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ScienceDirect Journal of Nutritional Biochemistry 25 (2014) 95 – 103

REVIEWS: CURRENT TOPICS

The impact of dietary fatty acids on macrophage cholesterol homeostasis☆ Milessa da Silva Afonso a , Gabriela Castilho a , Maria Silvia Ferrari Lavrador a , Marisa Passarelli a , Edna Regina Nakandakare a , Simão Augusto Lottenberg b, Ana Maria Lottenberg a,⁎ a

Lipids Laboratory (LIM10), Faculty of Medical Sciences of the University of Sao Paulo, Sao Paulo, Brazil b Endocrinology Service from of the Clinical Hospital of the University of São Paulo, Sao Paulo, Brazil

Received 9 February 2013; received in revised form 11 September 2013; accepted 3 October 2013

Abstract The impact of dietary fatty acids in atherosclerosis development may be partially attributed to their effect on macrophage cholesterol homeostasis. This process is the result of interplay between cholesterol uptake and efflux, which are permeated by inflammation and oxidative stress. Although saturated fatty acids (SAFAs) do not influence cholesterol efflux, they trigger endoplasmic reticulum stress, which culminates in increased lectin-like oxidized LDL (oxLDL) receptor (LOX1) expression and, consequently, oxLDL uptake, leading to apoptosis. Unsaturated fatty acids prevent most SAFAs-mediated deleterious effects and are generally associated with reduced cholesterol efflux, although α-linolenic acid increases cholesterol export. Trans fatty acids increase macrophage cholesterol content by reducing ABCA-1 expression, leading to strong atherosclerotic plaque formation. As isomers of conjugated linoleic acid (CLAs) are strong PPAR gamma ligands, they induce cluster of differentiation (CD36) expression, increasing intracellular cholesterol content. Considering the multiple effects of fatty acids on intracellular signaling pathways, the purpose of this review is to address the role of dietary fat in several mechanisms that control macrophage lipid content, which can determine the fate of atherosclerotic lesions. © 2014 Elsevier Inc. All rights reserved. Keywords: Macrophage; Fatty acids; Cholesterol uptake; Cholesterol efflux

1. Introduction In recent decades, cardiovascular diseases have become the leading cause of morbidity and mortality in developed countries and are increasingly prevalente in developing nations [1]. In particular, atherosclerosis is characterized as a chronic inflammatory disease that is initiated by the subendothelial retention of low-density lipoprotein (LDL), followed by physicochemical modifications such as oxidation. Macrophages located in the arterial intima layer present several scavenger receptors that uptake modified LDL. This contributes to intracellular cholesterol accumulation and inflammation and leads to monocyte recruitment [2,3]. Atherosclerotic lesions are characterized by progressive macrophage lipid accumulation, which leads to foam cell formation. All of the steps in these processes are mediated, in part, by dietary fatty acids. Lipid-laden macrophages release many chemoattractants and inflammatory mediators, leading to lesion progression. Later stages of atherosclerosis are marked by rupture-susceptible lesions that are ☆ Author contributions: Afonso MS, Castilho G, Lavrador MSF, Nakandakare ER, Passarelli M Lottenberg SA, Lottenberg AM were responsible for developing and writing of the whole review. ⁎ Corresponding author. Endocrinology Service from of the Clinical Hospital of the University of São Paulo, CEP: 01246–000 Sao Paulo, Brazil. Tel./ fax: +55 11 30621255. E-mail address: [email protected] (A.M. Lottenberg).

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prone to events such as arterial occlusion and atherothrombotic processes that culminate in apoptosis and the formation of a necrotic core [4]. Macrophage lipid homeostasis is determined by the balance between cholesterol uptake and the efflux of excess cholesterol to extracellular acceptors such as high density lipoprotein (HDL) and apolipoprotein A-I (apo A-I) [5]. Cholesterol is delivered to these cells mainly from modified lipoproteins as well as in a minor amount from native LDL. The accumulation of lipids within macrophagic cells is detrimental to arterial wall thickening because these cells engulf cholesterol depending on the amount of modified lipoproteins available and the expression of scavenger receptors. Lipid-laden macrophages ultimately generate foam cells, a hallmark of the early stages of atherosclerosis. In this scenario, the ATP-binding transporters A-1 (ABCA-1) and G-1 (ABCG-1) play a central role in maintaining the macrophage lipid content by driving reverse cholesterol transport (RCT), an antiatherogenic system that promotes the trafficking of excess cholesterol from arterial wall macrophages to the liver for excretion in the bile and feces [6]. According to clinical and epidemiological studies, dietary fat plays an important role in the plasma cholesterol concentration and also modulates several steps in RCT [7–9]. However, the specific role of each fatty acid in the signaling pathways that modulate macrophage cholesterol homeostasis remains elusive. In addition, depending on the experimental protocol, fatty acids exert multiple effects that can be dose-dependent and/or tissue specific and also related to the

