biophysics, Boston University School of Medicine, Boston, MA 02118, United Statesb Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, United States

a r t i c l e i n f o

Article history:Received 30 March 2016Accepted 11 May 2016

Keywords:Fatty acidSurface plasmon resonanceLipid uptakeTransport CD36Low-density lipoprotein

x.doi.org/10.1016/j.plefa.2016.05.00578/& 2016 Elsevier Ltd. All rights reserved.

viations: oxLDL, oxidized LDL; FA, fatty acid;magnetic resonance; TG, triglyceride; MβCD,tty acid-binding protein; SPR, surface plasmo0E,12Z-octadecadienoic acid; Dii-oxLDL, 1,1′dndocarbocyanineperchlorate-oxLDLwork was supported by the American DiabeteJAH).esponding author.ail address: [email protected] (J.A. Hamilton

e cite this article as: A.G. Jay, J.A. Haing and their..., Prostaglandins Leukot

a b s t r a c t

The scavenger receptor CD36 binds numerous small biomolecules, including fatty acids, and even largeligands such as oxidized LDL, for which it is considered a receptor. Although CD36 has often been pos-tulated to “transport” fatty acids across the plasma membrane, fatty acids translocation (mass transportor kinetics) was not affected by expression of CD36 in HEK293 cells; however, esterification of fatty acids(cellular uptake) was increased. These recent results from our lab are consistent with the establishedmechanism of fatty acid entry into cells by passive diffusion (flip-flop) and also with the well-docu-mented enhancement of uptake of fatty acids by CD36 in other cell types. A fascinating new discovery isthat CD36 has multiple fatty acid binding sites on the extracellular domain of CD36. As illuminated bynew methodologies that we have applied, these sites have high affinity and exhibit rapid exchange withthe medium. In an initial study of functional consequences of binding, several dietary fatty acids en-hanced uptake of oxidized LDL into HEK293 cells expressing CD36. This is the first established link be-tween physical binding of fatty acids and a function of CD36, and has implications for obesity andatherosclerosis. New methods as those used in our study could also be applied to elucidate otherfunctional roles of fatty acid binding to CD36.

& 2016 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12. CD36 ‘transport’ of unesterified fatty acids through the plasma membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13. CD36 binding to unesterified fatty acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34. The alteration of biological activities of CD36 via unesterified fatty acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

1. Introduction

Highly expressed in adipocytes, myocytes, macrophages,

2D, two-dimensional; NMR,methyl-beta cyclodextrin;n resonance; HODE, 9S-hy-ioctadecyl3,3,3′,3′ tetra-

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milton, The enigmatic memrienes Essent. Fatty Acids (2

endothelial cells, and a host of other cell types [1], CD36 has beenshown to play important roles in lipid metabolism and is oftenassociated with obesity-related complications [2]. One of the well-established roles of CD36 is binding of oxidized LDL (oxLDL),which promotes accumulation of cholesterol in atheroscleroticplaques [3] and inflammatory responses in certain cells. The mostwidely discussed role of CD36 is in unesterified fatty acid (FA)binding and uptake [4], although the mechanisms of FA uptake arenot well understood. Here we discuss possible connections be-tween both of these CD36 properties; oxLDL interactions and FAinteractions: how they may be functionally related and maybeeven complementary.

brane fatty acid transporter CD36: New insights into fatty acid016), http://dx.doi.org/10.1016/j.plefa.2016.05.005i

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Fig. 1. Fatty acid ‘transport’. Early schematic of CD36 structure including fatty acid uptake via increased lipid droplet formation. Figure modified from [22].

Fig. 2. NewMeasurements of Fatty Acid Binding to CD36 extracellular domain by surface plasmon resonance (SPR). Proteins are bound to an activated SPR chip surface. Next,analytes such as fatty acid complexed with methyl-β-cyclodextrin are pulsed through microfluidics channels and across these bound proteins, including CD36, albumin, andIgG [29].

A.G. Jay, J.A. Hamilton / Prostaglandins, Leukotrienes and Essential Fatty Acids ∎ (∎∎∎∎) ∎∎∎–∎∎∎2

2. CD36 ‘transport’ of unesterified fatty acids through theplasma membrane

Early studies described CD36 as a “necessary transporter” ofunesterified FA through the plasma membrane [5–7]. One of theoriginal postulates was that CD36 could act as an anion trans-porter in the plasma membrane since aqueous unbound fatty acidsexist as anions [7]. This flippase mechanism is not essential be-cause measurements of the carboxyl ionization state in the