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animal model utilized. Furthermore, the same fatty acid may exhibit different actions in similar metabolic contexts. Therefore, this review explores the role of different fatty acids in the cellular and molecular mechanisms involved in macrophage cholesterol balance, which is an important factor in the initiation, progression and outcome of atherosclerotic lesions. 1.1. Macrophage cholesterol homeostasis Intracellular lipid content is tightly regulated to maintain cellular function and mitosis and to avoid lipotoxicity. The cellular cholesterol content is modulated by sterol regulatory element binding protein (SREBP)-dependent gene regulation, including cholesterol biosynthesis and expression of the LDL receptor. In addition, the intracellular lipid concentration is determined by the HDL-mediated cholesterol and oxysterol efflux. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is the key regulatory enzyme in endogenous cholesterol biosynthesis and converts acetate to mevalonate [10]. The transcriptional regulation of HMG-CoA reductase and the modulation of the native-LDL receptor (B-E) are influenced by the intracellular cholesterol and oxysterol concentrations. These molecules inhibit the proteolytic cleavage of SREBP-2, blocking the transport of its active transcription factor. During sterol depletion, a sensitive domain of the complex that keeps SREBP anchored to the endoplasmic reticulum (ER) releases the transcription factor for nuclear binding, where it induces HMG-CoA reductase and B-E receptor gene expression [11]. The ER is also a resident site for other proteins involved in cellular lipid metabolism. When the load of intracellular free cholesterol rises above the physiological concentration, the ER resident enzyme acyl-coenzyme A: cholesterol acyltransferase (ACAT) esterifies the excess cholesterol, creating a pool of lipids that is maintained in an inert form within lipid droplets in macrophages. However, neutral cholesterol ester hydrolase (nCEH) can reconvert esterified cholesterol into free cholesterol upon sterol demand [10]. Many cells rely on the transcriptional and translational modulation of the B-E receptor by SREBP-2 and proprotein convertase subtilisin/kexin type 9, which increases B-E receptor degradation. By contrast, macrophages have few B-E receptors on their surface, even though these cells present many scavenger receptors, such as the lectin-like oxLDL (LOX-1), the cluster of differentiation 36 (CD36), and the class A and class B scavenger receptors [SRA and scavenger receptor class B type 1 (SR-BI), respectively] [2]. These receptors are quite promiscuous and bind a wide range of molecules, such as advanced glycation end products, oxidized lipids, inflammatory mediators and chemically modified peptides. RCT is particularly relevant for adjusting the intracellular lipid content in arterial wall macrophages. In this regard, RCT acts to prevent atherosclerosis and, disturbances in one or many steps of this pathway are usually related to the premature development of atherosclerosis in humans and animal models [6]. Cholesterol efflux from arterial macrophages occurs by active mechanisms involving ABC transporters. ABCA-1 is a cell membrane protein that requires energy released by ATP hydrolysis to export cholesterol to the outer leaflet of the cell membrane and then to lipidpoor apoA-I and pre-beta HDL. ABCA-1 is up-regulated by oxysterols such as 22-hydroxycholesterol and 24,25-hydroxycholesterol, which activate liver X receptor (LXR). The latter protein dimerizes with retinoic X receptor (RXR) and interacts with a DR-4 sequence in the ABCA-1 gene promoter to induce gene transactivation [12]. ABCA-1 protein content is mostly regulated by post-transcriptional mechanisms that regulate its half-life, including degradation by the ubiquitin–proteasome system, lysosomal degradation through the endosomal sorting complex required for transport pathway and surface proteases [13]. The half-life of ABCA-1 is extended in the plasma membrane by its interaction with apo A-I, which promotes receptor dephosphorylation

of a PEST (proline, glutamic acid, serine, threonine) sequence, impairing ABCA-1 degradation by proteases such as calpains [14]. Studies have revealed a synergistic role for ABCA-1 and ABCG-1 in cholesterol export. ABCG-1 can also remove cholesterol by interacting with large mature HDL particles. In addition, ABCG-1 exports oxysterols, thus minimizing their deleterious effects in macrophages, for instance, by reducing inflammation and apoptosis, which are related to plaque instability and rupture. Likewise, ABCA-1 and ABCG1 are up-regulated by LXR/RXR [15]. In addition to the active process, intracellular cholesterol can be exported to HDL by a passive mechanism involving scavenger receptor class B type 1 (SR-BI). This receptor is located in cholesteroland sphingomyelin-rich domains in the plasma membrane called caveolae and forms a hydrophobic channel through which cholesterol crosses the membrane. This transport depends on the HDL composition of phospholipids and cholesterol and on a membrane composition that favors a concentration gradient that propels cholesterol across the membrane [10]. The exact contribution of SRBI to macrophage RCT is not fully recognized, and studies using animal models have revealed that this receptor does not seem to contribute to RCT in vivo. However, several findings have suggested that SR-BI has a role in the uptake of esterified cholesterol from HDL in the liver during the final step of RCT. In mice, the overexpression of SR-BI is related to the decreased development of atherosclerosis despite elevated plasma HDL cholesterol levels, and SR-BI knockout mice present the opposite effect, displaying increased atherosclerosis despite the presence of high levels of HDL cholesterol [15]. In cholesterol-overloaded macrophages, ABCA-1 contributes to most of the cholesterol export, with a minor contribution ascribed to ABCG-1 and even less to SR-BI [16]. The main mechanisms involved in macrophage cholesterol homeostasis are presented in Fig. 1. 1.2. Fatty acids and macrophage cholesterol uptake The degree of saturation and the amount of fatty acids in the diet can modulate not only the plasma lipoprotein concentration but also cellular cholesterol uptake [7,8,17]. 1.2.1. Lectin-like oxLDL receptor (LOX1) It is well established that palmitic acid is positively associated with atherosclerotic plaque development [18]. A recent work conducted in RAW 264.7 and THP-1-derived macrophages demonstrated that different concentrations of palmitic acid were able to increase LOX1 expression, an effect that was not obtained with other scavenger receptors (CD36, SR-A, SR-BI and CD68) [18]. In the same study, the authors showed that palmitic acid induced LOX-1 expression is mediated by the oxidative stress/mitogen activated protein kinase (p38 MAPK) pathway. In fact, when these cells were treated with the antioxidant n-acetylcysteine and a specific MAPK inhibitor, the deleterious effect of palmitic acid was reversed. Subsequent data from the same group revealed that palmitic acid-mediated LOX-1 expression is also associated with ER stress, which is characterized by an accumulation of misfolded peptides [19]. The overload of misfolded proteins leads to an adaptive response known as the unfolded protein response, which counts with metabolic sensors, such as protein kinase-like ER kinase (PERK), inositol requiring kinase/endonuclease-1 (IRE-1) and activating transcription factor-6, or ultimately leads to pro-apoptotic signaling [20]. 1.2.2. Endoplasmic reticulum stress and apoptosis Prolonged ER stress is detrimental in macrophages and is present in many steps of atherogenesis, including foam cell formation. Moreover, in advanced lesions, ER-stressed macrophages are prone to apoptosis, leading to less stable plaques, which correlate with

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Fig. 1. Macrophage cholesterol homeostasis. Arterial wall macrophages display scavenger receptors on their surface (CD36, SR-A, and LOX-1), which uptake modified LDL, increasing the intracellular cholesterol and oxysterol content. The uptake of native LDL contributes minimally to the balance of cholesterol in macrophages. Upon lipid accumulation, free cholesterol is esterified via ACAT and stored in an inert form in lipid droplets. Oxysterols are strong ligands of LXR, which heterodimerizes with RXR, leading to ABCA-1 and ABCG-1 gene transcription. Cholesterol can also be exported by aqueous diffusion, which is mediated by SR-BI. This receptor can also take up esterified cholesterol by selective uptake. apo A-I removes cholesterol by interacting with ABCA-1, while HDL removes cholesterol by interacting with ABCG-1. The cholesterol acceptors apo A-I and HDL drive cholesterol from the periphery to the liver for bile secretion via reverse cholesterol transport.

higher rupture events [21]. Under experimental conditions, ER stress may be pharmacologically induced by thapsigargin, which disrupts intracellular calcium homeostasis through sarcoendoplasmic Ca2+ ATPase inhibition [22]. Although SAFAs are closely associated with deleterious events in atherosclerosis development, there is a body of evidence that assigns an anti-atherogenic status to unsaturated fatty acids (UFAs). A new proposed mechanism regarding atherosclerosis development and prevention exerted by UFAs is its action in preventing the expression of the scavenger receptor LOX-1 in macrophages treated with SAFAs [18,19]. According to Ishiyama et al. (2011), oleic and linoleic acids can attenuate ER stress in SAFA treated macrophages, which suppresses LOX-1 expression. In addition, UFAs also suppress thapsigargin-induced LOX-1 up-regulation, with reduced activation of ER stress markers; all of these events culminate in decreased oxLDL uptake [19]. Regarding ER stress-mediated apoptosis, SAFAs increase expression of IRE-1α, PERK, binding immunoglobulin protein (Bip), phospho-c-Jun N-terminal kinase (JNK-p), X-box binding protein-1 and C/EBP homologous protein (chop), as demonstrated in mouse peritoneal macrophages treated with stearic acid. These changes were further correlated with an increase in caspase 3 cleavage and TUNELpositive cells, emphasizing the effect of SAFAs on macrophage apoptosis [23,24]. In mice, macrophage apoptosis mediated by thapsigargin plus SAFA is dependent on CD36 and TLR2 [25]. A few groups have shown that macrophage programmed cell death is a defense mechanism that reduces both the number of lesional macrophagic cells and their autocrine and paracrine effects, which ultimately leads to necrosis [26]. A recent study has

demonstrated that palmitic acid induces macrophage death via ROS generation mediated by kinase receptor-interacting protein 1 (RIP1), a key signaling molecule for necrosis [27]. Although SAFAs are involved in macrophage apoptosis, in some situations, depending on the type of UFAs, they can also promote cellular death. For instance, arachidonic acid esterified with 7ketocholesterol (7-KC) by ACAT-1 is considered a candidate molecule involved in apoptotic pathways [28]. In the apoptotic process, arachidonic acid is released by the action of phospholipase A2 (PLA2), which is activated by oxysterol (7-KC)/oxLDL [29,30]. Although arachidonic acid induces these events, it comprises b10% of the total 7-KC esters, while palmitic and oleic acids contribute ~21% and ~71% of the total 7-KC esters, respectively [28]. Therefore, it is possible to conclude that although arachidonic acid represents a minor amount of total 7-KC esters, it can induce apoptosis by oxLDL/ 7-KC-mediated PLA2 activation. 1.2.3. Cluster of differentiation 36 (CD36) Regarding CD36, products of lipid peroxidation, such as reactive aldehydes, induce its expression via nuclear factor erythroid-2related factor 2 or peroxisome proliferator-activated receptor gamma (PPARγ). These effects are directly associated with the generation of lipid-laden plaques, thus creating a positive feedback loop between lipid peroxidation and CD36 macrophage expression [31–33]. In fact, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and their oxidized metabolites increased CD36 expression in THP-1 macrophages, an effect not observed in macrophages treated with lauric, myristic, palmitic and stearic acids [34]. On the other hand, compared to oleic acid, palmitic acid increases CD36 expression,