Please cite this article as: A.G. Jay, J.A. Hamilton, The enigmatic membinding and their..., Prostaglandins Leukotrienes Essent. Fatty Acids (2

membrane interface showed that fatty acids are about 50% un-ionized at physiological pH's [8]. The same biophysical propertyalso made a permease mechanism unlikely because the lipid bi-layer has a high permeability to un-ionized fatty acids [9]. Manycell types expressing CD36 exhibit a substantial increase in un-esterified FA incorporation into intracellular lipids compared tocells without CD36, as demonstrated in CD36 knockout experi-ments [10]. However, most of these studies measure metabolicproducts (“uptake”), and comparisons between different types of





Fig. 3. EA (trans-oleic acid)), HODE (9S-hydroxy-10E,12Z-octadecadienoic acid), PA(palmitic acid), and DHA (docosahexaenoic acid) binding to CD36 and HAS Re-presentative SPR data is shown, using specific fatty acids from structurally distinctclasses of fatty acids. HODE is the only fatty acid that does not bind to CD36. Figuremodified from [29].

A.G. Jay, J.A. Hamilton / Prostaglandins, Leukotrienes and Essential Fatty Acids ∎ (∎∎∎∎) ∎∎∎–∎∎∎ 3

cells are further complicated by the presence of many factors thatinfluence metabolism or transport, including the presence of otherputative membrane transport proteins and intracellular enzymelevels [11].

When CD36 is expressed in cells, some studies have showndecreases in unesterified FA uptake in the presence of small mo-lecule “inhibitors” [12]. Although these findings have been inter-preted as a disruption of a CD36 ‘transport’ mechanism, thesemolecules could also inhibit metabolism, as found in a new studyin our lab (unpublished). The continuing discussions and debatesabout the roles of CD36 were summarized recently in the article“Commentary of the Fatty Acid Wars: The Translocationists versus


the Diffusionists” [4].The low aqueous solubility of long chain FA has been a major

impediment in the extensive work to clarify the roles of CD36 inboth binding of FA and uptake. FA uptake studies have often uti-lized albumin to better solubilize and deliver unesterified FA to thecells and membranes [7,12,13]. However, the affinity of albuminfor FA is very high, and competition of albumin and CD36 for FAbinding alters the amount of FA that partitions to plasma mem-brane, which complicates the interpretation of results [8,14].

Additional ambiguities of interpretation arise from ‘transport’studies performed over the course of minutes or hours, duringwhich metabolism can play a major role in overall uptake. Fur-thermore, some assays were also performed with procedures thatseparate the donor and acceptors, which can alter FA distribution.

To exclude these technical deficiencies and address the ques-tions of (i) how unesterified FA might cross protein-free plasmamembrane bilayers and (ii) whether CD36 is truly necessary fortransporting unesterified FA, our group developed novel fluor-escent, real-time, FA uptake experiments. First, studies were doneby adding exogenous unesterified natural FA directly without al-bumin as a delivery vehicle to protein-free unilamellar vesicles[15–19]. The experimental design was to perform real-time fluor-escence spectroscopy measurements after adding FA to phospho-lipid vesicles with a pH-sensitive dye (pyranine or BCECF) en-trapped inside. After addition of FA, a decrease was observed influorescence intensity of the pH-sensitive dyes. This pH-changewas the result of the “flip” of un-ionized FA from the outer leafletto the inner leaflet of the plasma membrane, followed by a protonrelease, which occurred within ms–s [15]. In other vesicle andcellular experiments, our group also characterized the delivery ofFA via methyl-beta cyclodextrin (MβCD). At μM concentrations,MβCD is able to rapidly deliver and extract unesterified fatty acidsfrom plasma membranes [20], again within sec. MβCD eliminatedthe complications of using albumin for FA delivery and gave re-sults similar to those with unbound FA alone.

Applications of the new approaches above to several cell typesdemonstrated passive diffusion of FA across the plasma membrane[16] but did not address potential roles or contributions of CD36 tomembrane “transport”. To address this directly using well-con-trolled experiments, we compared FA transport and uptake in thesame cell type (HEK293) with or without CD36 expression. Thesecells metabolize FA slowly, allowing a “window” free of contribu-tions (and complications of interpretation) from metabolism forobserving the movement of FA across the membrane after theiraddition to the exogenous medium. FA flip-flop was found to occurwithin sec, and CD36 expression did not enhance this rapid FAflip-flop rate [21]. However, CD36 did play a significant role inenhancing overall FA uptake. Cells with CD36 exhibited an in-creased rate of intracellular esterification, primarily into trigly-cerides, over a time course of min-hr. The lack of dependence ofthe FA transmembrane movement on the presence of CD36 andthe separate processes of membrane transport and intracellularmetabolism are summarized schematically in Fig. 1.