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as demonstrated in a study performed in human U937 and THP-1 macrophages, culminating in an increased uptake of oxLDL [35]. The authors claim that the effect of SAFAs on CD36 is mediated by ceramides because the pharmacological inhibition of de novo ceramide synthesis by fumonisin B1 impairs the expression of this receptor in macrophages. Thus, divergent responses may occur depending on the type of fatty acid analyzed. The isomers of conjugated linoleic acid (CLA) also regulate transcription in many cell types. These fatty acids are a group of naturally occurring isomers of linoleic acid (C18:2, ω-6) that differ in the position or geometry of their double bonds. The predominant biologically active isomers are cis9, trans11-CLA (c9, t11-CLA) and trans10, cis12-CLA (t10, c12-CLA), which are the major isomers used in many studies, although minor isomers such as trans 9, trans 11-CLA (t9, t11-CLA) have also been studied [36,37]. In THP-1 foam cells, the isomers c9,t11-CLA and t10,c12-CLA increase intracellular esterified cholesterol content compared to linoleic acid, although c9,t11-CLA has not changed and t10, c12-CLA has reduced CD36 expression [38]. The same effect of CLA isomers on CD36 expression was observed in a study conducted in RAW 267.4 macrophages; however, no increase in the amount of esterified cholesterol within the macrophages was observed when compared to linoleic acid treatment [39]. These contradictory effects of CLAs on cholesterol ester content were further described by the authors as a possible regulatory effect of CLAs on ACAT and/or nCEH [39]. Although there are no studies indicating the action of Trans fatty acids (TFAs) on the scavanger receptors, an in vitro study demonstrated that elaidic acid did not increase the macrophage lipid content compared to oleic, linoleic and α-linolenic acids [40]. This effect may be attributed to the fact that fatty acid loaded macrophages were more enriched in triglycerides than in esterified cholesterol. However, a recent study conducted in LDLr knockout mice revealed that elaidic acid increased the lesion area, macrophage infiltration and arterial total cholesterol content [41]. These divergent results may be attributed to the fact that the in vitro model did not mimic the in vivo conditions to which monocytes/macrophages are exposed [40]. This limitation was discussed by the authors of this important in vitro study. 1.2.4. ACAT and selective uptake The accumulation of free cholesterol in macrophages leads to cytotoxic events that can trigger several pathological consequences, such as oxidative stress and inflammation (Table 1) [42], and these deleterious conditions are prevented by ACAT1 activity. Oleic acid is the preferential substrate for ACAT1 for the production of esterified cholesterol, and this fatty acid also accumulates in triglyceride molecules via diacylglycerol acyltransferase [43–45]. By contrast, palmitic acid accumulates in the diacylglycerol, monoacylglycerol, and phospholipid fractions. Although these fatty acids (palmitic and oleic acids) have different roles in cholesterol esterification, they display similar uptake in J774 macrophages [44]. In agreement, THP1-derived macrophages treated with LDL from primates that were fed high-fat diets containing oleic acid (MUFA) displayed a higher accumulation of total and esterified cholesterol compared to cells treated with LDL from primates that were fed SAFA (palmitic acid), UFA ω-6 (linoleic acid) or UFA ω-3 (EPA and DHA) [46]. However, without macrophage cholesterol loading, UFAs, such as linolenic and oleic acid, also induced cholesterol esterification compared to cells treated with stearic acid or EPA, although only oleic acid displays specificity as a substrate for ACAT1 [43]. However, in THP-1 macrophage-derived foam cells, EPA increases ACAT1 mRNA levels, which was not observed for linoleic acid [47]. These data confirm the differential impact of each fatty acid depending on the lesion stage. One possible explanation for why polyunsaturated fatty acids did not induce cholesterol accumulation is that they increase cholesterol

ester hydrolysis, providing free cholesterol for extracellular efflux in culture medium depleted of triglycerides [46]. Dietary fatty acids may also induce selective uptake from circulating LDL particles by SR-BI, which removes specific components of the lipoprotein, such as esterified cholesterol [48]. UFAs stimulate LDL uptake in J774 macrophages by increasing LDL receptor activity, which is attributed to a decrease in intracellular cholesterol content as a result of increased ACAT activity; in turn, LDL uptake and cholesterol synthesis are up-regulated to supply the plasma membrane with lipids [49]. Regarding the effect of fatty acids on the uptake of esterified cholesterol derived from LDL, oleic acid and EPA increased selective uptake in an ACAT-dependent mechanism, because inhibition of ACAT activity reduced this effect by 50% [50]. In addition, SAFAs seem to increase the selective uptake of esterified cholesterol in vivo, which is positively associated with an increase in plasma cholesterol and lipase lipoprotein (LPL) concentrations, since this enzyme promotes a bridge that facilitates the entry of esterified cholesterol into macrophages [8]. These data were confirmed by Chang et al. (2009), who demonstrated that ω-3 fatty acids from fish oil abolished SAFA-mediated selective uptake and therefore reduced cholesterol ester deposition and LPL expression in the aortic intima [51]. Kämmerer et al. [52] demonstrated that oxidized lipids, such as the hydroxylated derivative of linoleic acid (13-HODE), have atheroprotective effects. However, the classical deleterious events triggered by lipid peroxidation, such as oxidative stress and inflammation, must be addressed. Indeed, 13-HODE, which is a PPARγ ligand, displayed deleterious effects related to the stimulation of CD36 expression and, therefore, cholesterol uptake [53]. In addition, Itoh et al. (2008) stated that PPARγ can bind simultaneously to two oxidized molecules, 9- and 13-HODE, implying that the two different fatty acids may provide a graded response to varying compositions of the cellular pool of fatty acid ligands [52,54]. Although PPARγ transactivation stimulates cholesterol uptake, it also presents anti-inflammatory properties. Moreover, PPARγ agonists modulate genes involved in whole lipid metabolism, including genes involved not only in lipid uptake, such as CD36, but also in cholesterol efflux, including liver X receptor α (LXRα) [33,55]. 1.3. Fatty acids and macrophage cholesterol efflux Cholesterol efflux is a unique mechanism in maintaining macrophage lipid homeostasis, and dietary fatty acids also modulate several signaling pathways involved in cholesterol export to extracellular acceptors (Table 2). 1.3.1. ATP-binding cassette A-1 (ABCA-1) and G-1 (ABCG1) A well-referenced study from Wang and Oram (2002) stated that UFAs (oleic, palmitoleic, linoleic and arachidonic acids) inhibit the export of cholesterol and phospholipids from macrophages because they increase ABCA-1 degradation [56]. The authors also demonstrated that the effect of UFAs on ABCA-1 was a post-translational event, as the mRNA levels and methionine incorporation into ABCA-1 were unaltered. In fact, several authors have affirmed that ABCA-1 is widely modulated at the post-translational level [57–59]. The molecular mechanisms of ABCA-1 degradation upon exposure to UFAs have been addressed and include the increased phosphorylation of the serine residues of the ABCA-1 peptide, which destabilizes the protein structure and facilitates its degradation [60]. Another group also described UFA-mediated ABCA-1 degradation as a mechanism that is dependent on fatty acid thioesterification of acylCoA by the enzyme acyl-CoA synthetase 1 (ACSL1). These results were obtained in peritoneal macrophages from ACSL1 knockout mice

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Table 1 Fatty acids and macrophage cholesterol uptake Study design

Fatty acid concentration

Effects on gene expression and cholesterol efflux

Reference

RAW 264.7 and THP-1-derived macrophages RAW 264.7 and THP-1 macrophages

200 μM palmitic, oleic, linoleic, palmitoleic, myristic and lauric acids 200 μM palmitic, oleic or linoleic acids

[18,19]

RAW 267.4 macrophages with acLDL

50 μmol/L of CLA isomers (c9, t11 and t10,c12) or linoleic acid 10 μM lauric, myristic, palmitic, stearic, oleic acid, linoleic, arachidonic, EPA, DHA 100 μmol/L oleic, elaidic (trans), linoleic, α-linolenic acids 100 μM c9, t11-CLA, t10,c12-CLA, linoleic acid and stearic acid

Palmitic acid increase LOX-1 expression and oxLDL uptake as compared to oleic, linoleic, palmitoleic, myristic, and lauric acids Palmitic acid increases LOX-1 expression and oxLDL uptake. However, these events are prevented when palmitic acid is incubated with oleic or linoleic acids Increase CD36 expression; Did not increase the amount of esterified cholesterol as compared to linoleic acid UFAs, especially EPA and DHA, increase CD36 expression which was not observed for SFAs Elaidic acid do not exert any adverse effect on macrophage lipid content as compared to the others t10,c12-CLA and c9, t11-CLA increase CD36 expression and t10,c12-CLA increases intracellular esterified cholesterol content as compared to linoleic acid MUFA-diet promotes higher total and esterified cholesterol accumulation as compared to SAFA, ω-6 PUFA and ω-3 PUFA

THP-1 and human monocyte-derived macrophages THP-1 THP-1 foam cells

THP-1-derived macrophages incubated with LDL from primates THP-1 macrophages-derived foam cells THP-1 macrophages without cholesterol loading Human U937 and THP-1 macrophages

SAFA (29% as palmitic acid), MUFA (58% as oleic acid), ω-6 PUFA (58% as linoleic acid), ω-3 PUFA (9% EPA + 7% of DHA) 100 μM linoleic acid, EPA or CLA 300 μM oleic, linoleic, arachidonic, stearic acids and EPA 300 μM palmitic acid, 50 μM oleic acid or 300 μM palmitic plus 50 μM oleic acid