3. CD36 binding to unesterified fatty acids

Although, according to our studies, CD36 does not enhance thereal-time uptake rate of FA, CD36 does contain a fatty acid-bindingprotein (FABP) homology domain that is reported to bind one FAmolecule, based mostly on indirect evidence [22–24]. The 3D so-lution structure of several FABP family members using multi-dimensional NMR spectroscopy, including our own studies [24],which have shown that the FA is completely enclosed in a beta





Fig. 4. Binding and uptake of oxidized LDL in cells in the presence of fatty acids. Without CD36 expression in HEK293 cells, there is very little Dii-oxLDL binding/uptake (a–d).When CD36 is expressed, fatty acids that bind CD36 via SPR were also found to increase Dii-oxLDL binding/uptake in cells (e.g. PA, in e–h). HODE neither bound CD36 on SPRnor increased Dii-oxLDL (not shown) but DHA also did not bind CD36 on SPR but did not increase Dii-oxLDL binding/uptake (i–l). 3D confocal microscopy uptake data notshown. Figure modified from [29].


barrel. In contrast to FABP, FA binds to albumin at multiple sites inhelical regions [25], and solution state two-dimensional (2D) nu-clear magnetic resonance (NMR) spectroscopy has revealed 9 sites[26]. Unfortunately, NMR methods have not been applied to CD36to achieve such characterization, and would be technicallydifficult.

Past focus with CD36 has been on one binding site for FA, al-though one study of purified CD36 solubilized in Triton X-100suggested the possibility of 2 binding sites. We hypothesized thatthe large and complex ectodomain of CD36 that contains the FABPhomology domain (Fig. 1), might bind several FA molecules. Weapplied the method of surface plasmon resonance, which facil-itates characterization of kinetics, affinity, and specificity of ligandinteractions without detergent solubilization and separation pro-cedures as previously used [22,27]. CD36 is an excellent protein forthis study because the attachment sites simulate the two trans-membrane domains that anchor the protein in the membrane andthe native structure in the ectodomain can be preserved. Toovercome the poor solubility of FA in buffer, we solubilized FA inMβCD (which rapidly delivers FA as described above). MβCD: FAwas then utilized, flowing over the CD36 ectodomain attached tothe chip, as illustrated (Fig. 2). In these experiments, structurallydistinct FA (saturated, monounsaturated [cis and trans], poly-unsaturated, and oxidized) were all tested. Association and dis-sociation binding curves were generated for the first time for CD36in this manner using SPR.

In addition to CD36, these FA binding experiments were doneside-by-side with albumin as a positive control and IgG as a ne-gative control. Albumin bound all FA types with rapid associationand high affinity, as expected since with its multiple binding sitesthat can accommodate many different types of FA. CD36 also


bound all of the FA types except HODE (9S-hydroxy-10E, 12Z-oc-tadecadienoic acid). In addition, CD36 bound FA with comparablerapid kinetic on/off rates to albumin but generally with decreasedaffinity and/or binding sites compared to albumin.

Fig. 3 illustrates our SPR binding data for four FA: oleic, pal-mitic, DHA, and HODE. Interestingly, the data strongly suggestedthat CD36 contains multiple FA binding sites [28] as shown by thesimilarity in binding curves of CD36 compared to albumin and thedose dependency. And while CD36 does not necessarily have asmany FA binding sites as albumin, it is likely to have more thanone. However, binding of several molecules of FA to CD36 wouldnot be sufficient to enhance mass transport of FA through themembrane, even while they could affect the structure and phy-siological functions of CD36. If CD36 does not enhance real-timeFA uptake across the plasma membrane, is there a physiologicalrole for its ability to bind most types of unesterified FA?

4. The alteration of biological activities of CD36 via un-esterified fatty acids

Our group began to investigate the possible role of FA in theuptake of oxLDL using cells with and without CD36 expression andfluorescent labeled human oxLDL [Dii-oxLDL (1,1′dioctade-cyl3,3,3′,3′ tetramethylindocarbocyanineperchlorate-oxLDL)], iso-lated from plasma and oxidized chemically.

We first found that serum-free media leads to very little Dii-oxLDL binding in HEK293 cells with or without CD36 expression[28]. These experiments showed an expected low fluorescencesignal from (i) background fluorescence, (ii) FA already bound tothe oxLDL and (iii) possible low expression levels of other





Fig. 5. Oxidized LDL binding directly to recombinant CD36 is significantly amplifiedby addition of fatty acid. Oleic acid (OA) complexed to MβCD bound human serumalbumin (HSA) on SPR as expected (A) and bound to CD36 (B). Next, oxLDL boundCD36 via SPR as expected (C). The presence of 150 μM OA (complexed to MβCD)added with the Dii-oxLDL gave a strikingly large increase in the R.U., indicatingincreased binding to CD36 despite the relatively small contribution of OA by mo-lecular weight. Figure modified from [29].


scavenger receptors. Interestingly however, with serum-contain-ing media, the cells expressing CD36 gave a significantly higherDii-oxLDL signal. The CD36-dependent Dii-oxLDL uptake and itsenhancement by FA was also verified using 3-D confocal micro-scopy. In addition, charcoal-stripped serum, with all hydrophobicmolecules removed, including FA, gave rise to extremely lowbinding levels of Dii-oxLDL comparable to serum-free media whenCD36 was expressed [28].