J774 macrophages

303 μM oleic acid

J774

300 μM oleic acid and EPA

J774 macrophages C57BL/6 mice fed high-fat diets

15-30 μg/mL oleic and palmitic acids High fat diet (42% total calories from fat) enriched in SAFA (71% coconut oil) or fish oil (50% menhaden oil) Chow (4.5% fat) or high saturated fat diet (21% fat being 71% from coconut oil) 40% of energy as fat (PUFA, SAFA and Trans fatty acids)

C57BL/6 and apoE knockout mice fed a chow or high fat diet LDLr knockout mice fed a high fat diet

treated with oleic and linoleic acids, which were protected from ABCA-1 degradation, leading to a higher apo A-I-mediated cholesterol efflux rate [61]. In addition, UFAs reduce ABCA-1 promoter activity mediated by 22-(R)-hydroxycholesterol, a well-known LXR agonist [62]. These data indicate that UFAs compete with oxysterols to bind to LXR and therefore impair ABCA-1 transcription. In fact, the same group demonstrated in RAW 264.7 macrophages transfected with wild type h-ABCG1 and the ABCA1 promoter that EPA reduces promoter activity compared to bovine serum albumin (BSA)-treated control cells. However, in cells with a mutated DR4 promoter, to which the transcriptional inducers LXR and RXR bind, this effect was not observed, clearly indicating that UFAs suppress the transcription of the ABCG-1 and ABCA-1 genes through a mechanism that involves the binding of LXR/RXR to their promoters [63]. Because oxysterols act as LXR ligands and therefore increase ABCA-1 and ABCG-1 expression, researchers have turned their attention to identifying an LXR agonist. However, LXR is also involved in SREBP-1c and SCD-1 transcription, which favors fatty acid synthesis and desaturation, leading to the generation of UFAs that could further cleave and destabilize ABCA-1 and alter its transcription pattern [56]. By contrast, recent studies conducted in macrophage-derived foam cells (MDFCs) have demonstrated that α-linolenic acid increases cholesterol efflux by transactivation of the farnesoid X receptor (FXR), which reduces SREBP-1c and SCD-1 transcription via LXR [64]. The authors claim that the α-linolenic acid-mediated decrease in SCD-1 expression reduces the generation of MUFAs, thus diminishing the loading of substrate for cholesterol ester synthesis and allowing free cholesterol to be transported along the plasma membrane to extracellular acceptors [64]. It is important to emphasize that the authors compared cells treated with increasing concentrations of α-linolenic acid to cells treated with BSA alone

EPA increased the ACAT1 mRNA levels as compared to linoleic and CLA Oleic, linoleic and arachidonic acids induce cholesterol esterification as compared to cells treated with stearic acid or EPA Palmitic acid increases CD36 expression and oxLDL uptake as compared to oleic acid alone or co-incubation of palmitic and oleic acids Oleic acid stimulates LDL uptake, increases LDL receptor activity and ACAT activity as compared to control cells Oleic acid and EPA increase selective uptake via ACAT activity as compared to stearic acid or control cells Cellular uptake of radiolabeled oleate and palmitate was similar ω-3 fatty acids reduce selective uptake and therefore reduced CE deposition and LPL expression in the aortic intima as compared to SAFA group SAFA diet increases selective uptake of CE as compared to chow groups Trans diet increases lesion area, macrophage infiltration and arterial TC content as compared to PUFA and SAFA

[19]

[39] [34] [40] [38]

[46]

[47] [43] [35]

[49] [50] [44] [51]

[8] [41]

instead of cells treated with other fatty acids with different degrees of saturation. However, in another study of cholesterol efflux in macrophages exposed to different fatty acids, α-linolenic acid was shown to increase cholesterol exportation compared with palmitic, oleic and linoleic acids [65]. ABCA-1 may also be transcriptionally regulated by histone deacetylases (HDACs). The HDAC inhibitor trichostatin A can reverse the impaired expression of ABCA-1 and ABCG-1 in cells treated with UFAs [66]. However, these studies were conducted in vitro, and some used high concentrations of fatty acids to mimic individuals suffering from metabolic syndrome and insulin resistance, conditions in which free fatty acid concentrations are elevated. The capacity of macrophages isolated from mice fed high-fat diets supplemented with fish oil to export cholesterol was not improved compared with macrophages isolated from mice fed a soybean oil enriched diet. However, macrophages isolated from coconut oil-fed mice had decreased cholesterol efflux as compared to macrophages from fish oil-fed mice. No difference was observed in the cholesterol efflux from peritoneal macrophages between the low and high soybean oil groups [67]. SAFAs do not seem to directly destabilize or degrade ABCA-1, although they can induce ER stress, which is associated with decreased ABCA-1 protein levels [62,63,68]. SAFAs can induce ABCG-1 transcription independently of LXR/RXR, [63]. However, when exposed to SCD-1, long chain saturated fatty acids, such as palmitic acid and stearic acid, become unsaturated, which can lead to transporter degradation [69]. In monocyte-derived human macrophages, elaidic acid (trans-9, 18:1) reduces ABCA-1-mediated cholesterol efflux in comparison to vaccenic acid (trans-11, 18:1). In addition, the impact of elaidic acid was more severe in cholesterol-enriched macrophages compared to cells with normal cholesterol concentrations [70]. On the other hand,

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Table 2 Fatty acids and macrophage cholesterol efflux Study design

Fatty acid concentration

Effects on gene expression and cholesterol efflux

Reference

RAW267.4 macrophages

500 μM/L oleic, 100 μM/L EPA or 500 μM/L palmitic acid

[62]

RAW264.7 and THP-1 macrophages transfected with wild type h-ABCG1 and the ABCA1 promoter RAW 264.7 macrophages

100 μM oleic, linoleic, EPA, arachidonic, palmitic and stearic acids

Oleic acid and EPA reduce ABCA-1 mRNA and ABCA-1 promoter activity mediated by 22-(R)-hydroxycholesterol, a well-known LXR agonist, as compared to palmitic acid and untreated cells Reduce promoter activity compared to bovine serum albumin (BSA)-treated control cells, palmitic and stearic treated cells.