Selected results of our confocal imaging of Dii-oxLDL bindingand uptake into HEK293 cells are illustrated Fig. 4. In the controlexperiment with empty vector (Fig. 4A), there is essentially nofluorescence signal even after adding 500 mM oleic acid. In con-trast, in cells expressing CD36 the addition of unesterified FAbound to MβCD into serum-free media ‘rescued’ the CD36-de-pendent Dii-oxLDL binding and uptake in a dose responsivemanner for saturated and monounsaturated FA as well as trans FA[28]. Further, addition of 250 μM palmitic acid resulted in high


levels of uptake of Dii-oxLDL, as shown in Fig. 4B, panel f and h.DHA was the exception and addition of an even higher dose

of the n-3 FA DHA did not promote Dii-oxLDL uptake. Other than13-HODE, which we showed does not bind to CD36 in our SPRanalysis, the only FA that did not stimulate uptake were n-3 FA(even though n-3 bound CD36 according to SPR; something that13-HODE did not do). The concentrations of FA used in our studywere high but within physiological FA concentrations, and theplasma of diabetics and obese persons can contain even higherlevels [8].

The SPR method, which can also detect binding of very largeligands, was next applied to attempt to observe binding of oxLDLto the CD36 extracellular domain. Fig. 5 compares experiments inwhich SPR was applied to study the Dii-oxLDL with and withoutadded oleic acid. As shown in Fig. 5, not only was it possible todetect binding of the lipoprotein ligand (Fig. 5C), the methodelegantly demonstrated significantly enhanced binding of Dii-oxLDL in the presence of oleic acid (Fig. 5D).

Taken together, our novel findings establish a link betweenphysical binding of FA to CD36 and a function of CD36. We hy-pothesize that unesterified FA may bind to CD36, which enhancesbinding of oxLDL to the receptor. This hypothesis is supported withour recent new results for HODE and DHA. HODE was found to notbind to CD36 by SPR, and did not increase Dii-oxLDL uptake, evenat relatively high doses. All the other FA tested gave binding andincreased uptake, except DHA. DHA was shown to bind avidly toCD36 by SPR but gave no increase in Dii-oxLDL, indicating an in-hibitory characteristic of DHA on CD36 oxLDL binding and uptake.

Fig. 6 shows the new crystal structure of the super familymember and close analogue of CD36, LIMP-2, which revealed de-tails that are relevant to oxLDL binding and uptake of lipids fromthe bound LDL [29]. The major lipid transported by LDL is cho-lesteryl ester, a very non-polar molecule that cannot desorb fromthe lipoprotein and pass through an aqueous phase. The LIMP-2structure has interconnected binding hydrophobic cavities whichwould channel this lipid into the cell without contact with water.Consistent with this proposed mechanism, the width of thechannel is similar to the cross sectional area of the steroid ring[30]. The structure is not consistent with a channel for transport ofFA. A recent proposed model of FA binding to the FABP homologydomain and channeling through the protein to deliver FA to cells,is not necessary for the more water soluble amphipathic FAstructure, which desorbs from its carriers into the aqueous phase(Fig. 1). Our new studies have shown that CD36 is not an “essen-tial” FA transporter: FA diffused through the lipid bilayer of theHEK293 cells with or without CD36, and a single FA binding sitecould not compete with the multiple sites in the bilayer. A moredetailed analysis of the crystal structure may reveal regions on theectodomain that bind FA, as predicted by our SPR analysis.

5. Conclusions

There is increasing consensus that CD36 is not essential formass transport or translocation of FA into cells. However, manycells have not been characterized rigorously by appropriatemethods to determine contributions of CD36 to FA translocation,separated from metabolism, and indirect roles for CD36 such asaltering enhancing membrane permeability in some cells remainsto be investigated. The new data on direct binding of FA to theectodomain of CD36 and their effects on oxLDL uptake into cellsuggest that binding to CD36 might alter the docking of oxLDL intothe binding pocket, as discussed in [28]. Regardless of the precisemechanism, the findings have implications for the prevalent dis-eases of obesity, type 2 diabetes and atherosclerosis. Our newstudies should stimulate applications of novel approaches to





Fig. 6. The proposed ectodomain of CD36 based on LIMP-2 homology crystal structure. The proposed structure of the ectodomain of CD36, based on the LIMP-2 homologycrystal structure highlighting the CD36 ligand ‘cavity’. Figure modified from [30].


understand functional roles of FA and may shed light on me-chanisms for new proposed functions of CD36, such as mechan-isms promoting insulin resistance [30] and taste functions [31].

Acknowledgements

We thank Grace Yee for help in preparation of the manuscript.

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