RAW 264.7 cells and THP-1 cells

100 μM t9,t11-CLA, c9, t11-CLA and t10, c12-CLA isomers 100 μM linoleic acid, stearic acid, c9,t11-CLA and t10,c12-CLA 100 μM t9, t11-CLA vs. c9, t11-CLA

THP-1 foam cells THP-1 and Human primary monocytes

50 μmol/L c9, t11-CLA, t10, c12-CLA, linoleic acid vs. BSA treated cells

J774 and RAW267.4 macrophages

125 μM oleic, palmitoleic, linoleic, arachidonic, stearic and palmitic acids

J774 macrophages

125 μM Palmitic, stearic, palmitoleic, oleic, linoleic and arachidonic acids 70 μM elaidic, vaccenic and palmitic acids and BSA-treated cells A 30% fat diet enriched with TRANS, SAFA or PUFA

Monocyte-derived human macrophages Mouse peritoneal macrophages incubated with HDL from young normocholesterolemic subjects fed a normal fat diet Peritoneal Macrophages from mice fed a high fat diet

Peritoneal macrophages from ACSL1 knockout mice MDFCs

High fat containing fish, soybean or coconut oils (42% energy from fat with 37% energy from soybean oil, hydrogenated coconut oil, and menhaden oil) 225 μM oleic and linoleic acids

25, 50 and 100 μM of α-linolenic acid and BSA treated cells

recent data have demonstrated, in J774 macrophages, that elaidic acid did not reduce ABCA-1-mediated cholesterol efflux when compared to monounsaturated fatty acid [71]. However, in this later study, the authors compared the effect of TFA to MUFA, which is known by reducing ABCA-1 macrophage content. In another study conducted by our group, LDLr-KO mice fed a high fat diet enriched in TFAs, had increased ABCA-1 protein content in the lesion area as compared to SAFAs and PUFAs fed animals. It is important to note that this result was attributed to a higher atherosclerotic plaque formation and, therefore, to a higher macrophage content [41]. Therefore, the critical analysis of these data reinforces the need of a carefully conducted interpretation in order to extract the right idea from each study concerning TFA effects in macrophages. Considering HDL isolated from young normocholesterolemic subjects who consumed a recommended amount of fat (25–30% of their total calorie intake) enriched with TFA, no impairment in the ability of HDL to remove cholesterol from cholesterol-loaded macrophages was found in comparison to HDL from subjects who consumed diets that were enriched in UFAs or SAFAs [72]. Together, these data suggest that the atherogenic effects of fatty acids depend on the previous metabolic status of the subject and on the origin of the fatty acid. Elaidic acid is a product of the industrial hydrogenation of vegetable oil and is atherogenic, whereas vaccenic acid, which is naturally synthesized by bacterial fermentation in ruminants, is apparently harmless. In macrophage colony stimulating factor-differentiated monocyte derived macrophage and THP-1 macrophages, t9,t11-CLA activates SREBP and ABCG1 compared to the c9,t11-CLA and t10,c12-CLA isomers [37]. It was subsequently shown in THP-1-derived macrophages that this isomer is able to induce the target genes of LXRα,

Increase expression of ABCA-1 and LXR Decrease intracellular concentration of esterified cholesterol, which is accompanied by increased HDL-mediated cholesterol efflux t9,t11-CLA activates ABCG1 as compared to the c9, t11-CLA and t10, c12-CLA isomers t10, c12-CLA increases esterified cholesterol and decreases free cholesterol concentration as compared to linoleic acid t9, t11-CLA induces ABC transporters and increases the cellular cholesterol efflux to both HDL and apo A-I Oleic, palmitoleic, linoleic and arachidonic acids inhibit the export of cholesterol and phospholipids from macrophages because they increase ABCA-1 post-translationally degradation as compared to stearic and palmitic acids. As a result of SCD-1 activation, SAFAs become UFAs which can lead to transporter degradation Reduces ABCA-1-mediated cholesterol efflux specially in cholesterol-enriched macrophages There were no difference on the macrophage cholesterol efflux among the groups studied

[63]

[39]

[37] [38] [72] [56,60]

[69] [70] [71]

Coconut oil decreased cholesterol efflux as compared to fish oil; No differences in cholesterol efflux between fish and soybean oil

[67]

ACSL1 knockout mice were protected from ABCA-1 degradation, leading to a higher apo A-I-mediated cholesterol efflux rate as compared to cells from wild type mice α-linolenic acid increases cholesterol efflux by transactivation of the farnesoid X receptor

[61]

[64]

which includes genes encoding the ABC transporters and lipogenic enzymes. Consequently, this isomer increases the cellular cholesterol efflux to both HDL and apo A-I, in contrast to c9,t11-CLA [73]. However, RAW 264.7 macrophages treated with c9,t11-CLA or t10, c12-CLA had higher expression of ABCA-1 and LXRα and, consequently, an increase in the cholesterol efflux compared to control cells that were not treated with fatty acids [39]. However, other studies conducted in macrophages have also demonstrated that CLAs lead to an increase in intracellular reactive oxygen species and isoprostane (8-epi-prostaglandin F2αIII) production, which promotes apoptosis, as assessed by Annexin V staining [74,75]. When the systemic effect of CLA is analyzed, its beneficial effect is questionable. In fact, obese men challenged with a CLA-enriched diet had increased oxidative stress and inflammatory insult, as evidenced by their urinary isoprostane concentrations and plasma reactive C protein levels, respectively [76]. Therefore, there is no consensus regarding the effects of CLAs on macrophage cholesterol export. 1.3.2. PPARs and ACAT CLAs are described as strong PPARα and PPARγ ligands [77], and Hirakata et al. (2004) demonstrated that PPARγ activation increases nCEH and inhibits ACAT expression [78]. Together, these mechanisms increase the availability of free cholesterol to extracellular acceptors. In fact, macrophages treated with c9, t11-CLA or t10, c12-CLA show a decrease in the intracellular concentration of esterified cholesterol, accompanied by increased HDL-mediated cholesterol efflux. Increased cholesterol efflux is most likely a consequence of stimulated ABCA-1 expression. When studying the effects of CLA on cholesterol efflux, control cells that were incubated in fatty acid-free medium were used for comparison, which is not appropriate for comparing the

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101

Fig. 2. Fatty acids and macrophage lipid homeostasis. SAFAs induce oxLDL uptake by elevating LOX-1 and CD36 expression, and increased lipid content leads to macrophage-ER stress, which ultimately triggers apoptotic pathways. SAFAs can induce the expression of ABCG-1 but not ABCA-1. However, as SAFAs are converted to UFAs by SCD-1 they further contribute to ABCA-1 destabilization. UFAs can diminish both ABC transporters and can also induce CD36 expression and the generation of oxidized lipids, even though UFAs alleviate ER stress and SAFAs-mediated LOX-1 expression. The beneficial effects of UFAs involve the release of free cholesterol for extracellular exportation through induction of nCEH and promoting free cholesterol esterification by ACAT1 stimulation in foam cells. TFAs reduce apo A-I-mediated cholesterol efflux by reducing ABCA-1 expression. c9,t11-CLA and t10,c12-CLA are PPARγ ligands that can induce CD36 expression and, consequently, oxLDL uptake. The effects of CLAs on esterified cholesterol (EC) content as well as on LXR-ABC transporters are contradictory: stimulatory or neutral.

effects of different fatty acids. Moreover, linoleic acid decreases both the ABCA-1 protein content and cholesterol export, which makes comparisons with other fatty acids, such as CLAs, difficult [39]. The fatty acid concentrations that were utilized in this study were considerably high, and the authors emphasized that it is challenging to extrapolate their cellular model to humans. Analysis of the cellular fatty acid composition demonstrated that CLAs were incorporated while MUFAs were reduced in macrophages, which could contribute to the lower level of esterified cholesterol in the CLA-treated macrophages because MUFAs are specific ACAT substrates [39,43]. Foam cells exposed to CLAs exhibited increased esterified cholesterol, while only t10,c12-CLA was able to reduce the concentration of free cholesterol, an effect that reduces the availability of lipids to extracellular acceptors, although no difference was observed in the apo A-I-mediated cholesterol efflux [38]. A summary of the data regarding the effects of dietary fatty acids on macrophage cholesterol homeostasis is presented in Fig. 2. 2. Conclusion Experimental studies of the impact of dietary fatty acids on macrophage cholesterol homeostasis do not necessarily reflect the roles of these fatty acids in the whole organism. Not all of the data indicating beneficial or detrimental effects of macrophage lipid accumulation are coincident with the cardiovascular endpoints stated in important clinical and epidemiological studies, which have results that support essential nutritional guidelines. Even when experimental protocols utilize the same fatty acid, divergent results may be

attributed to the macrophage cell lineage, cholesterol loading, concentration of the fatty acid in the culture medium and presence or absence of transcriptional nuclear agonists that may induce different responses in the pathophysiological background concerning metabolic stress. SAFAs impair macrophage cholesterol homeostasis because of their deleterious effects on scavenger receptor expression, ER stress, inflammation and apoptosis. UFAs counteract most of these SAFAmediated mechanisms, although they are also shown to reduce cholesterol efflux. CLAs induce the expression of CD36, an important scavenger receptor involved in atherosclerosis development. TFAs enrich macrophages in cholesterol, impair ABCA-1 expression and elicit inflammation. The differential effects found in cells exposed to fatty acids can result from a series of macrophage adaptive responses that act synergistically to maintain cholesterol balance and harmonic intracellular mechanisms. In this regard, advances in cellular and molecular biology in the field of nutrition will facilitate the elucidation and comprehension of important phenomena involved in the effects of different fatty acids on foam cell formation and atherosclerotic outcomes.

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